Abschlussarbeiten

Habilitation
Promotion
Master-Arbeit
Bachelor-Arbeit
Diplomarbeit

Habilitation

Jahre: 2020 | 2017 | 2016 | 2014 | 2005

2020

Centrifugal microfluidic unit operations and applications
2020, Nils Paust
Erstgutachter: Prof. Yoon-Kyoung Cho, School of Life Sciences, UNIST, Ulsan / Korea
Zweitgutachter: Prof. Anja Boisen, Technical University of Denmark, Kgs. Lyngby / Dänemark
Kurzfassung

2017

Towards multi-scale tailoring of electrochemical energy applications
2017, Simon Thiele
Kurzfassung

2016

Bioelektrochemische Systeme – vom energieautarken Implantat zur nachhaltigen Bioproduktion
2016, Sven Kerzenmacher
Kurzfassung

2014

Advanced lab-on-a-chip technology platforms for leveraging point-of-care nucleic acid testing
2014, Felix von Stetten
Kurzfassung

2005

Centrifugal Microfluidics for Lab-on-a-Chip and Micro Process Engineering
2005, Jens Ducrée
Kurzfassung

Promotion

Jahre: 2024 | 2023 | 2022 | 2021 | 2020 | 2019 | 2018 | 2017 | 2016 | 2015 | 2014 | 2013 | 2012 | 2011 | 2010 | 2009 | 2008 | 2007 | 2006 | 2000

2024

A Novel Multianalyte Lateral Flow Immunoassay for the Simultaneous Detection of Protein and Nucleic Acid Biomarkers
2024, Anna Klebes
Erstgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK - Chemie und Physik von Grenzflächen
Kurzfassung
Investigation of salt precipitation and water management in zero-gap CO2 electrolyzers producing CO
2024, Joey Disch
Erstgutachter: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Ingo Krossing, Institut für Anorganische und Analytische Chemie Albert-Ludwigs-Universität Freiburg
Kurzfassung

2023

Development and quantitative assessment of generic processes to enable 3D-bioprinting of arti cial tissue
2023, Fritz Koch
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Günter Finkenzeller, Uniklinik Freiburg
Kurzfassung
Electrode engineering for anion-exchange membrane water electrolyzers
2023, Susanne Koch
Erstgutachter: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung/Elektrochemische Energiesysteme
Zweitgutachter: Prof. Dr. Stefan Glunz, INATECH, Freiburg
Kurzfassung
Fantastic nanoparticles and where to find them: Advanced electron microscopy for the investigation of novel electrocatalysts
2023, Philipp Heizmann
Erstgutachter: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Anna Fischer, Institut für Anorganische und Analytische Chemie, Uni Freiburg
Kurzfassung
Optimization and characterization of proton-exchange membrane fuel cells based on novel hydrocarbon ionomers
2023, Hien Nguyen
Erstgutachter: Dr. Severin Vierrath, EES-Group, IMTEK
Zweitgutachter: Prof. Dr. Thomas Hanemann, IMTEK - Werkstoffprozesstechnik
Kurzfassung

2022

Automation of library preparation for next generation sequencing by centrifugal microfluidics
2022, Jacob Hess
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Melanie Börries, Institut für Medizinische Bioinformatik und Systemmedizin, Uni Freiburg
Kurzfassung
Bound-free phase detection immunoassay compatible with nucleic acid amplification point-of-care devices
2022, Benita Johannson
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
Kurzfassung
Characterization of Membrane Electrode Assemblies for Vanadium Redox Flow Batteries
2022, Brian Shanahan
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
Kurzfassung
Detection and Enumeration of Bacteria by Pump-free Digital Droplet Assays
2022, Yu-Ting Kao
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Piotr Garstecki, ICHF, Warschau
Kurzfassung
Liquid Biopsy for colorectal cancer – from cfDNA extraction to the quantification of point mutations by multiplexed ddPCR
2022, Franziska Schlenker
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: PD Dr. Cornelia Eckert, Charite Universitätsmedizin Berlin
Kurzfassung
Microfluidic Automation of Sample Preparation for Mass Spectrometry Based Proteomics
2022, Niklas Klatt
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Oliver Schilling, Inst. Für Molekulare Medizin, Uni Freiburg
Kurzfassung

2021

Fabrication of hollow 3D tissue models with drop-on-demand bioprinting and controlled cellular self-assembly
2021, Kevin Tröndle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Soeren Lienkamp, Universität Zürich, Schweiz
Kurzfassung
Microfluidic system integration for droplet based digital nucleic acid testing
2021, Martin Schulz
Erstgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK - Chemie und Physik von Grenzflächen
Kurzfassung
Normally-closed dispensing valve and in-line pressure and flow sensor fabricated in low-cost polymer technology
2021, Sabrina Kartmann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Joost Lötters, University of Twente
Kurzfassung

2020

Development of Novel Electrode Concepts for Proton Exchange Membrane Water Electrolyzers
2020, Melanie Bühler
Erstgutachter: Prof. Dr. Stefan Glunz, INATECH, Uni Freiburg
Zweitgutachter: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
Kurzfassung
Mediator displacement LAMP: A novel method for universal sequence-specific detection of isothermal nucleic acid amplification
2020, Lisa Becherer
Erstgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK - Chemie und Physik von Grenzflächen
KurzfassungNucleic acid amplification tests (NAATs) are an essential diagnostic tool throughout the field of life sciences, including clinical applications, food quality control, and environmental monitoring. In the last 20 years, polymerase chain reaction (PCR) has been steadily replaced by isothermal alternatives. These are especially suitable for point-of-need (PON) applications. Loop-mediated isothermal amplification (LAMP) stands out as a particularly robust and highly sensitive method. Special attention is given to the sequence-specific, fluorescence-based detection of LAMP, which allows the highly specific and simultaneous detection of various targets (multiplexing). However, current state-of-the-art methods suffer from complex probe design and elaborate optimization work, which is required for the detection of different targets due to the use of target-specific fluorogenic probes. The principal objectives of this thesis are first, to develop an improved sequence-specific detection method for LAMP with a simplified probe design, second, to verify its analytical performance and third, to validate the method by using it to analyze clinical samples. Furthermore, the feasibility of transferring the novel method to two other platforms, digital nucleic acid testing and electrochemical nucleic acid testing, is investigated. A novel method was successfully developed and named mediator displacement (MD) LAMP. MD LAMP stands out from other state-of-the-art methods because of its use of unique, target-independent fluorogenic reporter molecules. The working principle involves a non-fluorogenic MD probe which features a primer bound to a generic mediator. Mediator displacement occurs during the amplification of target DNA. Fluorescence signal generation is then induced by the interaction between the displaced mediator and a universal reporter molecule. The universal mediator-reporter set can be used to detect various targets. Simplified probe design was demonstrated by the example of a reverse transcription (RT)-LAMP of human immunodeficiency virus (HIV)-1 RNA. The time required for MD probe design was first compared with the time needed to create a state-of-the-art molecular beacon (MD: 10 minutes, molecular beacon: 3–4 hours). Moreover, HIV-1 MD RT-LAMP surpassed molecular beacon-based HIV-1 RT-LAMP, with times to positive 4.1 ± 0.1 minutes shorter iv (16–20 minutes for 103–106 HIV RNA copies per reaction) and double the signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, molecular beacon: 2.7 ± 0.4; n = 15). The analytical performance parameters were equally good for both detection methods (limit of detection for MD: 132 HIV RNA copies per reaction, molecular beacon: 141 HIV RNA copies per reaction; linearity for MD and molecular beacon: R2 = 0.94). Furthermore, multiplex detection of two different targets was demonstrated in a biplex MD RT-LAMP for the simultaneous detection of HIV-1 and human T-lymphotropic virus (HTLV)-1. To present an additional possible application, the biplex RT-LAMP of HIV-1 and HTLV-1 was transferred to a centrifugal microfluidic platform for digital nucleic acid amplification in droplets. Digital NAATs allow precise quantification without the need for standard curves. Compared to existing viral load measurements, the novel assay excelled through the simultaneous and quantitative detection of relevant coinfections. The universal mediator-reporter sets were successfully used for the detection of a second clinically relevant target panel, which is associated with yaws, a neglected tropical disease. For the molecular detection of yaws, a biplex MD LAMP of Treponema pallidum and Haemophilus ducreyi (TPHD-LAMP) was designed, optimized and clinically validated with 293 patient samples. The samples originated from individuals with yaws-like skin lesions in Ghana and Papua New Guinea. So far, no isothermal alternatives to PCR which enable differentiation between T. pallidum, which causes yaws, and H. ducreyi, which is responsible for skin lesions of similar appearance, are available. TPHD-LAMP revealed high diagnostic sensitivities and specificities for T. pallidum (84.7 % and 95.7 %) and H. ducreyi (91.6 % and 84.8 %) compared to TaqMan singleplex qPCR. TPHD-LAMP has become the focus of a large clinical trial conducted in three yaws endemic African countries and linked to the goal of supporting national yaws eradication programmes. So far, the analytical performance of MD LAMP has been verified, the feasibility of a digital MD RT-LAMP has been shown, and MD LAMP has been clinically validated with patient samples. To emphasize the versatility of this novel method, its compatibility with real-time electrochemical NAATs was also demonstrated. Microarray-based electrochemical detection facilitates the simultaneous detection of multiple targets by using spatially-resolved measurement with the help of immobilized probes. In addition, it enables the use of very small and therefore portable devices. To exploit these benefits, MD LAMP was adapted to electrochemical detection by immobilizing universal reporter molecules on an electrode microarray. The feasibility of electrochemical MD LAMP was demonstrated for real-time detection of T. pallidum and end-point detection of HIV-1. Rapid signal increase was observed in real time after 15 minutes for 10³ T. pallidum DNA copies per reaction. Electrochemical MD LAMP enables the use of universal electrode microarrays and could thus bring economic advantages with regard to large-scale fabrication. v This thesis concludes with a critical review of the methods and the method-related instrumentation for the sequence-specific detection of LAMP, which are based on a diverse variety of sensing techniques. The multitude of methods is systematically classified and critically evaluated according to a catalogue of criteria covering analytical performance, handling of complex samples, multiplexing, and quantification. The most widespread sensing technique is based on fluorescence detection and is used in around half of the discussed methods. A particular highlight is the universal character of a few fluorescence-based methods, which use generic probes applicable to different targets.
Microfluidic Automation of Multiplex Mediator Probe PCR for Leukaemia MRD Monitoring
2020, Peter Jülg
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: PD Dr. Cornelia Eckert, Charité, Berlin
KurzfassungMinimal residual disease (MRD) describes the remaining amount of cancerous cells among healthy cells during or after therapy. The monitoring of MRD over time provides valuable information on therapy effectiveness and is therefore an essential component of personalised medicine for leukaemia patients. The diagnostically relevant MRD level can be as low as one cancerous cell among 100,000 total cells, corresponding to a sensitivity of 10-5. This requires sensitive molecular methods such as quantitative real-time polymerase chain reaction (qPCR) which target on cancer-specific gene sequences. Unfortunately, these gene sequences can be unstable, especially under intensive or targeted therapy. As a consequence, multi-target quantification is essential for comprehensive MRD monitoring. Additionally, the serial dilution procedure for qPCR standards is considered a weak point since it must be repeated at each MRD quantification time point. The accuracy of this manually executed liquid handling procedure can directly influence the MRD result and, consequently, the clinical decision. In summary, MRD monitoring is a great example how personalised diagnostics can drastically improve patient outcome, but nonetheless an improvement of the underlying qPCR technique towards multiplexing and automation is crucial. This thesis aims to extend the line of research around MRD monitoring providing a solution for automated, multi-target qPCR. Three goals are pursued: (1) centrifugal microfluidic automation of serial dilutions, (2) microfluidic integration of an easy-to-multiplex assay (Mediator Probe PCR), and (3) the combination of both findings to realise automated, multi-target MRD monitoring inside a single LabDisk cartridge. The LabDisk is a centrifugal microfluidic platform with the ability to automate complex molecular diagnostic workflows. Centrifugal and other forces are employed for liquid manipulation and enable metering, transfer, mixing, and numerous other unit operations. The accompanying LabDisk Player does not only provide spinning, but also thermocycling and fluorescence readout, as required for the pursued qPCR integration. To achieve the first goal of this thesis which represents the automation of high-dynamic-range serial dilutions, a new centrifugal microfluidic unit operation is introduced: fill-level-coupled temperature change rate valving (FLC-TCR). In combination with shake-mode mixing, inward pumping, and other unit operations, the FLC-TCR valving is implemented into a microfluidic process chain for automation of theoretically unlimited serial dilutions (DilutionDisk). In contrast to the state-of-the-art, the FLC-TCR principle enables time-independent, serial valving during rotation (f = 7 Hz). As such, it represents a key solution for the reliable handling of wetting reagents such as qPCR mixes (typ. contact angle 40° - 70°). For verification of the principle DNA template dilutions of 10-1 to 10-5 are generated in a background of unspecific DNA and vi Mediator Probe PCR mastermix. For evaluation in a qPCR reference device, the automated dilutions are extracted from the DilutionDisk and compared with manual dilutions. Isolated DNA from leukaemia patient cell samples, which contain the fusion gene ETV6-RUNX1, is employed as target sequence. The automated dilutions are at least as accurate (R² ≥ 99.9%) as the manual reference dilutions (R² ≥ 99.7%). When considering the manual effort, which constitutes the risk of inducing liquid handling errors, the number of manual operations is reduced from 25 steps down to two steps (-92%). To eliminate the transfer step between the serial dilution generation (LabDisk Player 1) and the qPCR reference device (Rotor-Gene Q), the Mediator Probe PCR assay must be adapted for thermocycling inside a LabDisk. For this integration task, a centrifugal microfluidic 72-well-plate analogue is designed (ReactionDisk), which enables efficient and systematic parameter variation. The developed ReactionDisk generates 10 μl reaction triplicates from 24 inlet chambers at high metering accuracy (V = 10.1 ± 0.1 μl). During the assay integration different polymer materials, thermocycling dynamics, and further parameters are screened and optimised. For manually prepared ETV6-RUNX1 DNA dilutions from 10-2 to 10-4 the final parameter set yields a PCR efficiency of E = 99% inside the LabDisk Player, compared to E = 98% inside the Rotor-Gene Q reference run. In another experiment the limit of detection (LoD) is shown to be as low as LoD = 10-5, corresponding to a single target DNA template per reaction. To complete the assay integration, the exchangeability of target sequences is shown by assaying another leukaemia-specific target (Vd2Dd3) and a reference target (beta-globin) under identical PCR conditions while still reaching clinically required reaction efficiencies (E ≥ 86%). Finally, the principle for automated serial dilution (DilutionDisk) and the findings on Mediator Probe PCR assay integration (ReactionDisk) are combined to realise the automated, multi-target MRD monitoring (MRD Disk). Starting from isolated DNA samples the MRD Disk features simultaneous monitoring of four gene targets per run. The panel design comprises two reference genes (fixed for all patients) and two MRD markers (individual for each patient). The MRD Disk is shown to reach clinically required linearities (R² ≥ 98.1%) and efficiencies (E ≥ 83%) for both the reference targets (albumin and beta-globin) as well as the exemplary MRD targets (VH3D3D5JH3 and VkIkde), respectively. In a direct comparison with manually pipetted, gold standard assays, the MRD Disk with Multiplex Mediator Probe PCR assays achieves the same limit of quantification of LoQ = 10-4 in duplex standard curves (six out of six) than the gold standard in singleplex (four out of four). When evaluating the sensitivity, the MRD Disk yields the aspired level of LoD = 10-5 even in a higher number of standard curves (six out of six) than the gold standard (two out of four). Comparing manual effort, the MRD Disk reduces overall hands-on time for one multi-target MRD monitoring from about 35 minutes to less than five minutes (-86%). In summary, the combination of a generic disk layout with an easy-to-adapt, pre-disk prepared target panel, makes the MRD Disk a promising tool for standardisation of patient-individual qPCR assays. This thesis demonstrates the ability of the MRD Disk-automated Multiplex Mediator Probe PCR to replace the current gold standard technique for leukaemia MRD monitoring. The presented results set the base for an upcoming diagnostic study at the Charité - Universitätsmedizin Berlin. Future investigations will focus on a higher degree of reaction multiplexing (4-plex) and a platform transfer on a certifiable processing device (LabDisk Player 2). In addition to the demonstrated application of leukaemia MRD monitoring, the universal character of the multiplexed Mediator Probe PCR and the adaptability of the microfluidic dilution structure make the presented system an attractive candidate for further qPCR applications.
Morphology Analysis and Development of Electrodes for Polymer Electrolyte Membrane Water Electrolyzers and Fuel Cells
2020, Friedemann Hegge
Erstgutachter: Prof. Dr. Stefan Glunz, INATECH, Uni Freiburg
Zweitgutachter: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
KurzfassungSustainably produced hydrogen as a flexible carbon free energy carrier has a huge potential to reduce greenhouse gas emissions in the sectors energy, transport and industry. Polymer electrolyte membrane water electrolyzers (PEMWE) and polymer electrolyte membrane fuel cells (PEMFC) are key technologies for the production of hydrogen and the use of hydrogen to generate electricity. This work is focusing on two research and development topics. In the first part, porous catalyst layers (or electrodes) of PEMWEs and PEMFCs are investigated morphologically and electrochemically, with the aim of contributing to a better theoretical understanding of the relationship between morphology and performance. In the second part, a PEMWE electrode with a novel architecture is developed, which mitigates practically relevant microstructure related conversion losses. The main part of this work consists of three peer review publications (two published and one submitted). The main results are as follows. In the electrochemical modelling of PEMWEs, morphological properties of the electrodes have mostly been described by approximate bulk parameters. In chapter 4.1 a state-of-theart PEMWE electrode is reconstructed using a combination of focused ion beam scanning electron microscope (FIB-SEM) tomography and modelling of the ionomer phase. Depending on the ionomer content, transport and microstructure parameters are calculated. At an assumed ionomer content of approx. 50% of the pore volume (approx. 10 wt%) the tortuosities of the ionomer phase and the pore space are 3.5 and 6.7 respectively. The corresponding phase fractions are 26 % and 29 %. The catalyst surface area estimated from the tomography (1.0 m²/g) is considerably lower than literature values, indicating a roughness below FIB-SEM resolution. In the future, extending electrochemical models with the calculated microstructure parameters enables deeper insight into the relation between the structure of an electrolyzer anode and the electrochemical performance. Chapter 4.2 deals with the influence of morphological changes due to carbon corrosion on the performance losses of PEMFCs. Corrosion of the cathode carbon support significantly influences the lifespan of PEMFC electrodes for both mobile and stationary applications. To investigate the impact, membrane electrode assemblies (MEAs) were aged with accelerated stress tests and examined. From the polarization measurements of the aged samples it was calculated that approximately 80 % of the performance loss is due to increased mass transport losses. A combination of tomography and electrochemical measurements showed that the diffusion resistance of the pore space of the cathode does not play a significant role (3 to 4 sm-1) with respect to the performance loss. The largest portion (50 %) is attributed to an increased local transport resistance due to a loss of active catalyst surface (roughness factor drops from 282 to 169). A portion of 44 % is attributed to a change in water management, while 6 % remain unexplained. Chapter 5.1 describes the development of a novel hybrid PEMWE anode which combines IrOx nanofibers with an IrOx nanoparticle catalyst layer. With this novel electrode design, the overall catalyst loading (particles and fibers) could be significantly reduced in comparison to a conventional electrode without sacrificing performance and durability. Both improvements are attributed to the combination of an improved electrical contact of the IrOx nanofibers and the large catalyst surface of the IrOx nanoparticles. In spite of an ultra-low overall catalyst loading of 0.2 mgIr/cm², a cell with a hybrid layer shows equal performance as a state-of-the-art cell with 1.2 mgIr/cm² and clearly outperforms identically loaded reference cells with pure IrOx nanoparticle and pure nanofiber anodes (4 A/cm² at a cell voltage of 1.83 V). The fabrication of the nanofiber interlayer is compatible with scalable, established processes. Hence, the novel electrode architecture has the potential to reduce the catalyst loading, improve the performance and thereby significantly reduce the hydrogen production costs of PEM water electrolyzers.
Novel polymer electrolyte membrane compositions for electrolysis and fuel cell systems
2020, Carolin Klose
Erstgutachter: Prof. Dr. Stefan Glunz, INATECH, Uni Freiburg
Zweitgutachter: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
Kurzfassung
Reduction of system complexity in centrifugal microfluidics by magnetophoresis at continuous rotation and thermo-pneumatic bubble mixing
2020, Sebastian Hin
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Bastain Rapp, IMTEK - Prozesstechnologie
KurzfassungCentrifugal microfluidics is one among several microfluidic platforms to miniaturize, integrate, parallelize and automate biochemical assays for the point-of-need (PON). This work aims to reduce system complexity in molecular diagnostics on the centrifugal microfluidic platform, while maintaining integration density. Predominant applications cover the detection of pathogens causing infectious diseases, food safety or monitoring of cancer therapies. Moving complex workflows from a centralized lab to the PON is challenging, because it requires the integration of all process steps and reagents into a single cartridge (here: the LabDisk), including a sample interface, pre-storage and release of all required reagents, purification of nucleic acids, mixing with amplification reagents and performing the final analysis, allowing for an evident decision. In this framework, this thesis focusses on the reduction of complexity of two unit operations, to increase overall system robustness and performance: Automated nucleic acid extraction and dead-volume free mixing of eluted nucleic acids with dry reagents. To maintain full control over liquid menisci in a rotating system, and thus, to increase the system’s robustness, continuous rotation is required for all processing steps. This applies especially for the highly wetting buffers in nucleic acid extraction. To provide continuous rotation during nucleic acid extraction, I introduce the concept of “magnetophoresis at continuous rotation”. A stationary magnetic field was provided by four permanent magnets located above the LabDisk, exerting a radially inwards pointing magnetic force on magnetic beads in the LabDisk. Magnetophoresis in radial inward direction was achieved in the present setup at a critical frequency of 5 Hz followed by azimuthal bead transfer to the adjacent chamber, which occurred by means of particle inertia, supported by deflection structures. By this, the beads were transported three times in azimuthal direction along four adjacent microfluidic chambers holding the extraction buffers. The process was verified by extracting nucleic acid from mosquito pools, whereas eluates from LabDisk and reference extractions (in tube) showed comparable results in terms of protein contamination (LabDisk: A260/A280 = 1.6±0.04; n = 4 vs. reference: A260/A280 = 1.8±0.17; n = 3) and threshold values (Ct) in real-time RT-PCR (LabDisk: Ct = 17.9±1.6, n = 4) falling in the 95 % confidence interval (CI) of the reference (Ct = 19.3±1.7, 95 % CI = [17.5 - 21.0], n = 3). Notably, ethanol Abstract iv concentration was quite high in the obtained eluate (cEthanol ~10 % (v/v)), here, corresponding to 18 μL ethanol carry-over. Consequently, for the first time, I comprehensively investigated ethanol carry-over during nucleic acid extraction in the LabDisk, aiming to reduce it below an inhibitory level for downstream analysis (cEthanol, threshold ≤ 2.5 % (v/v)). While I could not observe significant carry-over of ethanol within the magnetic bead cluster, diffusion of ethanol vapor in the gas phase and condensation at the eluate was identified as the dominating effect. In the utilized LabDisk, ethanol carry-over by diffusion was measured to be as high as 2.1 nL s-1, 2.6 nL s-1 and 4.1 nL s-1 at 25 °C, 35 °C and 45 °C, respectively. To reduce the diffusion of ethanol vapor into the eluate passive countermeasures were taken. These included a reduction of the cross-section of the diffusion path (cEthanol = 4.2±0.5 % (v/v), equal to 7.6 μL carry-over, n = 3) and the introduction of a dynamic pressure pump inducing gas convection in the cartridge (cEthanol = 0.2±0.3 % (v/v), equal to 0.36 μL carry-over, n = 3). In batch mode mixing, dead volumes during rehydration and mixing of lyophilized reagents are a big challenge, resulting in concentration gradients and thus inconsistent reaction conditions in downstream reactions. In this work, I introduce “thermo-pneumatic bubble mixing”, to compensate dead-volumes in downstream channels without the need for additional means. A high fluidic resistance vent channel acts as a low-pass filter with respect to pressure changes in the downstream air volume allowing for overpressure generation at high positive temperature change rates. Overpressure leads to bubble formation, pushing liquid from the downstream channel back into the mixing chamber. The concept was verified by rehydrating lyophilized reagents for loop-mediated isothermal amplification (LAMP). Mixing efficiency was characterized by the mean coefficient of variation (CV̅̅̅̅, n = 4 LabDisks) of the time-to-positivity (tp) of the LAMP reactions in 11 replicate reaction chambers on the LabDisk, resulting in a CV̅̅̅̅ reduction from 18.5 % to 3.3 %. In summary, the presented unit operations are a major achievement towards less complex, and more robust integration. This strongly extends the toolbox for centrifugal microfluidic assay integration in molecular diagnostics. The work simplified system complexity by consequently exploiting the inherent advantages of the centrifugal microfluidic platform, while maintaining integration density.
Segmentation and computational analysis of the 3D porous microstructures in Li-ion cells
2020, Rico Moroni
Erstgutachter: Prof. Dr. Lars Pastewka, IMTEK - Simulation
Zweitgutachter: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
Kurzfassung

2019

Characterization and optimization of anodes for microbial electrochemical technologies treating wastewater
2019, Joana Madjarov
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
Zweitgutachter: Prof. Uwe Schröder, TU Braunschweig
Kurzfassung
Frequenzgesteuertes Öffnen von Stickpacks in zentrifugalen Mikroanalysesystemen
2019, Daniel Baumann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jens-Peter Majschak, TU Dresden
Kurzfassung
Handling of Microparticles for Assay Automation in Centrifugal Microfluidics
2019, Yunpeng Zhao
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Ulrike Wallrabe, IMTEK - Mikroaktorik
Kurzfassung
Highly parallel microbioreactors for cell line development based on well plates with functional lids
2019, Cheng-Han Tsai
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Joost Lötters, University of Twente, Niederlande
Kurzfassung
LabSlice XL – A centrifugal microfluidic cartridge for the automated bio-chemical processing of industrial process water
2019, Stefan Burger
Erstgutachter: Prof. Dr. Roland Zengerle, Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Anja Boisen, Technical University of Denmark
Kurzfassung
Lumped model simulation for the fast development of robust centrifugal-microfluidic lab-on-a-chip systems
2019, Ingmar Schwarz
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jens Ducrée, Dublin City University, Irland
Kurzfassung
Pneumatic operations in centrifugal microfluidics - An enabling technology for assay automation
2019, Steffen Zehnle
Erstgutachter: Prof. Dr. Roland Zengerle, Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Ulrike Wallrabe, Mikroaktorik
Kurzfassung
Screening of electrode materials for microbial fuel cells operating with S. oneidensis and G. sulfurreducens
2019, Elena Kipf
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. J. Gescher, KIT Karlsruhe
Kurzfassung
Single-cell dispensing for isolation and analysis of individual mammalian and microbial cells
2019, Julian Riba
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: PD Dr. Heiko Becker, Uniklinik Freiburg
Kurzfassung
StarJet printheads for printing molten solder jets at 320°C and molten aluminum alloy droplets at 950°C
2019, Björn Gerdes
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jürgen Wilde, IMTEK / Aufbau- und Verbindungstechnik
KurzfassungThis thesis presents research based on the StarJet technology which is a means of directly printing molten metal. StarJet printheads can be used for printing of metal microdroplets in drop-on-demand mode, or continuous molten metal jets. For the first time, the technology is used for printing of functional metallizations based on solder. Moreover, the design, fabrication and evaluation of a new printhead developed for drop-on-demand printing of molten aluminum alloys at up to 950 °C and the metallurgical analysis of printed metal droplets are presented. The StarJet technology is based on an interchangeable nozzle chip that features a star-shaped orifice which confines droplets and jets in the orifice by capillary forces before they are ejected. It further features a heatable, pneumatically driven printhead. Short pressure pulses on a reservoir, controlled by a solenoid valve, lead to the ejection of metal. The so-called rinse gas flows through the chip’s orifice, supports droplet formation and surrounds the droplets after ejection. Prior to this work, the technology was mainly used to print molten solder microdroplets at up to 350 °C with nozzle chips fabricated by deep reactive ion etching from silicon. In this work, a new operation mode of the solder printhead is demonstrated and evaluated for the first time. It is used for printing of electrically conductive – and therefore functional – metallizations via StarJet. The so-called JetMode enables the direct printing of stable molten solder microjets, featuring minimum diameters of 50 µm. A StarJet printhead for molten solder is integrated in a modified industrial printing platform that enables fast printhead displacement velocities of up to 1 m/s. With this machine integration, direct printing of interconnections on printed circuit boards and moreover direct printing of the front side metallization of silicon solar cells are presented. Printed lines exhibit a linear current-voltage characteristic, making them feasible for establishing electrical interconnections. By printing of molten metal jets on prefabricated solar cells, energy conversion efficiencies of up to 18.2% are demonstrated. Moreover, a small module of four cells, fully metallized via StarJet technology is presented featuring an energy conversion efficiency of 9.2 %. Until today, the StarJet technology is the only method that enables direct printing of silicon solar cell front side metallizations from molten metal. The metallization is functional without further processing, potentially saving process steps compared to established screen-printing processes. Moreover, research on drop-on-demand printing of metal microdroplets from high melting alloys is carried out in this work. The StarJet technology is used for the first time as a means of directly printing aluminum-rich alloys. The high process temperatures of up to 950 °C and the chemical aggressivity of molten aluminum require the development of a printhead specifically designed for printing this material class. A hybrid printhead, consisting of stainless steel and ceramic materials is designed and fabricated for this purpose. A major challenge is the design and fabrication of star-shaped nozzle chips that can withstand the temperatures and are chemically inert toward aluminum melts. Despite extensive research, the aforementioned silicon nozzle chips cannot be effectively protected from being dissolved by aluminum alloys until today. Therefore, nozzle chips fabricated by laser ablation from silicon carbide are used for the first time. They enable direct printing of molten aluminum alloys via StarJet at up to 950 °C. The StarJet printhead is the only printhead for molten aluminum alloys that does not require an inert atmosphere around it, making it potentially feasible for in-line fabrication processes. Droplet printing of the aluminum alloy AlSi12 is demonstrated with different nozzle orifice diameters which mainly determine the droplet diameter. Printed AlSi12 droplets of 700 µm diameter exhibit a standard deviation of 1 %. Further, the smallest aluminum alloy droplets printed in drop-on-demand as of yet, are presented, featuring mean diameters of 176 µm. The main limitation of the droplet generation regarding droplet diameters is most likely the fabrication process of the silicon carbide nozzle chips. For small orifice diameters, the complex geometry of the nozzle chip is difficult to fabricate. Free-standing 3D objects e. g. walls or a flexible spring that consist of printed AlSi12 microdroplets featuring approximately 700 µm diameter are presented in this work. Objects are printed without support structures. For the first time, a metallurgical analysis of droplets printed via StarJet is presented. It comprises an analysis of the metallic microstructure, investigated by optical microscopy and energy dispersive X-ray tomography. AlSi12 droplets exhibit short dendrites (approx. 5 − 10 µm), indicating high undercooling during solidification, potentially leading to a hardening of the printed material. Moreover, formation of oxides on the droplets’ surface is critical as the StarJet printhead is operated at ambient atmosphere. Auger electron spectroscopy reveals a thin oxide layer on printed AlSi12 droplets of approximately 110 nm compared to about 50 nm native oxide, making the process potentially feasible for additive manufacturing.
Towards abiotic glucose fuel cells as powering concepts for medical implants and sensors
2019, Maxi Frei
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
Kurzfassung

2018

Needle-free trans-endoscopic micro injection for flexible endoscopy
2018, Klaus Mutschler
Erstgutachter: Prof. Dr. Roland Zengerle, Anwendungsentwicklung
Kurzfassung

2017

Advances in Fabrication and Characterization of Functional Layers in Polymer Electrolyte Fuel Cells
2017, Matthias Klingele
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Richard Hanke-Rauschenbach, IfES - Leibniz Universität Hannover
KurzfassungHydrogen fueled polymer electrolyte fuel cells (PEFCs) efficiently convert the chemical energy of hydrogen oxidation and oxygen reduction into usable electrical power without generating harmful emissions. Polymer electrolyte fuel cells are therefore considered as key-technology for the global energy transition towards sustainable and renewable energy carriers and energy converters. The functional unit of a polymer electrolyte fuel cell is the membrane electrode assembly (MEA), consisting of an ion exchange membrane sandwiched between nano-porous anode and cathode catalyst layers (ACL and CCL). This thesis presents advances in the fabrication of membrane electrode assemblies (MEAs) (1), novel metal-free materials for application in polymer electrolyte fuel cell cathode catalyst layers (PEFC CCLs) (2), and a method for reliably calculating transport parameters in conventional polymer electrolyte fuel cell catalyst layers (3). (1) Conventionally, membrane electrode assemblies (MEAs) are formed starting with a free standing proton exchange membrane (PEM) foil which is coated with an anode and cathode catalyst layer (ACL and CCL) on each side, respectively. The so obtained MEA is conventionally referred to as ‘catalyst coated membrane’ (CCM). This thesis presents a novel manufacturing route for membrane electrode assemblies, in which polymeric electrolyte is deposited directly onto anode and cathode catalyst layers to work as a proton exchange membrane. This route allows the fabrication of MEAs with membranes as thin as 12 μm. This leads to a power increase of an acidic polymer electrolyte fuel cell (PEFC) by a factor of 2.3 when compared to a conventional PEFC using a 20 μm thin membrane-foil (chapter 2.1). It is shown that mainly the reduced low frequency resistance at high power densities, as determined by electrochemical impedance spectroscopy, is responsible for this increase in cell power (chapter 2.2). The reduction in low frequency resistance can be attributed to a facilitated oxygen diffusion through the porous layers of a PEFC with directly deposited membrane. Besides these advantages, it is further shown that consecutive application of catalyst ink and polymeric electrolyte provides a fast and versatile method for whole MEA manufacturing on both lab- and industry-scale. (2) The CCL in state-of-the-art PEFCs contains 80 % of the overall needed platinum, and is therefore the major cost driver of such devices. In this thesis, sulfur doped reduced graphene oxide (S-rGO) is presented as a cost-effective alternative cathode catalyst material for the oxygen reduction reaction (chapter 3.2). It is shown that S-rGO forms well defined single-layer graphene flakes, efficiently doped with sulfur atoms. The catalytic activity of S-rGO is shown in rotating disc electrode measurements and in working acidic and alkaline PEFCs. S-rGO is theorized to hold unpaired carbon electrons as the single catalytically active sites. With the intention of creating polarized carbon atoms as additional active sites for higher catalytic activity, S-rGO was further doped with nitrogen and fluorine to yield F,N,S-rGO (chapter 3.3). A PEFC with F,N,S-rGO as porous cathode catalyst layer outperforms a S-rGO reference fuel cell by at least a factor of 2. By benchmarking these results, as well as existing literature, to the cost per power ratio of state-of-the-art platinum PEFCs, it is shown that PEFCs which are constructed with doped carbon materials as porous cathode catalyst layers still provide by a factor of 10 too little volumetric power density for being economically relevant (chapter 3.1). This can be linked to a relatively lower charge transfer number and catalytic activity of doped carbon as catalyst in acidic electrolyte when compared to platinum. (3)The nano-morphology of the CCL is crucial for sufficient oxygen supply and water removal to and from the catalytically active sites. Typically, the nano-morphology is imaged by focused ion beam (FIB) / scanning electron microscopy (SEM) tomography (FIB-SEMt). This technology works on the basis of subsequent FIB milling and SEM imaging. Next to the resolution of the SEM, the FIB milling distance leads to severe coarsening of a FIB-SEMt-based CCL reconstruction. In this thesis it is shown that this morphology adulteration leads to errors in the calculated transport parameters of the investigated CCL and a strategy for compensating these errors is provided (chapter 4.1).
Automated microfluidic nucleic acid analysis for single-cell and sample-to-answer applications
2017, Fabian Stumpf
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
KurzfassungThe scope of this thesis is the development of automated microfluidic nucleic acid analysis for single-cell and sample-to-answer applications with the long-term goal to provide microfluidic, analytical and diagnostic tools for the fast-growing field of personalized medicine. The first part of this thesis describes three developments in the field of single-cell analysis wherefore microfluidic miniaturization and automation provides major benefits for the required single-cell handling and isolation. Single-cell analysis allows identifying rare differences within a cell population which are often related to diseases and has thus developed into a key topic in cell biology with future applications in personalized medicine, tumor identification and tumor discovery. As there is only one DNA double-strand of each chromosome and therefore unique sequences are contained in a single cell, an amplification step is required prior to further downstream analysis like sequencing of the nucleic acids. In this thesis, single-cell PCR with human B cells as sample was chosen as application to demonstrate amplification of single cell contained nucleic acids. Thermal cell lysis is applied to release the DNA from single cells without usage of PCR inhibiting lysis reagents and no need for additional extraction steps. For the first time, the novel approach of singe-cell solid-phase PCR (SP-PCR) is demonstrated. The parallelization of amplification is achieved by distribution of cells, suspended in PCR mixture, into 104,000 wells of a commercial sequencing chip. The nucleic acids of the single cells are amplified inside the 18.5 pl wells and synthesized onto a surface by an immobilized primer and thus are accessible for further downstream analysis. The validation of the single-cell SP-PCR was applied with 10,000 as well as with 50,000 human B cells per sequencing chip with a detection rate of up to 66 % demonstrating successful SP-PCR on the single-cell level. The second single-cell PCR development was realized in polydimethylsiloxane (PDMS) chips enabling droplet-based microfluidics for automated generation of single cell containing droplets, acting as individual PCR reaction volumes. Semi-contact-writing is demonstrated as a novel method for on-demand fabrication of polymer molds for casting these PDMS chips in a flexible, time and cost-efficient manner. This method is ideally suited for rapid chip layout iterations which are required for assay integration into microfluidic systems. No cleanroom facilities are required and fabrication time from scratch to ready-to-use PDMS-chip is less than 5 hours. The PDMS chips were used for successful demonstration of droplet-based flow-through PCR with single-cell sensitivity. In this novel concept, cell containing PCR mixture was used to encapsulate single cells into droplets which are guided over temperature zones for flow-through PCR amplification. The PCR amplification was identified by gel electrophoresis showing comparable results of the on-chip droplet sample and the reference sample amplified in a standard PCR thermocycler. The third single-cell PCR application was developed to prevent multiple-cell events and enable a confirmed single-cell PCR. Inkjet-like printing was employed to isolate individual B cells and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The implemented and automated workflow for single-cell isolation is robust and easy to use. PCR of single-cell DNA was performed directly from single B cells with 33 % success rate (N = 197) and Cq values of 36.3 ± 2.5. Additionally, single-cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of > 1000. This facilitated subsequent PCR yielding a success rate of 64 % (N = 33) which will allow more sophisticated downstream analysis like sequencing. Analyses of single cells are also applied for infectious disease diagnostics. However, in time-critical situations or settings without access to a centralized laboratory, molecular diagnostics can be applied for a fast, decentralized and patient-side point-of-care (POC) diagnostics. Microfluidic lab-on-a-chip systems enable miniaturization, automation and integration of the complex workflows for pathogen detection and the feasibility to be operated in a portable device. Such miniaturized personalized healthcare tools for the use at the point-of-care require user-friendly and minimal handling steps and must provide the same diagnostic performance compared to centralized laboratories. The second part of this thesis presents the fully automated sample-to-answer detection of respiratory pathogens in a centrifugal microfluidic LabDisk with complete prestorage of reagents. Thus, the initial sample supply remains the only manual handling step. The self-contained LabDisk automates by centrifugal microfluidics all necessary process chains for PCR-based pathogen detection: pathogen lysis, magnetic bead based nucleic acid extraction, aliquoting of the eluate into 8 reaction cavities and real-time reverse transcription PCR (RT-PCR). Prestored reagents comprise air-dried specific primers and fluorescence probes, lyophilized RT-PCR mastermix and stick-packaged liquid reagents for nucleic acid extraction. Employing two different release frequencies for the stick-packaged liquid reagents enables on-demand release of highly wetting extraction buffers, such as sequential release of lysis and binding buffer. Microfluidic process-flow was successful in 54 out of 55 tested LabDisks. Successful detection of the respiratory pathogen influenza A H3N2 virus is demonstrated in a total of 18 LabDisks with sample concentrations down to 2.39 × 104 viral RNA copies per ml, which is in the range of clinical relevance. Finally, real patient samples were tested and fully automated sample-to-answer multiplex detection of respiratory pathogens was successfully demonstrated and even multiple pathogens within a sample were detected. The detection of the respiratory pathogen and the internal control in the same reaction cavity demonstrated the first successful 2-plex real-time amplification in one reaction cavity on a LabDisk. The experiments were applied in a 2 kg portable, laptop controlled point-of-care device. The turnaround time of the complete analysis from sample-to-answer was less than 3.5 hours.
Direct membrane deposition as novel fabrication technique for high performance fuel cells
2017, Matthias Breitwieser
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Stefan Glunz, Fraunhofer ISE
Kurzfassung
Microfluidic Apps for centrifugal thermocyclers at constant rotational frequency
2017, Mark Keller
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
KurzfassungLab-on-a-Chip systems can advance laboratory workflows through automation in a miniaturized format. However, their commercialization has been exceedingly slow. One potential shortcut to market is offered by Microfluidic Apps, i.e. cartridges which can be processed on existing off-the-shelf instruments instead of requiring custom devices. Present Microfluidic Apps, however, have not yet succeeded in fully complying with the constraints of their off-the-shelf processing devices, thereby limiting their success. This thesis presents the development of three novel Microfluidic Apps for operation on off-the-shelf rotary thermocyclers, i.e. on standard devices for nucleic acid analysis via real-time polymerase chain reaction (PCR). The Microfluidic Apps are driven by new unit operations, i.e. basic microfluidic functionalities, to comply with the constraint of off-the-shelf rotary thermocyclers. Off-the-shelf rotary thermocyclers offer global temperature control and may in theory drive centrifugal microfluidics by their rotation, which is however in all cases constant and for the majority at low 400 rpm. State-of-the-art unit operations of centrifugal microfluidic platforms are unsuitable to be operated under these conditions. Consequently, compatible unit operations were developed using novel centrifugo-thermopneumatic (CTP) liquid transport by thermal gas compression/expansion (chapters 3 and 7). Chapter 3 introduces CTP siphon valving, which was demonstrated to reliably transfer a sample liquid downstream in all cases (n = 35). In addition, CTP two-step aliquoting is presented, which was proven to meter this liquid into aliquots of 18.2 ± 1.2 μL (n = 8). Thermal characteristics of the latter were found to be in good agreement with a newly developed analytical model (R2 = 0.99, n = 3). The automation of real-time PCR of Escherichia coli DNA by the concatenation of both unit operations with pre-stored primers and probes showed similar PCR efficiency, repeatability, and reproducibility as reference reactions in conventional PCR tubes, meanwhile reducing 25 error-prone pipetting steps to a single loading step with subsequent automated aliquoting. CTP wax valving is introduced in chapter 7, which is also based on CTP liquid transport. Uniquely for wax valves, it is fabricated by pick-and-place, self-aligns during processing, allows arbitrary temperatures below its activation temperature and triggers valving at temperature decrease after it was activated once. For valving, the wax solidifies and blocks an air vent, thereby enclosing an air volume for CTP liquid transport. Investigations on different polyethylene glycols (PEG) as wax material showed the highest reliability in solidification for PEG 6000/8000 (80/20 wt% ratio). Design studies allowed for the implementation of the CTP wax valve for valving after PCR thermocycling on an off-the-shelf rotary thermocycler. Finally, besides CTP liquid transport, another novel unit operation for liquid transport by temperature change rates (TCR) is presented in chapter 8. TCR actuated siphon valving is implemented by introducing a fluidic resistance to an air vent of a fluidic structure downstream of a siphon channel. Temperature decrease thermally induces a temporary air underpressure inside the downstream structure, which is slowly compensated through the air vent. This underpressure pulls liquid against a centrifugal counterpressure, possibly above the siphon crest to trigger valving. For layout, an analytical model is provided and verified for the prediction of thermally induced underpressure up to 1.92 kPa with an accuracy of ± 0.17 kPa. Underpressure is demonstrated to increase with an increase in TCR and/or fluidic resistance. On the basis of these unit operations, chapters 4 to 6 introduce newly developed Microfluidic Apps for operation on the off-the-shelf rotary thermocycler Rotor-Gene Q (QIAGEN, Germany). First, the TB-Slice, based on CTP siphon valving and two-stage aliquoting, is presented in chapter 4. It detects mutations in the inhA, katG, and rpoB (two loci) genes, which mediate resistances of Mycobacterium tuberculosis complex against isoniazid (INH) and rifampin (RMP). Using next generation sequencing (NGS) as a reference, 95.9 % of 91 tested patient samples (excluding 4.5 % (fluidic) failures) could be correctly classified as drug-sensitive / -resistant. The diagnostic sensitivity / specificity of the TB-Slice was 79.3 % / 96.8 % for INH and 100.0 % / 94.7 % for RMP using drug susceptibility testing as a reference and proved highly comparable with reactions in conventional tubes using NGS as reference (inhA: 100.0 / 101.6 %; katG: 99.6 / 100.0 %; rpoB1: 104.5 / 99.3 %; rpoB2: 90.5 / 100.0 %). Second, the FTMS-Slice, i.e. a fluidic system for temperature measurement under rotation, is demonstrated as proof of principle in chapter 5. The FTMS-Slice utilizes CTP fluid transport to displace fluorescent liquid into a detection cavity at a calculated “measured” temperature. Measured temperatures (66.4 °C, 70.3 °C, 73.3 °C, 75.8 °C, n = 1) were in good agreement with temperatures indicated by the Rotor-Gene Q (65 °C, 70 °C, 73 °C, 76 °C). Finally, the Forensic-Slice is introduced in chapter 6 for the identification of animal groups in forensics. The Forensic-Slice, partly based on CTP two-stage aliquoting, features the pre-storage of reagents, universal PCR pre-amplification of genes encoding 12S rRNA and cytochrome b (cytb), animal-group-specific PCR main-amplifications, and melt curve analysis for differentiation. 92.2 % of the tests performed were recognized as fluidically failure-free sample handling and used for evaluation. Analytical sensitivity (20 pg/sample for the majority of groups), specificity, and detection of minor components in mixtures (e.g. 1 % of human or pig for 12S rRNA, 5 % of pig and 10 % of human for cytb) were characterized. In summary, six novel unit operations for centrifugal microfluidic platforms were developed, which can be operated at constant and low rotational frequency and actuated by global temperature control. On their basis, three Microfluidic Apps on off-the-shelf rotary thermocyclers were developed, which for the first time render compatible with their constraints, which in turn is expected to pave the way for their commercialization.
Morphological and Electrochemical Investigation of Polymer Electrolyte Fuel Cell Components
2017, Severin Vierrath
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Richard Hanke-Rauschenbach, IfES - Leibniz Universität Hannover
KurzfassungIn this thesis devices for electrochemical energy conversion and storage are investigated with tomographic and electrochemical methods with a focus on polymer electrolyte fuel cells. The first part of the work presents ZnO-contrasting via atomic layer deposition for focused ion beam - scanning electron microscopy (FIB-SEM) tomography, which for the first time enables reliable and fast reconstruction of nanoporous carbon-based materials. The potential of ZnO-contrasted FIB-SEM tomography is illustrated with various findings: the calculated O2 diffusivities (23.1 - 25.4 x 10-7 m2s-1) and porosity (65%) of a fuel cell catalyst layer are in good agreement with experimental values from literature, while they deviate strongly when obtained from state-of-the-art reconstructions. Furthermore, filling with ZnO permits the identification of large Pt clusters inside the catalyst layer, which were estimated to reduce the catalyst surface area by 9%. A similar material system, the carbon binder domain (CBD) of a LiCoO2 battery cathode, was reconstructed for the first time thanks to ZnO-contrasting possibly ending the era of virtually created CBDs via stochastic-based reconstruction. The calculated porosity (57%), tortuosities and size distributions lead to two findings: first, the inhomogeneity of the CBD reduces electronic conductivity by up to 30%, and second, swelling of the PVDF binder (75 vol%) reduces ion transport in the pores of the CBD significantly. ZnO-contrasting is further used to compare a fuel cell microporous layer and catalyst layer, to demonstrate the pore space connectivity of carbon nanowalls synthesized by plasma enhanced chemical vapor deposition and to investigate the morphology of supercapacitor materials. Most likely ZnO-infiltration also aids to preserve the original structure of the sample during FIB-SEM tomography by increasing mechanical stability and thermal conductivity. Being the missing piece to reliably v reconstruct large volumes ZnO-contrasting is believed to pave the way for industrial application of FIB-SEM nanotomography. In the second part of the thesis the reasons for the increased power density of fuel cells fabricated with the in-house developed direct membrane deposition (DMD) are systematically investigated. The reasons for the increased power density were found to be a 50% reduction in ionic resistances of the polymer electrolyte membrane and mass transport resistance of the oxygen diffusion compared to conventional fuel cells. The reduced mass transport losses, which are responsible for 90% of the increase in power density (at the maximum power point of the reference), are attributed to an increased cathode water removal through the membrane due to a thinner membrane, differences in catalyst layer morphology and an increased membrane surface.
Single-Cell Handling using Drop-on-Demand Printing and Impedance Cytometry
2017, Jonas Schöndube
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Ulrike Wallrabe, IMTEK - Mikroaktorik
KurzfassungDie vorliegende Dissertation behandelt technische Ansätze zur Handhabung individueller lebender Zellen mittels drop-on-demand Drucken und Einzelzellerkennung. Ein Schwerpunkt liegt dabei auf der Impedanzzytometrie als Messverfahren für die Einzelzellerkennung. Das Feld der Einzelzell-Analyse verspricht, neue Erkenntnisse in der Zellbiologie zu ermöglichen. Die Analyse von großen Zellpopulationen (typischerweise mehrere tausend Zellen oder mehr) als ein Ensemble verdeckt den Blick auf Phänomene oder Entwicklungen, die nur bei wenigen Zellen auftreten. Die Analyse von Populationen liefert lediglich Durchschnittswerte. Die Einzelzell-Analyse hingegen erlaubt die Identifikation von Mustern innerhalb einer solchen Population oder das Verfolgen von bestimmten Entwicklungen, die mit nur einer Zelle beginnen (wie z.B. die Karzinogenese). Konventionellen Methoden zur Einzelzell-Handhabung fehlt es oft an nötiger Automatisierung oder sicherer Kontrolle über einzelne Zellen. Ziel dieser Dissertation ist die Evaluation des Potentials der Dispensierung von Pikoliter-Tropfen, die einzelne Zellen enthalten, als Werkzeug für die effiziente Einzelzell-Analyse. Mikro uidische Chips wurden verwendet, um Tropfen von Zellsuspensionen zu dispensieren. Optische und elektrische Messverfahren wurden eingesetzt, um die Zellen vor dem Ausstoß zu detektieren. Impedanzzytometrie, als elektrisches Messverfahren, erlaubt es, die Impedanz kleiner Volumina von Zellsuspensionen (ca. 50 µm x 55 µm x 70 µm) zu messen. Diese Impedanz hängt dabei von der Zahl der Zellen im Messvolumen ab. Die Kombination von drop-on-demand Dispensierung und Zellerkennung erlaubt es, den Prozess der Verkapselung einzelner Zellen in frei- iegenden Tropfen zu kontrollieren. Nach einem Überblick über den aktuellen Stand der Technik, werden drei experimentelle Projekte beschrieben, die eine optische Zellerkennung verwenden. Zwei dieser Projekte stellen Anwendungen der Einzelzell - Drucktechnologie vor: Tissue Engineering und Einzelzell-Genomik. Für das Tissue Engineering wird ein Instrument vorgestellt, dass drei-dimensionale Hydrogelstrukturen, mit einer Auflösung von ca. 500 µm und einem Aspektverhältnis von 1:4 (Höhe:Breite), drucken kann. Dasselbe Instrument kann im Anschluss eine kontrollierte Anzahl an Zellen in diese Hydrogelstrukturen dispensieren. Für die Einzelzell-Genomik wurde gezeigt, dass der Einzelzell-Drucker angewandt werden kann um direkte PCR von genomischer DNA einzelner Zellen zu erreichen (Erfolgsrate 33 %, N=197). Weiterhin wurde gezeigt, dass eine Whole Genome Amplification einzelner Zellen, die mit dem Einzelzell-Drucker prozessiert wurden, durchgeführt werden kann. Das dritte Projekt mit optischer Zellerkennung beschreibt einen Ansatz zur Verbesserung der Zellerkennung mittels der Aufreihung der Zellen oder Mikrokugeln (als Zellmodell; engl. Beads) im Chip durch Akustophorese. Dies erlaubte es den Verlust von Beads, als Zellmodell, während des Prozesses von 52 % +/- 6 % auf 28 % +/- 1 % zu reduzieren. Der Hauptteil dieser Arbeit handelt von der Einzelzell-Handhabung mithilfe von drop-on-demand Drucken (500 pl - 800 pl Tropfenvolumen) und Impedanzzytometrie. Die Zellsuspension strömt dabei in einen Mikrochip und passiert einen Impedanzsensor. Das Impedanzsignal wird in Echtzeit analysiert, Zellen werden detektiert und deren Geschwindigkeit indirekt bestimmt. Mithilfe dieser Information kann der Dispensiervorgang im passenden Moment gestartet werden, d.h. wenn sich die Zelle an der Düse befindet. Dieser Prozess wurde mit Beads systematisch charakterisiert und eine Einzel-Bead-Effizienz von 73 % +/- 11 % wurde gezeigt. Zwei verschiedene Zelltypen wurden verwendet, um qualitativ zu zeigen, dass die Zellviabilität während der Erkennung und des Druckens erhalten bleiben kann. Schließlich wurde das Impedanzsignal verwendet um in zwei Machbarkeitsstudien Zellen und Beads zu sortieren. Beads unterschiedlicher Größen (10 µm und 20 µm im Durchmesser) konnten semi-automatisch differenziert werden. Dabei wurden Sortierreinheiten von 74 % (N=35) und 91 % (N=58) gezeigt. Anschließend konnte das Sortieren von Beads und Zellen mithilfe von drop-on-demand Drucken und Impedanzzytometrie erstmals durchgeführt werden. Multifrequenzmessungen wurden eingesetzt um Klassifikationseffizienzen von bis zu 92% (N=152) und geschätzte Sortierreinheiten zwischen 86 % (N=23) und 100 % (N=21) zu erreichen. Es wird erwartet, dass der Bedarf an Werkzeugen für die Handhabung einzelner Zellen in den nächsten Jahren steigen wird. Die Ansätze die in dieser Arbeit präsentiert werden haben das Potential zu Verbesserung der Effizienz und der Qualität von Einzelzellanalysen beizutragen. Das Drucken einzelner Zellen ist dabei eine vielseitige Möglichkeit zur Automatisierung der Zellisolation und die Impedanzzytometrie könnte in der Zukunft ein markierungsfreies Sortieren von Zellen ermöglichen. Momentane Limitationen sind unter anderen die Sensitivität und die Spezifizität der Impedanzmessung, sowie der Durchsatz der Zellisolation.
Tomography-based Analysis of Battery, Electrolyser and Fuel Cell Microstructures
2017, Lukas Zielke
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof.in Dr.-Ing. Ellen Ivers-Tiffée , KIT Karlsruhe
KurzfassungIn this thesis, it is shown how imaging by X-ray tomography (Xt) and focused ion beam / scanning electron microscopy tomography (FIB/SEMt), image processing, and subsequent calculations can be used to quantify morphological-, transport- and degradation parameters in porous structures used in energy devices. The investigated features range from approximately 300 micrometres to approximately 10 nanometres. A special focus lies on the role of the porous carbon and binder domains (CBD), as they are used in PEM electrolysers, PEM fuel cells, lithium ion batteries and many more energy devices. During this thesis, there were several challenges that were identified and partially overcome, such as: 1) The CBD cannot be reliably segmented using Xt, if the investigated electrode contains heavy metal atoms, e.g. Co. A solution is presented, via a statistical modelling approach where the CBD is virtually inserted in the active material framework from X-ray tomography. This approach also provided understanding as to how changes in CBD morphology influence e.g. Li-ion transport in such electrodes. 2) It was unclear how the three-dimensional morphology of lithium sulphur electrodes changes when the battery is cycled. It is shown in this thesis that Xt can be used to quantify such morpho-logical changes and that not only the sulphur particles do change size, distribution and form with increased cycling, but that the morphology of the CBD changes as well. When using three-dimensional carbon paper as a current collector, the CBD clogs the top of the current collector. This partially clogged surface than acts as a barrier to sulphur, increasing sulphur retention in the electrode. 3) Mechanical stress during lithiation of Silicon electrodes changes the electrodes morphology. It is shown that these stresses have caused a partial contact loss of single Si particles to the CBD or the ionically conducting electrolyte phase. The high image contrast between pore, CBD and silicon particles have allowed for a deeper analysis of the battery, finding that particles which have less than 40% of their surface area still covered by CBD do not experience lithiation related fracturing. 4) There were no studies on the three-dimensional morphology of an interface between a catalyst layer (CL) and a micro porous layer (MPL) in a fuel cell in their application relevant state. By using a combination of FIB/SEMt and ALD, such an interface was reconstructed and it was shown that its morphology represents a transitional region, rather than a barrier between the CL and the MPL.

2016

A parallel dispensing system for an improved front surface metallization of silicon solar cells
2016, Maximilian Pospischil
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Norbert Willenbacher, KIT, Karlsruhe
KurzfassungWithin this thesis, the dispensing technology was enhanced to a high throughput alternative for industrial front side metallization in silicon (Si) Photovoltaic (PV) production lines. Starting with a single nozzle dispensing setup based on commercially available products, the technology was stepwise enhanced to various multi-nozzle systems that were specially developed to meet process requirements of today’s production sites and continuously demonstrate physical and economical advantages compared to fast emerging, established screen printing technology. The main task included the application of commercially available silver (Ag) sinter pastes on industrial solar cells, predominantly p-type Czochralski (Cz) grown Si wafers (156 · 156mm2) that were mostly delivered from industrial suppliers after deposition of the anti-reflective coating (ARC). For both, the development of the multi-nozzle print heads by means of computational fluid dynamics (CFD), as well as for the optimization of the geometrical shape of dispensed contact geometries the rheology of the involved printing pastes turned out to be of major importance for the progress of this thesis. The most important rheological parameter in terms of the resulting contact shape on the wafer turned out to be the yield stress τy. Analytical considerations further revealed that despite yield stresses in the range of τy ≈ [600; 3200]Pa, surface effects play an important role during the free flow phase of dispensing thread including wetting behaviour at the nozzle tip and on the wafer surface. It was found that the aspect ratio (AR) depends linear on the product of yield stress τy and nozzle diameter D divided by the surface tension Γ of the paste. With the pendant thread approach, a new method was developed that allows for the extraction of a dynamic extensional yield stress as well as the corresponding surface tension of highly filled pastes (Ca >> 1).
Advanced Centrifugal Microfluidics: Timing, Aliquoting and Volume Reduction
2016, Frank Schwemmer
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jens Ducrée, Dublin City University, Dublin/Irland
KurzfassungDiagnostics and analytics can be fundamentally improved by automation, miniaturization and parallelization. A tool to enable automation, miniaturization and parallelization highly efficiently in compact and easy to use instruments is centrifugal microfluidics. With recent advances the field of centrifugal microfluidics has become more attractive than ever for the integration of complex laboratory workflows. This thesis introduces advanced centrifugal microfluidic unit operations and process chains for fluidic automation in three areas of centrifugal microfluidics: - Volume reduction, allowing handling of volumes down to 40 nl within a new process chain for aliquoting and combination of three liquids in 20 different target ratios. The process chain is applied for preparation of protein samples in small-angle X-ray scattering (SAXS). - Parallel multi-liquid aliquoting, which allows the parallel aliquoting of two liquids and subsequent pairwise combination of these aliquots in a single fluidic layer, and - a new unit operation for timing of pumping and valving events, largely independent of the rotational frequency protocol. The ability to handle nanoliter volumes is one of the key selling points of microfluidics as a field. However, most centrifugal microfluidic cartridges handle liquids in the microliter range. Reduction of volumes has the potential to both reduce the use of expensive sample material and increase the number of tests per cartridge. In chapter 4, this thesis presents a centrifugal microfluidic LabDisk for protein structure analysis via small-angle X-ray scattering on synchrotron beamlines. One LabDisk prepares 120 different measurement conditions, grouped into six dilution matrices. Each dilution matrix: (1) featuring the automatic generation of 20 different measurement conditions from three input liquids and (2) requiring only 2.5 μl of protein solution per dilution matrix, which corresponds to a tenfold reduction in sample volume in comparison to the state of the art. The total hands-on time for preparation of 120 different measurement conditions is less than 5 min. Read-out is performed on disk within the synchrotron beamline P12 at EMBL Hamburg (PETRA III, DESY). This work (1) demonstrates aliquoting of 40 nl aliquots for five different liquids typically used in SAXS and (2) confirms the fluidic performance of aliquoting, merging, mixing and read-out from SAXS experiments (2.7–4.4% CV of protein concentration). The LabDisk for SAXS is applied for basic analysis methods, such as the measurement of the radius of gyration, and advanced analysis methods, such as the ab initio calculation of 3D models. Such experiments can be performed by a non-expert, since the only manual handling step for sample preparation remains the filling of the LabDisk for SAXS with regular pipettes. The new platform has the potential to introduce routine high-throughput SAXS screening of protein structures with minimal input volumes to the regular operation of synchrotron beamlines. The second innovation presented focuses on the precise and highly reproducible metering and aliquoting of liquids, which is a pre-requisite for many analytical applications, e.g. the previously discussed analysis of protein structures. So far no solution has existed for assays that require simultaneous aliquoting of multiple liquids and the subsequent combination of aliquots in a single fluidic layer. In chapter 5, this work introduces the centrifugo-pneumatic multi-liquid aliquoting designed for parallel aliquoting and pairwise combination of multiple liquids. All pumping and aliquoting steps are based on a combination of centrifugal forces and pneumatic forces. The pneumatic forces are thereby provided intrinsically by centrifugal transport of the assay liquids into dead-end chambers to compress the enclosed air. As an example, the unit operation is demonstrated for the simultaneous aliquoting of (1) a common assay reagent into 20x 5 μl aliquots and (2) five different sample liquids, each into four 5 μl aliquots. Subsequently, the reagent and sample aliquots are simultaneously transported and combined into 20 collection chambers. All coefficients of variation for metered volumes were between 0.4–1.0% for intra-run variation and 0.5–1.2% for inter-run variation. The aliquoting structure is compatible with common assay reagents with a wide range of liquid and material properties, demonstrated here for contact angles between 20 and 60°, densities between 789 and 1855 kg·m−3 and viscosities between 0.89 and 4.1 mPa·s. The centrifugo-pneumatic multi-liquid aliquoting is implemented as a passive fluidic structure into a single fluidic layer. The last innovation introduced focuses on the precise, and in large part rotational frequency protocol independent, timing of microfluidic operations. Timing of microfluidic operations is essential for the automation of complex laboratory workflows, in particular for the timed supply of samples and reagents. In chapter 6, this thesis presents a new unit operation for timed valving and pumping in centrifugal microfluidics. It is based on the temporary storage of pneumatic energy and the time-delayed sudden release of said energy. The timer is loaded at a relatively higher spinning frequency. The countdown is started by reducing to a relatively lower release frequency, at which the timer is released after a pre-defined delay time. Timing is demonstrated for (1) the sequential release of 4 liquids at times of (2.7 ± 0.2) s, (14.0 ± 0.5) s, (43.4 ± 1.0) s and (133.8 ± 2.3) s, (2) timed valving of typical assay reagents (contact angles 36–78°, viscosities 0.9–5.6 mPa·s) and (3) on-demand valving of liquids from 4 inlet chambers in any user-defined sequence controlled by the spinning protocol. The microfluidic timer is compatible with all wetting properties and viscosities of common assay reagents and does neither require external means nor coatings. In summary, three new centrifugal microfluidic operations are presented for the integration of complex laboratory workflows. The fluidic operations introduced aim to benefit the development of test carriers that are user-friendly, offer robust fluidics, can be fabricated using scalable fabrication technologies and do not require surface coatings or external means.
Centrifugal microfluidics for nucleic acid analysis at the Point-of-Care
2016, Oliver Strohmeier
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jens Ducrée, Dublin City University, Dublin/Irland
KurzfassungThe analysis of nucleic acids is an essential pre-requisite for initialisation of evidence based therapy of many genetic, inherited or infectious diseases in which the presentation of the clinical symptoms is non-specific or certain sequence patterns on the nucleic acid strand are associated with a pathological indication. Compared to conventional culture based methods, nucleic acid analysis provides an obvious benefit in specificity and reduction in time-to-result. In time-critical situations and in settings where access to centralized labora-tories for sample analysis is limited, nucleic acid analysis, conducted directly at the “point-of-care” would be desired. However, the complex workflow for analysis, typically comprising cell lysis, nucleic acid purification, amplification and detection, hinders the automation in portable systems and consequently only few point-of-care systems are available. Aim of this thesis was the development of a centrifugal microfluidic cartridge for nucleic acid analysis that potentially could be operated at the point-of-care. To achieve this goal, several sub-goals including the development of a process chains for nucleic acid extraction and for multiplex nucleic acid amplification were defined. These process chains were finally co-integrated on one cartridge for sample-to-answer nucleic acid analysis. Initially, a unit operation for transportation of magnetic beads between several microfluidic chambers was developed. Here, bead transport was solely depending on the azimuthal position of the cartridge with respect to a locally fixed, external permanent magnet, ‘transport magnet’. This implementation allowed an isoradial arrangement of the microfluidic chambers thereby circumventing one of the basic limitations for integration of complex assays on the centrifugal microfluidic cartridge: the arrangement of consequtive assay steps in radial direction from the cartridge center towards the rim. Yield of bead transport through three microfluidic chambers was measured to 82.6 % ± 3.6 %. With this unit operation, DNA from Listeria innocua lysates and lambda phages was purified with recoveries of up to 68 % ± 24 % and 43 % ± 10 %, respectively, compared to manual reference. The unit-operation was then completed by a second external permanent magnet, ‘collection magnet’, to collect beads in liquid volumes as large as 1 mL before they are transported out of the liquid into the adjacent microfluidic chamber by the ‘transport magnet’. This improved version was then used for nucleic acid extraction, comprising cell lysis and nucleic acid purification, from various target cells and pathogens spiked in different sample matrices such as whole blood, blood plasma and culture media. DNA has been extracted from logarithmic dilutions of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli with recoveries of 58.2 % - 98.5 % and 45.3% - 102.1 %, respecitively. RNA extraction from logarithmic dilutions of Rift Valley Fever virus spiked in blood plasma yielded between 29.5 % and 34.2 % compared to manual reference while recovery of human DNA from whole blood resulted in equivalent recoveries on-disk and in manual reference extraction ((10.1 ± 7.6) x 104 versus (10.2 ± 6.3) x 104 DNA copies). The process chain was conducted fully automatically within ~ 30 minutes including 15 minutes lysis time. Next, a centrifugal microfluidic disk-segment termed GeneSlice was developed for the detection of the 7 most relevant KRAS point mutations. KRAS mutations are prevalent in up to 40 % of all colorectal carcinomas and the mutation criticially effects cancer therapy. The detection was conducted by allele-specific real-time PCR exploiting the differences in amplification efficiencies between perfect or non-perfect primer match at the 3’ primer terminal. Allele-specific real-time PCR would provide a benefit in time to result (~ 2 hours) compared to dideoxy sequencing (~ 20 hours) currently conducted in routine diagnostics. Intra-chip standard deviation of Cq values for parallel amplification in all eight amplification chambers of a GeneSlice was negligible (Cq std. dev = 0.13 and 0.26) . To prove feasibility, DNA from 6 cell lines in replicates of 4 has been analysed on the GeneSlices resulting in correctly genotyped mutation in 23 out or 24 cases. Finally, DNA from two human colorectal carcinoma samples was analysed showing full consistency with results from sequencing. Furthermore, a process chain for the parallel real-time PCR based detection of DNA from six common food pathogens, including Salmonella, Listeria and Campylobacter, via real-time PCR was developed. The implementation of the process chain included integrated positive and no-template controls to be in compliance with the MIQE guidlines (Minimum information for Publication of Quantitative Real-Time PCR Experiments [1]). Additionally, a unit operation for sequential aliquoting of two liquids with highly different wetting characteristics into 14 aliquots with aliquoted volumes of 5.8 μL ± 0.3 μL (PCR mastermix) and 6.1 μL ± 0.8 μL (elution buffer) has been developed and integrated. In a qualitative operation mode quantities of 10 pg, 1 pg and 0.1 pg DNA of each food pathogen were detected successfully. In a quantitiatve mode, cartridge-integrated PCR standards were used to quantify 50 pg and 500 pg of Listeria monocytogenes DNA to 83 ± 17 pg and 540 ± 116 pg DNA while 50 and 500 pg of Staphylococcus aureus DNA were quantified to 48 ± 4 pg and 643 ± 211 pg. Finally, the process chains for nucleic acid extraction and multiplex amplification were co-integrated on one centrifugal microfluidic cartridge for full sample-to-answer nucleic acid analysis. The cartridge could be processed in a small and portable processing device (size: 25 x 17.5 x 8.5 cm3, weight < 2 kg). Instead of PCR, isothermal recombinase polymerase amplification (RPA) was used to amplify the DNA. Proof-of-principle was conducted for Bacillus anthracis, Yersinia pestis and Francisella tularensis spiked in blood plasma in less then 45 minuts from sample addition to positive amplification. However, amplification showed unreliable, hard-to-interpret fluorescence signals during real-time amplification. As the cause, the degradation of the RPA probe has been identified. Further potential for improvements includes the integration of liquid reagent prestorage on the cartridge. The prestorage of all reagents would reduce the number of manual preparation steps as the user only has to add the sample and start the analysis. However, reagent prestorage would require comprehensive shelf-live studies. These potential improvements would allow even untrained users to conduct complex nucleic acid analysis in point-of-care settings were access to medical diagnostics is currently not available.
Centrifugal step emulsification for digital droplet amplification
2016, Friedrich Schuler
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Piotr Garstecki, Polish Academy of Sciences, Warschau/Polen
KurzfassungMedical and environmental diagnostics often rely on absolute quantification of nucleic acids using digital nucleic acid amplification techniques. In recent years, several major companies have entered the digital amplification market and the number of applications has greatly increased. However, the existing digital amplification systems suffer from a low degree of integration and complicated handling steps. This thesis introduces centrifugal step emulsification as a new droplet generation method enabling droplet generation on a centrifugal microfluidic platform. In the generated droplets three digital nucleic acid amplification techniques are performed (polymerase chain reaction (PCR), recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)). The ability to produce large numbers of monodisperse compartments in the picoliter to nanoliter range is a key requirement for digital nucleic acid amplification. Centrifugal step emulsification offered robust and monodisperse droplet generation (coefficient of variation 1.4–2.2%) on a centrifugal microfluidic setup with only one flowing phase. The droplet diameter could be tuned in the range of 68–220 μm resulting in a droplet volume of 164 pl to 5.6 nl. The droplet volume was independent of rotational frequency in the region of 5–50 Hz. Moreover, centrifugal step emulsification allowed to produce water-in-oil-emulsions with an internal volume fraction of 97.2%. That is to the best of my knowledge the highest ratio reported so far for a microfluidic system. Parallelization allowed for droplet generation rates of up to 3.7 kHz, emulsifying 20 μl in less than 15 s. Only 2 pipetting steps are needed for droplet generation. Using a third pipetting step the dead volume could be reduced from ~1% to a value below the detection limit of ~0.5%. Centrifugal step emulsification was employed in digital droplet PCR (ddPCR) on a centrifugal microfluidic disk (DropDisk). For ddPCR on DropDisk the droplet generation, PCR and readout were integrated in one single disposable. The studied dynamic range was 5–5,000 cp. μl-1. A new unit operation called capillary driven self-alignment was developed that allowed to center >99% of the emulsion in the middle of a chamber to facilitate read out. Capillary driven bubble removal was developed to reliably and efficiently remove >99% of all bubbles from the system. The droplets generated by centrifugal step emulsification could also be used for isothermal digital amplification. On the DropDisk the first digital droplet RPA (ddRPA) was performed (studied dynamic range 100–1,000 cp. μl-1). The ddRPA allows for absolute quantification of DNA in 30 min which is four times faster than current commercially available ddPCR systems. To reduce entry barriers and enable widespread use of digital amplification, a digital droplet LAMP (ddLAMP) system was developed that relies exclusively on commercially available laboratory devices (studied dynamic range 15–1,500 cp. μl-1). The disposable (DropChip) has the size of a microscope slide, following the DIN ISO 8037-1 standard, and can hold two samples. The amplification is started by a thermal initiation step (warm start) and takes 60 min. The designs of DropDisk and DropChip are compatible with scalable manufacturing techniques such as hot embossing or injection molding and could be produced at low prices in the future. In order to reduce the cost for consumables in PCR and increase the throughput, a new multiplexing method for ddPCR and conventional PCR in tubes was developed. It allows to detect two targets in one color channel and was thus called monochrome multiplexing. The confidence levels for distinguishing two targets in one channel in conventional PCR were >99.7% if only one target was present in the reaction, and >68% if both targets were present simultaneously. The method requires only one additional measurement after 10 min of photobleaching and uses standard hydrolysis probes. In summary, droplet generation by centrifugal step emulsification can be employed in at least three digital nucleic acid amplification systems. Monochrome multiplexing enables the detection of more than one target DNA sequence with little additional effort using standard PCR assays. These new applications aim to pave the way for cost effective and sample-to-digital-answer systems which are easy to use.
Characterization and optimization of binding energies in mediator probe PCR enabling multiplex real-time DNA and RNA detection
2016, Simon Wadle
Erstgutachter: PD Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: PD. Dr. Fritz Schwarzmann, Uni Regensburg
KurzfassungMediator probe PCR (MP PCR) was introduced in 2012 as a novel assay principle for real-time nucleic acid testing (NAT). Most NAT formats use fluorogenic target-specific probes for signal generation, such as hydrolysis probe PCR or molecular beacon PCR. In contrast, MP PCR uses label-free target-specific probes (mediator probes, MP) in conjunction with universal fluorogenic reporter oligonucleotides (UR) for signal generation. During PCR, the MP is cleaved and releases a cleavage product, i.e. the mediator. The latter binds to the UR, is extended and leads to spatial separation of the fluorophore (F) and quencher (Q) moieties at the UR, resulting in an increase in the fluorescence signal. The UR can be optimized regarding the signal-to-noise ratio (SNR) upon target amplification independently of the sequence to be detected. To expand the applicability of MP PCR, this work has three major goals: 1) Improvement of MP PCR performance in terms of accuracy, precision, and detection limits, 2) demonstration of reverse-transcription MP PCR (RT-MP PCR) to enable RNA detection for the first time, and 3) development of first multiplex MP PCRs to detect up to five different targets in parallel. To reach these goals, a design-of-experiments (DOE) approach was used for the first time to characterize and optimize MP and UR binding energies in MP PCR. More, the design of the UR was investigated and optimized based on novel fluorescence signal characterization methods. The UR comprises a hairpin structure to bring F and Q into close proximity. A reduction in the distance between F and Q in the folded conformation of the UR from 4 nucleotides (UR4 nt) of state-of-the-art UR designs to 1 or 0 nt (UR1 nt/UR0 nt) led to a reduction in the background fluorescence signal (Fbackground) of 50-83%. Reduced Fbackground resulted in SNRs upon extension of the mediator that were up to 24-fold higher compared to the prior UR4 nt designs. With the novel UR1 nt designs, MP PCR excels gold standard HP PCR in the detection of a dilution series of human papillomavirus subtype 18 DNA (HPV18, 10-105 copies). Intra-assay precision / accuracy were 73-96% / 76-96%, and 59-97% / 69-92% using MP PCR and HP PCR for detection, respectively. For the detection of influenza virus B (InfB) RNA, the first demonstration of RT-MP PCR, the analytical limits of detection at 95% probability (LOD) (incl. 95% confidence interval) were 2 (2; 3) and 19 (11; 227) RNA copies (cp.) using RT-MP PCR and RT-HP PCR, respectively. The amount of released mediator was modeled for different MP and UR concentration ratios based on equilibrium constants. Earlier occupancy of 50% of UR with mediators by 1.3 PCR cycles was predicted for a 1.5x MP over UR excess compared to a 1.5x UR over MP excess (state-of-the-art guideline). This was verified with experimental data showing a reduction in the MP PCR Cq-value by up to eight cycles using the novel ratio. The first DOE-based characterization of the effect of MP design features on RT-MP PCR assay performance led to the following design guideline: the Gibb´s free energy change (ΔG°) of the heterodimer of UR and MP must be at a minimum 4 kcal∙mol-1 higher compared to ΔG° of the heterodimer of the target and MP. With this guideline, the LOD (95% confidence interval) of InfB and human metapneumovirus (hMPV) RT-MP PCR could be reduced from 8 (6; 23) to 5 (3; 13) and 29 (18; 165) to 8 (7; 11) cp. per reaction, respectively. Furthermore, this was the first time DOE has been applied to the design of real-time PCR probe sequences, which can lead to savings in experimental effort and costs during assay development. In the present study, the number of individual reactions required to receive the same statistical power of the analysis was reduced from theoretically 320 in a one-factor-at-a-time analysis to 180 with the DOE approach. The novel guidelines concerning UR fluorescence, and the MP and UR binding energies were applied to the detection of a panel of five different viruses causing respiratory tract infections. RT-MP PCR showed lower detection limits at 95% probability compared to RT-HP PCR in most cases: human adenovirus 7/7 cp. per reaction (RT-MP PCR/RT-HP PCR), influenza virus A 4/18, Inf B 5/8, hMPV 8/56, and respiratory syncytial virus 13/17. To increase the multiplexing degree beyond biplex assays developed prior this thesis, the aforementioned guidelines were adapted to five differently labeled URs, spanning the fluorescence detection channels of standard real-time PCR thermocyclers. Excellent performance of the developed UR set for multiplex MP PCR was observed for the detection of a panel of four different genes of methicillin-resistant Staphylococcus aureus (MRSA) and five different animal species in a food control assay. The latter pentaplex MP PCR performed equally well compared to a commercially available HP PCR assay concerning efficiency, linearity, accuracy and precision. In the case of MRSA detection, the LODs of the four different genes were 18, 33, 12 and 11 cp. with the quadruplex MP PCR instead of 99, 70, 52 and 51 cp. with the reference HP PCR. In summary, novel guidelines for the F-Q position on the UR and the binding efficiencies of the MP to the target and the UR were found to improve MP PCR performance and allowed for the first demonstration of RT-MP PCR. More, a constant set of five different fluorogenic reporters was designed and verified for multiplex MP PCR. Future use of this UR set in PCR mastermixes will simplify the development of real-time multiplex PCR, because no further fluorescence signal optimization is required. This work thus led to further exploitation of MP PCR’s potential in NAT, especially in comparison to HP PCR, RT-HP PCR and multiplex HP PCR.
Development of Genetically-encoded Biosensors with Microfluidic Large Scale Integration Technology
2016, Simon Ketterer
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Wilfried Weber, Biologie, Uni Freiburg
KurzfassungMetabolites play an indispensable role in cellular communication by co-determining cellular behavior. Pronounced examples of this link are the effects of metabolites pyruvate, glycine, and trehalose-6-phosphate on cancer, neuronal signal transmission and resistance to stress. Rapid, parallel, and quantitative determination of metabolite concentration has been a daunting challenge in analytical chemistry. Natural sensing systems in cells, however, are prevailing in macromolecular biology. Biological macromolecules are generally evolved to process the metabolic information essential for cellular needs. Reengineering the original function of biological macromolecules in order to generate genetically-encoded biosensors on analytical platforms for metabolite measurements in vitro and in vivo is the aim of the thesis. In detail, the biophysical properties of three types of signaling biological macromolecules – namely transcription factors, RNA aptamers and riboswitches – were investigated in their sensing capabilities by means of microfluidic technology. Biosensor technology is transformed by microfluidics because the realizable limits in terms of miniaturization, parallelization, automation and feasibility are pushed further by microfluidic technology. Microfluidic chips with large scale integration of functional elements were used as the analytical platform for generating hundreds of different biochemical reaction conditions. On these microfluidic devices, the fluorescence determined by the signaling macromolecules was measured in response to the different reaction conditions. Then the biophysical description of the measurement resulted in identification of optimal reaction conditions towards improving the potential of geneticallyencoded biosensors on microfluidic platforms. Summarizing, this thesis encompasses applied synthetic biology and microfluidic technology with the aim to measure cellular metabolic activity.
IntelliDrug - Controlled, oral drug delivery system as tooth implant
2016, Thorsten Göttsche
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK - Sensorik
KurzfassungIntelliDrug was the name of a project aiming the development of an Intelligent Drug delivery system that is implantable into the human oral cavity. Within the development of the system, two major novelties in the field of portable drug delivery systems were focused. One challenge was the investigation of a new drug delivery route to the body through the buccal mucosa. A second challenge was the technological realization of the miniaturized device itself. The main targeted application of the IntelliDrug system is the therapy of drug addiction with naltrexone and more general the long-term treatment of chronic diseases. Technological achievements The IntelliDrug system is a miniaturized drug delivery system for integration into an artificial denture. Here, it has to withstand punctual chewing loads of up to 250 N and the strongly corrosive environment in the oral cavity. Different concepts for the IntelliDrug system are presented and evaluated. The realized version of the system relies on a rigid drug reservoir, machined in stainless steel, which incorporates an osmotic pump, refillable with solid monolithic matrix drug pills. A microvalve is aimed to control the drug delivery to the buccal mucosa. A flow sensor allows measurements of the drug delivery rate while a fill-level sensor detects an empty drug reservoir. Control of the microvalve and read-out of the drug fill-level and the flow sensor signals are achieved by wireless IR-communication through the cheek. Upon application of the system to the oral cavity, i.e. a wet environment, the pressure inside the drug reservoir increases. With Naltrexone HCl as model drug, the maximum pressure inside the system raised up to 580 kPa. At open microvalve, the maximal pumping rate in a test setup measured 3.8 μl/h. A fluidic capacity inside the test setup and an appropriate switching of the microvalve allowed increased peak deliveries of 8 μl within less than 1 minute. Polypyrrole (PPy) was selected as actuator material of choice for the microvalve and accordingly evaluated regarding its actuation parameters in miniaturized setups. Different electrolytes, materials for the counter electrode and actuation potentials are considered. NaDBS (sodium dodecylbenzenesulfonate) was selected as electrolyte in the IntelliDrug system. Best actuator performance was obtained in a range of concentration from 0.1 M to 0.2 M. With silver as material for the counter electrode the actuation potentials could be kept below 1.2 V which avoids formation of gas by hydrolysis in the system. Three different valve approaches based on Polypyrrole (PPy) have been evaluated. A coated-wire tube valve, a wafer-level fabricated membrane valve and a micro-assembled membrane valve, both applying a PPy-goldbilayer membrane. The latter was evaluated for application in the IntelliDrug system. For the first time, a pressure-compensated separation between the electrolyte for actuating the PPy and the controlled drug solution allows a valve function that is independent from the controlled drug solution. At a pressure difference of 50 kPa in a measurement setup, the valve membrane was able to pull open upon actuation, allowing a flow-rate through the valve of 1.8 ml/h and repeatedly shut down the flow completely. While the opening and closing behaviour of the membrane is reproducible, the achieved flow-rate in open state was not clearly defined. In the presented configuration, a reliable valve function was limited to about 50 cycles of actuation. The long-term stability of the PPy-gold-bilayer membrane was identified as major issue and requires further research. For these reasons, in-vivo testing on humans was performed with a simplified system without valve in order not to endanger the validity of the achieved results. The housing into which the individual developed system components were integrated was machined in biocompatible stainless steel by wire discharge erosion. It is designed to resist a punctual load exerted by chewing of up to 250 N, leading to a maximum stress of 584 MPa and a maximum deformation of 10 μm. Therapeutic achievements In the course of animal trials with pigs that were conducted separately to the IntelliDrug system with a simplified delivery setup, the administration to the buccal mucosa of three different exemplary drugs, naltrexone, galantamine and insulin was evaluated regarding the achieved bioavailability. Naltrexone and galantamine were evaluated as drugs with increased therapeutic potential when delivered to the buccal mucosa compared to established per os administration (swallowing of a pill). The delivery of 10 mg of naltrexone at 80 mg/ml in 10 minutes showed a bioavailability of 64 % with a physiological favourable sustained release of the drug to the systemic circulation compared to the established per os delivery. Plasma levels of more than 10 times higher than the therapeutically necessary value of 2 ng/ml were achieved. The transbuccal delivery of 10 mg of galantamine at 10 mg/ml in 1hour showed a physiologically favourable profile over a period of 20 hours with a peak of 35 ng/ml after 2 hours of start of delivery. The achieved bioavailability measured 29 %. The administration to the buccal mucosa of 3 mg of insulin at 12 mg/ml in 10 minutes did not show detectable influences on the animals’ blood glucose levels. In the course of clinical trials with 12 healthy individuals (humans), the relative bioavailability of naltrexone, delivered by a simplified IntelliDrug system without valve, was calculated to be up to 25 times higher compared to the established per os delivery.
Microfluidic Large-scale Implementation of the Proximity Ligation Assay for High-content Signaling Studies on the Single-cell Level
2016, Matthias Blazek
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. M. Reth, MPI für Immunbiologie und Epigenetik
KurzfassungSignaling in biological cells is a complex and organized biochemical process, that allows the cells to maintain homeostasis and to adapt themselves to changing environmental conditions. Irregularity in cellular signaling is associated with diseases like diabetes and cancer. A majority of signaling processes, such as protein interaction and post-translational modfication is well investigated in terms of qualitative description. However, quantitative and dynamic data of signaling processes, as required for comprehensive understanding is missing. In addition, heterogeneity and cell-to-cell variations raise the demand for cell signaling studies on the level of individual cells. To retrieve such data, this thesis presents the successful implementation of the Proximity Ligation Assay (PLA) on microfluidic large-scale integration (mLSI) chips. The PLA is an in-situ analytical assay to detect concentration, location, post-translational modification and interaction of proteins with single-molecule resolution. It combines the specificity of antibody binding with the sensitivity of DNA-based signal amplification. The microfluidic implementation and automation leverages the PLA for highthroughput signaling studies. Performance advances achieved on the mLSI chips include (i) precise temporal and chemical control of 128 separated cell cultures, (ii) cell stimulation protocols with temporal resolutions down to 10 s, (iii) full automation of cell culturing (up to 96 h), sample preparation and PLA, (iv) multiplexing of up to eight targets in a single experiment, (v) reduction of reagent consumption by two orders of magnitude compared with standard procedures and (vi) a full automation of high content PLA image acquisition and analysis on the single-cell level. The on-chip PLA system was employed to characterize the Akt signaling cascade on two mouse fibroblast cell lines. The sequential phosphorylation response of PDGF/IGF receptors, Akt, GSK3beta, p70S6K, S6, Erk1/2 and mTOR as well as the translocation of FoxO3a was monitored upon stimulation with the growth factors PDGF and IGF-1. PLA results revealed characteristic phosphorylation times of the receptor proteins between 10 s and 40 s, of Akt and its direct targets between 30 s and 4 min and of indirect targets of Akt starting at 10 min after stimulation. Regarding the central protein kinase Akt, the order of activation of its both phosphorylation residues Ser-473 and Thr-308 could be resolved. In case of both stimulants, Thr- 308 preceded the phosphorylation of Ser-473 by 150 s (PDGF) and 20 s (IGF-1). Quantitative robustness of the results was confirmed (i) with help of a control cell line deficient for the Rictor protein and (ii) by a statistical analysis proposing a minimum sample size of 350 cells per assay condition. With the on-chip implementation of miniaturized and automated PLA, as presented in this thesis, the dynamics and the molecular order of phosphorylation reactions in the Akt signaling cascade could be quantitatively determined. The approach offers a biotechnical tool for dynamic cell signaling studies relevant for a broad range of applications.
Pneumatically actuated high temperature resistant printhead for molten metals based on the StarJet principle
2016, Nils Lass
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Holger Reinecke
KurzfassungThis thesis reports on the design and characterization of printheads that allow for generating molten metal micro droplets, based on the StarJet technology. The interest in 3D printing has strongly increased during the last few years and is also becoming more and more common in even more industrial sectors [1]. For the additive generation of three dimensional polymer structures different printing technologies have been presented and successfully established on the market. However, for the generation of metal structures only powder-based laser processes are available so far. Main disadvantages are the high investment costs and the large size of these machines, which renders access to new industrial sectors challenging. For that reason a printing technology, capable of directly printing metal structures from various raw materials, would enable to achieve a step-change in development. Beside the use as rapid prototyping system, such a printing technology could be utilized in numerous other fields of industry like e.g. the electronics or solar industry for the direct generation of various types of electrical interconnections. Core of the so-called StarJet printhead technology is a silicon nozzle chip comprising a star-shaped nozzle orifice which is fabricated by MEMS processes. The main advantages of the printhead result from the combination of this unique star-shaped nozzle design with a pneumatic actuation principle. Through the star-shaped channels an inert gas flow can be established which constricts the liquid metal column when it enters the star shaped nozzle orifice and hence facilitates a necking of the liquid column in that region. The gas flow further shields the liquid metal from oxidation as it gets separated into individual droplets. After tear off, the droplets stay centered by capillary forces resulting from the star- shaped structure until they are ejected. This so-called StarJet principle enables to generate droplets either individually actuated in Drop-on-Demand mode (DoD), or by applying a constant pressure on the reservoir resulting in a continuous droplet generation in the so called Continuous mode (CM). The StarJet principle is the first printing technology for liquid metal which provides those two operation modes. With the printhead version V2 and V3 this is demonstrated at frequencies up to 60 Hz in DoD and 10 kHz in CM mode. In both operation modes the generated droplets are highly uniform (CV < 3.8 %) and adjustable in size (diameters between 35 μm and 360 μm) by the type of nozzle chip orifice diameter. The nozzle chips are fabricated using silicon micromachining and especially employing Deep Reactive Ion Etching (DRIE). The fabrication process has been continuously optimized to ensure a high structure quality of the nozzle chip orifices. The current fabrication process enables the production of nozzle chips with a minimum feature size of 6 μm with an aspect ratio of up to 33. Applying this process enables the fabrication of chips with nozzle orifice diameters down to 30 μm. Staring from the printhead versions V0 and V1 which are regarded as State of the Art, two further printhead revisions V2 and V3 have been developed during this work. The final version V3 of the printhead is made of stainless steel providing a high mechanical and thermal stability and is fabricated by an adapted selective laser melting process. That allows for the fabrication of buried gas channels integrated in the sidewalls of the printhead. These channels enable the pre-heating of the required actuation gas streams to ensure a homogeneous operating temperature of the entire printhead during droplet ejection. In this work both operation modes of the printhead are extensively characterized at 320°C using solder as printing material. In this context the influence of the different actuation parameters, such as the temperature of the melt or the pressure values applied for actuating and rinsing the printhead, on the resulting droplet generation is investigated. It is found that the droplet generation frequency utilizing the continuous mode strongly depends on the amplitude of the actuation pressure. The droplet generation frequency can be controlled for each chip design within a certain frequency range in an almost linear manner, with a maximum coefficient of variation of 23%. The adjustable frequency range additionally strongly depends on the geometry of the reservoir outlet tubes (ROT). It was found that decreasing the fluidic resistance of the ROT as well as smaller nozzle orifice diameters lead to increasing droplet generation frequencies. For operating the printhead in Drop-on-Demand (DoD) mode a theoretical approximation is presented to calculate suitable actuation pressure values. According to this approximation the droplet generation process only depends on applied actuation pressure values and is independent of the duration of the applied pressure pulse, which is confirmed by the results of the characterization. It is found that the minimum actuation pressure required for generating droplets (with the same type of nozzle chip) in DoD mode is more than 5 times lower than for the droplet generation in continuous mode. The StarJet printhead is mounted into an axis system featuring a heatable substrate holder. With this printing system the influence of the individual printing parameters on the properties of the printed structures are studied. It is shown that the merging process of the printed droplets after impact strongly depends on the substrate temperature and the adjusted pitch values. By performing a parameter study parameters are found enabling the printing of three-dimensional structures with demonstrated edge lengths of up to 5 mm and an average 3D surface roughness of less than 10 μm. The printhead is evaluated at different operating temperatures using higher melting metal alloys. Supplying ZAMAK (Zn96Al4, melt = 420°C) at 550°C in combination with a nozzle chip featuring a nozzle chip diameter of nozzle = 144 μm enables the generation of ZAMAK droplets with droplet = 180 μm. However, due to chemical interaction of molten metal with silicon the nozzle chip is dissolved over time. Consequently, two approaches to passivate the nozzle chip are investigated to evaluate the feasibility of printing molten aluminum. First, the surface of the nozzle chip is coated with a zirconium dioxide layer and, second, a nitride layer is applied by chemical vapor deposition in a bulk process. Both methods can be used on unstructured surfaces to inhibit the dissolution of the silicon. However, neither of them can be applied without further optimization inside the bypass channels and nozzle grooves due to the small dimensions of the nozzle chip features. Hence, the final printing experiments with molten aluminum (melt = 650°C) at a temperature of 850 °C failed for this reason. Nevertheless, the experiment proves that printheads based on the StarJet principle are in general capable of being operated at higher temperatures and provide a good basis for further studies.
Pre-storage of reagents for nucleic acid analysis in unit-use quantities for integration in Lab-on-a-chip test carriers
2016, Markus Rombach
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK - Chemie und Physik von Grenzflächen
KurzfassungMolecular diagnostics is increasingly gaining importance for the detection of infectious diseases. Conventional analysis techniques like cell culturing are more and more supported by nucleic acid based methods since they enable a faster detection of causative pathogens. But above all molecular diagnostic methods allow for a decentralized and near-patient point-of-care (POC) diagnostic due to the possibility of miniaturization and full-automation. The knowledge in the field of microfluidics is a key factor for miniaturization and full-automation of clinically relevant diagnostic assay procedures, which also is reflected in the multiple so-called Lab-on-a-Chip (LOAC) test carriers that have been presented over the past decades. The majority of published LOAC test carriers focus on the miniaturization and automation of nucleic acid based pathogen detection, whereas the addition of required reagents is left out and still has to be done manually by trained staff. Ideally, a LOAC test carrier for POC diagnostics is self-contained featuring all required reagents pre-stored onboard, which continues to be a rarely addressed problem. This problem yet is demanding a solution preferably allowing for a low-cost production and being compatible for later mass production. Overall, this work presents four concepts for the pre-storage of reagents required for nucleic acid diagnostics allowing for being integrated into (centrifugal) microfluidic LOAC test carriers by focusing on the usage of techniques applicable for mass production. Reagents can be split into two sub-units according to their field of application: Reagents required for pre-analytical sample preparation and nucleic acid amplification testing (NAAT), respectively. The reagents for sample preparation used in this work come from the commercially available MagaZorb® DNA Mini-Prep Kit (Promega, USA) and comprise buffers and magnetic beads for the isolation of deoxyribonucleic acids (DNA) from a human sample, which were selected for reasons of simplified fluidic handling (free of highly wetting ethanol) and the large amount of clinically relevant sample types covered. All five buffers (lysis, binding, washing (2x) and elution buffers) of the extraction chemistry with volumes of 200 – 500 µL were compactly pre-stored in stacked stick-packs for reducing footprint consumption on the LOAC test carrier. Stick-packs were designed to controllably and simultaneously release their content by applying centrifugal pressures of pcent ≥ 100 kPa. Cross-contamination between buffers was prevented by integration of five downstream collection reservoirs covering each buffer volume. Simultaneous release and collection was successfully demonstrated in 16 consecutive runs. Magnetized cellulose beads, used for solid-phase capture of target DNA, were pre-stored using air-drying in a chemical scaffold consisting of PEG8000 chains. 20 µL MagaZorb® reagent (magnetic bead solution) were mixed with 10 – 20 µL PEG8000 (50%V/V in H2O), pipetted into the designated chamber on the LOAC test carrier and air-dried for 12 h under ambient conditions. Controlled release was realized by reconstituting the complex in the binding buffer matrix (VBB = 450 µL), which was successfully demonstrated in five independent runs. Pre-stored reagents for NAAT comprise unspecific and specific components for amplification and detection of target-specific DNA sequences by real-time polymerase chain reaction (real-time PCR) with the Thermus Aquaticus (Taq)-polymerase being the most challenging regarding pre-storage due to its labile 3D structure. Circumventing cost-intensive conventional freeze-drying, the possibility of air-drying of the glycerol-free AptaTaq-polymerase (Roche, Germany) stabilized by the disaccharide trehalose was investigated. 1 µL of a 2U/µL AptaTaq-polymerase solution were mixed with 4 µL of a 0.5 M trehalose solution and air-dried in a reaction tube at ambient conditions for 3 h. Accelerated stability testing at +70 °C over 7 days (approx. 6 months at 23 °C) followed by real-time PCR evaluation successfully proved the method to be suitable for pre-storage on LOAC test carriers. Finally, a method for conserving primers and differently labeled fluorogenic hydrolysis (i.e., TaqMan) probes at ambient conditions is studied. Primers and hydrolysis probes with four different fluorophore-quencher combinations (6-FAM–BHQ1, HEX–BHQ1, ROX–BHQ650, and Cy5–BHQ2) were mixed with trehalose and xanthan at final concentrations of 56 mM and 2.78 mM, respectively. Mixtures were air-dried at 23°C for 30 min on strips composed of cyclic olefin polymer (COP), a material widely used in the manufacturing of in vitro diagnostic (IVD) test carriers. After one year of storage, the functionality of the primers and fluorophore-quencher combinations was validated by real-time PCR, confirming their stability when stored in the presence of stabilizers, with the best results achieved using trehalose. This approach could be of great benefit for manufacturing IVD systems, for example, for genotyping applications based on multiplexing using different fluorescent dyes. In summary, all presented pre-storage concepts for the integration of different reagents allow for a large-scale production of low-cost LOAC test carriers. Stick-packaging is an already available technique used for mass production. The procedure of air-drying of the different reagents could further be accelerated by reducing humidity and/or environmental pressure and thus could be carried out simultaneously saving valuable time and, moreover, overall production costs.
Single-cell sorting and printing based on bright field microscopy and fluorescence detection
2016, André Groß
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Alexander Rohrbach, IMTEK - Bio- und Nanophotonik
KurzfassungThe fundamental units of each living being are cells. The endeavor to discover and understand the functional principles of cells has gone manifold ways in research. Cellular studies have been increasing in the past decades, aiming to reveal cells’ metabolism, inner structure, and signaling pathways. Nowadays, cells are commonly analyzed as populations which mean thousands or even millions of cells at once. Such analysis yields averaged results only and does not take cellular heterogeneity into account. The latter often is of greater interest than the population’s response itself, leading to the importance of single-cell analysis. The necessity for tools to isolate single cells from a population and extract them safely to a desired target is obvious. This thesis presents the development of a single-cell printer (SCP) to provide individual cells for single-cell applications. The SCP allows for fully automated, non-contact, inkjetlike printing of individual cells from suspension onto various substrates. The core of the instrument is a piezo-electric actuated, drop-on-demand inkjet dispenser chip with transparent top side. A bright field vision system is observing its nozzle. Image processing algorithms enable real-time detection and classification of cells inside the chip. Such, the system is able to forecast how many and which type of cell will be expelled within the next droplet to be generated. In combination with a high speed pneumatic shutter system this allows for rapid droplet sorting. Only droplets populated with exactly one cell are deposited onto the substrate, all void droplets or those populated with multiple cells are removed by the shutter system. Major part of the thesis is related to the development of the cell detection and classification algorithms controlling the automatic cell printing process. In more than 150 individual experiments more than 30’000 single cells were printed and the algorithms were constantly optimized. Furthermore, the developed algorithms are also suitable for effective detection and sorting of cells along morphological features like size and shape. The flexibility of the algorithms allows for isolation of individual cells that are presenting certain characteristics like a specific diameter and/or are specifically spherical. With these findings a growth study for monoclonal cell lines was conducted on three of the most prominent cell lines (CHO, HEK and L929) used world-wide in clonal cell line production with applications in drug screening, cytotoxicity testing and production of monoclonal antibodies. Using these sorting features, clonal growth efficiencies over 90 % could be achieved, which is a 50 % increase compared to standard methods. Second part of this thesis is the development of a single channel fluorescence detection setup and its integration into the SCP. Here, the attempt was to expand the functionality of the SCP from bright field imaging towards detection of fluorescent biomarkers. A mathematical model was developed to estimate sensitivity of the different laboratory setups and help to estimate the impact of certain improvements made throughout the development process. It could be demonstrated that the system is able to sort fluorescent cells by intensity and isolate them from non-fluorescent pools of cells. Finally three relevant applications from the field of cancer diagnostics could be demonstrated in cooperation with a project partner. Models of cervical cancer, cancer stem cells and thyroid cancer have been successfully processed with the fluorescent SCP prototype instrument.

2015

Contact-free dispensing for in-vitro diagnostics: Challenges of the reagent diversity on the performance of appropriate dispensing technologies
2015, Nadine Losleben
Erstgutachter: Prof. Dr. Roland Zengerle
Zweitgutachter: Prof. Dr. Gerald Urban
KurzfassungTrends in the laboratory diagnostic are mainly triggered by the improvement of the medical values of the offered test parameters and the increase of the test efficiency. Improving the test efficiency and the accompanying economic added value for the healthcare system aims to reduce the costs per result. Two mayor cost factors for the IVD tests are the consumption of reagents and disposables. Hence, the trend of the industry is towards the reduction of the needed reagent volumes in future IVD analyzers, which could be achieved by better detection methods and innovative dispensing systems. A most promising method to handle reagent volumes down to the sub-μl-range is the usage of microfluidic contact-free dispensing systems. In this account it has to be ensured that the dispensing system ejects the whole spectrum of IVD reagents precisely and accurately. Aims of this work are the description of the whole IVD reagent spectrum according their fluidic properties; the evaluation of selected dispensing systems regarding their applicability as IVD reagent dispenser; and the definition of operational modes to ensure a lossless fluid transfer from the dispensing nozzle into the reaction vessel. The required energy for a contact-free fluid breakup form the dispensing nozzle is mainly determined by the fluid properties density, viscosity and surface tension. Hence, in order to ensure a reliable fluid breakup for all IVD reagents, these fluid properties have to be known. These are not disclosed in the scientific literature thus far; hence their determination is one key element of this work. To achieve this objective, a representative set of 646 IVD reagents are selected, measured and arranged in a diagram: the fluid properties landscape. Out of this landscape, eight simple and easy to prepare model fluids are developed covering almost the whole property range of the IVD reagents (offered by the Roche Diagnostics GmbH). They are aimed to evaluate dispensing systems for their applicability of dispensing the IVD reagents within the desired performance. It is demonstrated in this work that the model fluids behave like real IVD reagents when the aforementioned fluid properties are almost identical. The differences in their chemical compositions do not influence the dispensing result significantly so that instead of an evaluation with all IVD reagents, only an evaluation with these eight model fluids is sufficient. The required volume range of present and future IVD analyzers are from the sub-μl to the μl-volume range. Dispensing technologies covering this range are primarily operated with either a pressure or a flow boundary condition. Thus, in this work one dispensing system for each group is selected exemplarily in order to evaluate its applicability as IVD reagent dispenser. The dispensing systems fulfilling the required precision and accuracy for all model fluids are the Vermes dosing system (pressure boundary condition) and the cartridge for dispensing a fluid (CDF) (flow boundary condition). The experimental results show that the robustness of the system with a flow boundary condition is better compared to that with a pressure boundary condition. For instance, for the CDF the volume variations are within-a-day ± 0.9 % and due to reassembling of the dispenser components ± 1.0 %, while these values are ± 1.3 % and ± 2.8 % for the Vermes dosing system (two sigma confidence interval). In order to assess the error-proneness of a dispensing system during operation in an IVD analyzer, all factors influencing the dosing performance have to be known. For the two selected dispensing systems they are described by the derived calculation model. This model presents the dispensed volume as a function of the fluid properties as well as the specific geometrical dimensions and the setting parameters of the dispensing system. A special challenge, hereby, is the theoretical system description of the complex dispensing system with a pressure boundary condition. This could be solved with the help of the fluidic network calculation including the pressure drop at a channel constriction. On the basis of the calculation models, error propagations by Taylor are performed for each of the selected systems. They present the total volume error in case that all influencing parameters vary maximal within their tolerance limits. Depending on the operational mode of the dispensing system at the analyzer, the influencing parameters can be different. Most promising for an IVD application is the CDF with a total volume error of ± 1.3 % (1 μl), if the system is primed, while it can be operated without a fluid or cartridge specific calibration. In comparison, the Vermes dosing system leads to a total volume error of ± 5 % (1 μl) for a primed system with a fluid and cartridge specific calibration by a maximal temperature variation of ± 1.9 °C. To profit from the good performance of a contact-free dispensing system in IVD applications, it has to be ensured that all reagents are transferred completely into the reaction vessel and take part in the detection reaction. The formation of satellites, bouncing droplets and foam in the vessel are only some examples of adverse events which may cause unacceptable wrong test results. Two countermeasures are presented in this work. Firstly, a new vessel shape is introduced featuring an inclined wall, a bigger vessel opening and a constriction at the bottom to avoid the formation of foam as well as the volume loss of already captured fluid. The second measure, which is based on theoretical and experimental studies of the satellite formation, describes the need of a carefully adjustment of the maximum distance between the end of the dispensing nozzle to the upper vessel opening in order to avoid volume losses due to satellites failing the vessel. The fluid properties (viscosity, surface tension), the surrounding as well as the length of the dispensing nozzle are investigated as parameters influencing the satellite formation. It is shown that the formation of satellites is favored by the combination of a low viscosity and a high surface tension. Thus, water builds the most satellites within the tested properties range of the model fluids. If water is used as fluid, satellites will not fail the vessel in a calm surrounding if the maximum nozzle-vessel distance is ≤ 30 mm (12 mm long nozzle) or ≤ 20 mm (2 mm long nozzle). In a more turbulent surrounding, as it occurs in laboratories, the nozzle-vessel distances should be shorter. Experiments show that for the longer nozzle a distance ≤ 20 mm is suitable, while for the shorter one satellites fail the vessel already at a distance of 10 mm.
Point-of-Care Detection of Botulinum Neurotoxins
2015, Thomas van Oordt
Erstgutachter: Prof. Dr. Roland Zengerle
Zweitgutachter: Prof. Dr. Gerald Urban
KurzfassungBotulinum neurotoxins (BoNT) are among the deadliest known toxins, are relatively easy to produce and have been incorporated into military weapons. Therefore they have been ranked as an agent with highest priority concerning threats of bioterrorism. In addition to the potential deliberate misuse of BoNT, natural outbreaks in farming and food manufacturing also constitute a typical point-of-care (POC) scenario for BoNT detection. In such a scenario fast (< 1 h), portable and automated, on-site detection of unknown BoNT types and forms is required. Existing POC systems for BoNT detection are suitable in specific fields of applications but are of limited use in such a scenario for several reasons. Possible disadvantages include: (i) the assay format does not allow for detection of a broad range of BoNT type and forms; (ii) the stage of developments are proof of concepts rather than thoroughly developed platforms; (iii) they are non-automated or (iv) have been developed on immobile laboratory devices; (v) important engineering aspects such as the reagent pre-storage or fabrication methods are neglected. In this thesis all these limitations are addressed, starting with the long-term storage and fabrication method: Reagent storage presents a major challenge, not only for BoNT detection but for POC detection in general. On-chip pre-storage and automated release of reagents would circumvent on-site pipetting steps which are otherwise necessary. This challenge is first addressed in this thesis with an ultrasonic welding parameter-study that allows for sealing reagents in aluminium / polyethylene (PE) composite film. These seals can be designed to open at defined burst pressures to release the reagents. The sealing time, pressure and amplitude were varied within the range of 100 – 400 ms, 50 – 250 kPa, and 12 – 24 μm, respectively. T-peel tests and electron micrographs revealed four different peel regimes, depending on the parameter combination: (I) Interlaminar peeling at low peel strength with uniform peeling along a weakly bonded PE lamination layer; (II) transition tearing at intermediate peel strength showing areas of interlaminar peeling and translaminar tearing; (III) translaminar tearing at high peel strength showing tears through the entire film; and, (IV) undefined tearing at varying tear strength occurring when vibration effects during welding lead to insufficient contact of the films or high pressures lead to a displacement of PE. This study allows the systematic adjustment of ultrasonic welding parameters for PE films. The parameter study was subsequently applied to tubular-shaped foil pouches called stick-packs. Stick-packaging has become a popular technology for food and drug packaging. In this study this technology has been miniaturized for use in lab-on-a-chip (LOAC) systems to pre-store and release liquid and dry reagents on-demand in a volume range of 80 – 500 μl. An integrated frangible seal enables the pressure-controlled release of reagents and simplifies the layout of LOAC systems, thereby making the package a functional microfluidic release unit. The frangible seal is adjusted to defined burst pressures ranging from 20 to 140 kPa. The applied ultrasonic welding process allows the packaging of temperature sensitive reagents. Stick-packs have been successfully tested applying recovery tests (where 99 % (STDV = 1 %) of 250 μl pre-stored liquid is released), long-term storage tests (where there is loss of < 0.5 % for simulated 2 years) and air transport simulation tests. The developed technology also enables the storage of a combination of liquid and dry reagents. It is a scalable technology suitable for rapid prototyping and low-cost mass production. In the final development a POC platform is presented that detects BoNT type A and E and all their subtypes and forms. A highly sensitive luciferase reporter assay has been automated by the centrifugal microfluidic LabDisk platform. The assay is based on the detection of BoNT’s proteolytic activity and generation of a bioluminescent signal. This signal is caused by the release of luciferase which is pre-bound to microbeads via a peptide linker cleaved by BoNT. It detected purified BoNT holotoxin, BoNT in complex with neurotoxin associated proteins, and the recombinant enzymatic BoNT light chain in buffer and whole milk in the concentration range of 8 pM – 6 nM with a limit of detection of 6 – 14 pM. The intra-disk, intra-day and inter-day variability were in the range of 1 – 13 %, 1 – 7 % and 10 – 13 %, respectively. The developed LabDisk assay correlated well with the conventional microwell plate assay. It is superior to the conventional BoNT assays in terms of portability, lesser sample requirement, lesser number of working steps and detection of broad spectrum of BoNT types and forms. It takes only 30 minutes for 7 tests in parallel, which is ideal for POC detection of BoNT in case of security threats and food monitoring. The developed microfluidic chip has further potential to perform more tests in parallel or even to incorporate additional sample pre-treatment. With the cleavable linker that is sensitive to serotypes A, E and all their subtypes and forms, the LabDisk already detects a broad range of the most common types causing human botulism. An additional cleavable linker for further serotypes could enable the detection of the entire BoNT spectrum. This would not require changing the fluidic design or handling of the LabDisk. Furthermore the platform developed in this study is not only a proof of concept but has been validated under conditions that reflect real POC requirements.
Using microorganisms culture supernatants to supply enzymes to biofuel cells and extend cathode lifetime
2015, Sabine Sané
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Johannes Gescher, KIT/IAB, Karlsruhe
KurzfassungBiofuel cells show great potential for the environment-friendly direct conversion of chemically stored energy into electricity. Microbial fuel cells use microorganisms and enzymatic fuel cells use purified enzymes as electro-catalysts. This omits the need to supply expensive noble metal catalysts. Microbial fuel cells typically have longer lifetimes than enzymatic fuel cells because microorganisms are able to regenerate, while enzymes usually degrade or become inactivated more quickly. The enzyme purification procedure is also time consuming and costly. However, higher current densities can typically be achieved with enzymatic fuel cells. The aim of the work described in this thesis was to combine the advantages of microbial (typically longer lifetimes) and enzymatic (typically higher current densities) fuel cells. Results described in this thesis show, for the first time, that supernatants of different enzyme-secreting microorganisms such as yeast, fungi and bacteria can be used to supply unpurified enzymes to the electrodes of biofuel cells without the addition of mediators. This obviates the need for elaborate and expensive enzyme purification, and simplifies the construction process. Furthermore, this thesis shows a first demonstration that with regular re-supply of fresh enzyme-containing culture supernatant, the lifetime of an enzymatic electrode can be extended. Therefore, the construction of a self-regenerating biofuel cell in which enzyme-secreting microorganisms continuously supply catalysts at the electrode could be feasible. At the cathode, laccase-containing supernatant (3.4 U mL-1 laccase, pH 5) of the fungus T. versicolor yielded the best cathode performance with current densities of 129 ± 19 μA cm-2 at 0.644 V vs. NHE. The achieved current density was even slightly higher than the current density recorded for cathodes supplied with purified laccase of the same activity. With laccase-containing supernatant (0.06 U mL-1 laccase, pH5) from the recombinant yeast Y. lipolytica YL4, a current density of 6.7 ± 0.4 μA cm−2 at 0.644 V vs. NHE was achieved. The use of a copper efflux oxidase (CueO)-containing supernatant (0.034 * 10-1 U mL-1 CueO, pH 5) of E.coliCueO resulted in a high current density of 119 ± 23 μA cm−2 , similar to the results achieved with supernatant of T. versicolor, but at a lower potential of 0.400 V vs. NHE, due to the lower redox potential of CueO. In contrast to laccase-containing supernatant, CueO-containing supernatant was also electro-catalytic active at pH 7.4 but at a 0.250 V lower potential at a current density of 100 μA cm-2 compared to its use at pH 5. At the anode, cellobiose dehydrogenase (CDH)-containing supernatant (0.08 U mL-1 CDH, pH 5) from the recombinant yeast Y. lipolytica YPC4 resulted in the highest current density of 39.1 ± 5.9 μA cm-2 at 0.600 V vs. NHE. A current density of 27.6 ± 1.3 μA cm-2 at 0.600 V vs. NHE, was achieved with supernatant of the fungus P. chrysosporium (0.12 U mL-1 CDH, pH 5). Also, complete biofuel cells solely supplied with microorganism culture supernatant were constructed and resulted in a maximum power density of 6.2 ± 1.2 μW cm-2 with laccase-containing supernatant from the fungus T. versicolor at the cathode and CDH-containing supernatant of the yeast Y. lipolytica YPC4 at the anode. The achieved power density was reduced by only around 50% compared to the use of purified enzymes, and was even in the same range as was reported for biofuel cells using purified laccase and CDH. Biofuel cells based on complete fungi (T. versicolor, laccase and P. chrysosporium, CDH) and complete yeast (Y. lipolytica YL4, laccase and Y. lipolytica YPC4, CDH) supernatants resulted in maximum power densities of about 5 μW cm-2 and 1 μW cm-2, respectively. Higher enzyme activity did not always lead to higher current densities. Components in the cultivation medium or secreted byproducts can reduce the electrode performance. Therefore, the possibility to use different microorganisms opens more options for their application at biofuel cells. The use of fungal supernatants is attractive for applications outside the laboratory where the use of genetically modified organism such as Y. lipolytica YL4 and YPC4 and E.coliCueO can be highly regulated. However, a continuous enzyme production might more easily be obtained with unicellular, planktonic yeast and bacteria. Finally, it was demonstrated that the lifetime of a cathode can be extended by at least 5-fold up to 120 days with the re-supply of fresh laccase-containing supernatant of T. versicolor at a continuous galvanostatic load of 50 μA cm-2. During the operation time, no gradual decay was recorded and the experiment had to be stopped due to technical reasons. Therefore, it is very likely that a longer lifetime is possible. Future work could concentrate on the construction of self-regenerating biofuel cells in which enzymes are supplied continuously at the electrodes by microorganisms. In this respect, it would be interesting to test if a lifetime extension is also possible by re-supplying enzyme containing supernatant of Y.lipolytica YL4 and YPC4, E. coliCueO or P. chrysosporium. Furthermore, the application of supernatants with increased enzyme activity could increase the achieved current density. Future work could also include the screening of alternative supernatants containing other enzymes such as bilirubin oxidase, which is electro-catalytically active at neutral pH. The combination of cathodes based on culture supernatant with a microbial anode would also be very interesting to investigate, because microbial anodes exhibit high current densities and have long lifetimes, but the cathode often limits the performance of complete microbial fuel cells.

2014

A Smart Reagent Dosing Cartridge for In-Vitro Diagnostics
2014, Stefan Borja Bammesberger
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Andreas Guber, KIT, Mikrofluidik
KurzfassungThis thesis reports about the development process of a “Smart Reagent Dosing” cartridge for in-vitro diagnostics. In-vitro diagnostics is the diagnosis of diseases in a test tube, i.e. in a controlled laboratory environment. This is performed in the form of biochemical assays. A comprehensive portfolio of typically used in-vitro diagnostic tests in professional diagnostics comprises nowadays about 600 different reagents that are handled by conventional pipetting procedures. The Smart Reagent Dosing cartridge aims to replace this pipetting technology by combining the capabilities of storing reagent in a reservoir, dispensing reagent into the test tube and featuring an online process control of the dispensing procedure. Hereby, the cartridge based approach differentiates between low cost components, which are assigned to the cartridge and more costly components which are assigned to the analyzer system. The cartridge-sided components are contaminated by the reagent and are therefore disposed after the entire reagent has been consumed, similar to the well-known cartridges for ink-jet printers. Components which are part of the analyzer system, in other words non-disposable components, are not contaminated by the reagent and therefore can be reused without requiring elaborate washing procedures. The Smart Reagent Dosing cartridge was developed within a structured and systematic development process which is described as part of this thesis. First, meaningful performance parameters are defined to enable an objective, comparable and differentiated evaluation of the dispensing performance of the many concepts of the Smart Reagent Dosing cartridge. These performance parameters are designed to be universally applicable for very different technological concepts as well as for other dispensing applications beyond the scope of in-vitro diagnostics. The superordinate goal here was to pave the way for an industry wide standard for the evaluation of dispensing performance. The development process as described here is subdivided into four distinct phases. During the research phase, 81 potential dispensing systems were identified, categorized and benchmarked. On this broad basis, 55 concepts of cartridges with different dispensing modules, reservoirs and sensor concepts were developed. These concepts were again categorized and evaluated theoretically in order to select the six most promising concepts. During the breadboard phase, these six concepts were realized as functional cartridge models and characterized experimentally based on the performance parameters defined previously. Finally, two prototypes were selected and developed further into a product near status. The main prototype, referred to as Pump’n Dispense prototype, features a non-contact dispensing cartridge for the calibration-free dosage of diverse biochemical reagents from the nanoliter to the microliter range. The patented system combines the advantages of a positive displacement syringe pump (responsible for defining the aliquot’s volume with high accuracy) with a highly dynamic, piezoelectric non-contact dispenser (providing kinetic energy to detach the liquid from the tip). A non-contact sensor monitors the dispensing process to enable an online process control. In order to further increase confidence and reliability for particularly critical biomedical applications, an optional closed loop control can prevent malfunctions. The dispensing performance of this prototype was characterized experimentally in the range of 0.25 to 10.00 μl using liquids of different rheological properties (viscosity 1 to 17mPas, surface tension 30 to 71 mN/m) without adjusting or calibrating the actuation parameters. The volumetric precision typically yielded a coefficient-of-variation (CV) of <3% and the accuracy was below ±10%. Spin-off results of the systematic development process include e.g. a patented solenoid microdispensing valve featuring a modular injection moldable design, which allows for low-cost fabrication and disposable use of all liquid-contaminated components. The valve enables high precision non-contact dispensing of liquid volumes down to the lower end of the submicroliter range comparable to high-end non-disposable microdispensing valves. The experimentally evaluated CV primarily was below 1% for volumes >2 ml and below 8% for volumes in the range of 0.1 to 2 ml. The developed technologies have the potential to contribute significantly to the improvement of biochemical liquid handling in terms of usability, miniaturization, cost reduction and safety for in-vitro diagnostics in particular as well as laboratory automation in general.
Combining polymer microfluidics with electrical functionality: Novel perspectives for the Bosch lab-on-chip platform
2014, Thomas Brettschneider
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Two phase flow drop formation in StarJet system: Simulations for molten metal and development of superhydrophobic nozzle for aqueous liquids dosage
2014, Artur Tropmann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jan Korvink, IMTEK
KurzfassungThe so-called StarJet technology which is presented in this work is capable of generating micro drops from nano- to picoliter range from liquid metal. The system includes a single or multiple heaters which sustain the liquid phase of molten metals in the reservoir and is subsequently actuated pneumatically. Applying an appropriately adjusted pneumatic pressure to the StarJet dispenser generates single micro drops automatically and continuously. These micro drops leave the star-shaped nozzle without any additional excitation techniques and are narrowly distributed in size. This thesis addresses two main topics: Firstly, an approach to model the unique drop generation method of the StarJet dispenser for liquid metals is conducted. Secondly, this unique drop generation method is expanded from molten, liquid metals to aqueous solutions. To achieve this, the star-shaped micro-nozzles are manufactured by a new process and afterwards characterized experimentally for continuous generation of monodisperse droplets. Pharmaceutical applications such as dry powder inhalers require microparticles with a narrow particle size distribution. Usually this particles are generated via spray drying but struggle with generation of monodisperse particles. The StarJet technology would be suitable for application in this field if it were possible to dispense aqueous liquids. With this in mind, the first challenge of this thesis was to fundamentally understand the working principle of the StarJet. This was achieved, by a combination of experimental studies and simulations using Computational Fluid Dynamics (CFD). Initially conducted experiments with liquid metal reveal different dispensing frequency ranges for nozzles with two different nozzle lengths (300μm and 560μm). Short nozzles generate drops with mean frequencies from about 500 to 2800 Hz; long nozzles generate drops at frequencies from 550 to 2500Hz. However, actuation pressures range from 90 − 125 hPa for short and 100 − 160 hPa for long nozzles, respectively. Generated drop sizes are measured by shadowgraphy and reveal maximum drop sizes of 330 μm for low actuation pressures, using short nozzles. Drop sizes of 260 μm are generated with high actuation pressures and long nozzles. All nozzle diameters are 183 μm. The experimental results further suggest, that the fluidic resistance of the starshaped nozzle is controlling the minimum actuation pressure. To a certain extent, that means that the lower the fluidic resistance the nozzle is, the lower the minimum actuation pressure becomes. Another observation concerning the so-called reservoir outlet tube (ROT) is also made. The ROT is a round shaped borehole which connects the reservoir and the inlet of the star-shaped nozzle. It is observed that an appropriate length and diameter of the ROT are required for proper functionality of the StarJet system. To gain further understanding of the working principle of drop generation within the star-shaped nozzle, CFD simulations of the fluid dynamics within the nozzle are performed: The approach utilizes 3D finite volume method (FVM) simulations. The model domain is divided in two parts. The first domain-part, responsible for the visualization of the two-phase flow within the nozzle is the fully meshed and discretized nozzle and adjacent parts from reservoir and bypass channels. Second domainpart describes remaining reservoir and gas channel geometries by lumped elements which include single phase flow only. Both domain-parts are coupled subsequently to simulate the realistic flow situation in the dispenser as good as possible. Results for drop dispensing frequency of the 3D FVM simulation (525 Hz) show good agreement to experimental results (550 Hz) for their respective low actuation pressures. However, the minimum actuation pressure in the simulation is 90 hPa, compared to the minimum actuation pressure in experiments of 100 hPa. Drop size in 3D FVM simulations is 218 μm and shows a discrepancy in diameter of about 32% compared to experimental observations. This result is explained by the implementation of the surface tension force damping in simulations. The set actuation pressure of 120 hPa results in jet formation and is also expected to be due to the implemented surface tension force damping, which is needed to stabilize liquid surface and reduce simulation times. Despite good quantitative results for low pressures, the 3D FVM simulation copes with disadvantages of extremely long simulation durations of several months. Still, these simulations shed light on the working principle of the dispenser and suggest an oscillating or pulsating flow in the ROT: The liquid first flows into the ROT and the nozzle, establishing a liquid plug in its center. While liquid in the upstream located ROT is decelerated and eventually stops flowing due to increased pressure at nozzle inlet, it is further dragged by the sheath gas flow in the downstream nozzle towards the nozzle outlet. Thus, a single droplet is torn off, leaves the nozzle and a new drop generation cycle occurs, when liquid repeatedly enters the nozzle. Furthermore, the 3D CFD model results are supplemented by a 2D model approach, which is promising in terms of very short calculation times. Both, the 3D, as well as the 2D model reveal a central role of the ROT for the working principle of the StarJet system. Thus, a clear design rule is derived from the simulations as well as experimental observations: The ROT has to allow the retraction of the meniscus of liquid which flows in it. The retraction of the liquid allows an oscillation or pulsation of liquid within the ROT. That means, that too short and wide ROTs or ROT surfaces exhibiting low liquid contact angle lack a sufficient Young-Laplace pressure to reverse the flow in the ROT. Also, too long ROTs lead to high inertia of liquid, preventing deceleration of the liquid column in the ROT and thus prevent continuous and periodic drop generation. The transfer of StarJet’s unique drop generation method from liquid metals to aqueous solutions is achieved by the lessons learned from the simulations performed: Firstly, the star-shaped nozzle must exhibit superhydrophobic properties at its surface and secondly, the ROT must allow for liquid retention. In consequence, a modified version of the StarJet printhead is built which provides a ROT milled from Polytetrafluoroethylene exhibiting high water contact angles of about 120°. However, the complete star-shaped nozzle demands for superhydrophobic surface properties with high advancing and receding contact angles to prevent pending drops at the nozzle outlet. Thus, several hydrophobization approaches are applied to the star shaped nozzles: Parylene, Teflon-Carbon-Black (TCB), plasma polymerization of a gaseous monomer and a composite material from Polytetrafluoroethylene (PTFE) and Polydimethylsiloxane (PDMS). As Parylene and plasma polymerization coatings of the silicone nozzles do not result in continuous and periodic drop generation, these coating methods are discarded and no longer pursued. TCB coated nozzles finally enabled continuous and periodic drop generation of aqueous liquids. With this coating, it is demonstrated that the StarJet system is capable of continuous and periodic drop generation from Polyvinylpyrrolidone( PVP)/water solutions with a viscosity of up to 105 mPas. Drop generation from solutions with higher PVP concentration (490 mPas) is also possible, but jet disintegration takes place outside of the nozzle. Additionally, dispensing of Mannitol/ water solution is performed. Subsequent drying of the microdrops reveals Mannitol particles with diameter sizes of about 25 μm, thus proving in principle that the StarJet system can be used in spray drying applications. As the concept utilizing TCB coating for the tiny structures on silicon chips is challenging due to clogging by coating and non-uniform coating issues, a new superhydrophobic composite material is developed. A unique process is established which allows to generate superhydrophobic properties on horizontal as well as vertical surfaces in only one processing step. This is achieved by implementing a composite material from incorporated PTFE nano-particles and a PDMS matrix. First, micro-structures of the nozzle are replicated in a double-casting process and initially enclosed nano-particles are excavated during a plasma etching process. This ensures uniform surface treatment and thus generates what can be discribed as a 3D superhydrophobicity, involving any surface that is treated by plasma.These surfaces generate advancing contact angles CAadv = 159°±4°and receding contact angles of CArec = 158°±3°. Finally, this study demonstrates that superhydrophobic nozzles from PDMS/PTFE are operable; hence generating droplets continuously and periodically at frequencies from 250 to 1300 Hz and sizes from 300 μm to 270 μm.
Visualization and Modelling of Porous Media in Electrochemical Cells
2014, Tobias Hutzenlaub
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungAssumptions about the basic microstructure of battery components form the starting point for most models predicting lithium-ion battery behaviour. Until now most models have relied on porous-electrode theory, which describes potential distribution in both liquid-filled pore space and solid phases by employing concentrated solution theory and Fickian diffusion. This approach simplifies the actual microstructure to a large degree by using artificially designed geometrical configurations such as a packing of spheres as calculation domains. This neglects effects that can only be described by considering an inhomogeneous layer with varying pore and particle size. In recent years, focused ion-beam scanning / electron microscopy (FIB/SEM) was developed to study microstructure. This method removes thin layers of electrode material and combines images of each layer to produce a three-dimensional reconstruction of the respective material investigated. This reconstruction can be used as a calculation domain for visualization based modeling instead of artificially designed domains. In the thesis presented, the FIB/SEM technique is for the first time employed to differentiate between all three constituent parts of a LiCoO2 based Li-ion battery cathode - (i) the active material consisting of LiCoO2 grains (ii) the carbon binder and (iii) the pore space - by applying a hybrid method of manual and grey-scale threshold segmentation (Chapter 2). Major achievement and difference to the state of the art is a three-phase reconstruction instead of a two-phase reconstruction which only distinguishes between LiCoO2 and pore space. After the segmentation process is completed, a resulting cuboid representing a cathode section with a volume of approximately 4500 µm3 is generated. Based on this geometrical configuration and including experimental data for the nanoporous carbon binder, a new method to compute tortuosity values for the pore space is developed (Chapter 3). Tortuosity values between 4.2 and 6.1 are derived. This method presents a clear advantage compared to the state of the art, as it is based on images of the actual microstructure. In the past, the most common approach to calculate tortuosity was by deriving it from porosity with the Bruggeman equation. This approach is valid for a packing of equally sized spheres but has been criticized for being inaccurate for real microstructure by battery scientists. The geometrical representation of the cathode generated in Chapter 2 is combined with an electrochemical model (Chapter 4). The model considers the electric potential and lithium/salt concentration distribution in both the liquid electrolyte and the solid active-material phases. A complete charging process from empty to full of the battery is calculated. In contrast to previously published tomography based electrochemical models, the carbon-binder phase is spatially resolved to provide a more realistic description of the electric potential. Carbon-binder coverage of the solid electrolyte interface (SEI) is studied. It impedes local surface reactions and thus affects lithium redistribution. For the considered cathode, the total surface to volume ratio of the SEI is reduced from 11.2×105 to 6.5×105 m2 m-3 when the carbon-binder phase is modelled explicitly. This leads to increased inhomogeneity of the lithium concentration in active-material grains during charging. Lithium/salt concentration in the electrolyte is studied, revealing gradients between 0.9 and 1.5 kmol m-3 depending on the distance to the separator. This is significant because the lithium/salt concentration directly affects the ion transport properties of the electrolyte. Improving the performance of the PEMFC cathode catalyst layer is of central importance in respect to broad market introduction of fuel cells. But due to small dimensions, most established experimental methods to generate information on porous material are not feasible. Consequently, a method based on 3D reconstruction is a possibility to calculate the data necessary for optimisation. In Chapter 5 an approximately 4.35 µm3 sized section of a PEMFC is reconstructed from FIB/SEM images. It is investigated how parameters such as specific surface area or diffusivity, calculated by employing this method, vary, depending on the resolution of the tomographic data. The resolution is varied between 3.4 and 108.8 nm voxel edge length by stepwise coarsening the original images. This provides information on the reliability of data generated by 3D reconstruction in regard to resolution dependency. The following results are derived: On the one hand, specific surface area displays a strong dependence on resolution, thus suggesting that this parameter was not very trustworthy in the first place. Specific surface area is reduced by 78% when changing the resolution from 3.4 to 108.8 nm voxel edge length. On the other hand, diffusivity at this length scale is very stable for all coarsening steps at approximately 2×10-6 m2 s-1 due to opposing trends for Knudsen and bulk diffusivity from which the overall diffusivity is calculated. An important aspect of improving the PEMFC cathode catalyst layer is optimizing its water management. A new modelling method to fill the pore space of a 3D reconstruction of a PEMFC cathode catalyst layer stepwise with water is presented (Chapter 6). It utilizes a sequence to fill the pores according to their size, going from small to large (hydrophilic) or vice versa (hydrophobic), until a predefined value of water saturation is reached. Both cases are compared. At 50% saturation, the hydrophobic case displays a twenty times larger reaction surface area than the hydrophilic case. This strongly favours hydrophobic CCLs to achieve higher power densities. Finally, magnifying lens and a µEye camera are used to study bubbles in DMFC transparent flow field channels (Chapter 7). Blocking bubble behaviour in a native, hydrophobic COC channel is compared to a channel coated with hydrophilic PDMAA-BP at average methanol solution velocities of up to 16.3 mm s-1. The same hydrophilic diffusion layer is used in both cases. The bubble in the hydrophilic channel shows almost no pinning and thus travels at approximately the same mean velocity as the fuel. In contrast to this, a blocking bubble in the hydrophobic channel pins to the channel wall at low mean fuel velocities and requires considerable fuel velocities to reach its tear-off point, which depends on the bubble length. The critical fuel velocities were in the range of 0.5 mm s-1 for the longest and 2 mm s-1 for the shortest bubble studied in this work. To improve the understanding of bubble dynamics, a CFD/analytical model is developed that describes fuel transport around CO2 bubbles. The model is validated and excellent agreement with the experimentally measured bubble velocity is achieved. The model is used to quantify the average fuel velocity of methanol solution bypassing a blocking bubble. This amounts to 74 mm s-1 for the smallest bubble in the hydrophobic channel investigated in this work, which is considerably higher than the average fuel velocity in the flow channel. With this in mind, the combination of a flow channel surface with a receding contact angle of ≥45◦ and a diffusion layer with a receding contact angle close to 0◦ is proposed. This configuration forces fuel to bypass blocking bubbles at a high relative velocity close to the diffusion layer and can mitigate mass transfer losses.

2013

Automatisches System zur Blutgerinnungszeitmessung für die extrakorporale Zirkulation
2013, Henning Höfemann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Hans Peter Wendel, Universitätsklinikum Tübingen
KurzfassungThis doctoral thesis reports on the measurement of blood coagulation time for extracorporeal circulation. The goal of this thesis is the development of a concept which allows for automated measurements with microliter sized volumes of whole blood and for integration as add-on into cardio-pulmonary-bypass systems and dialysis machines. The concept has to be capable of coagulation time measurements depending on heparin concentration and titration of high levels of heparin in whole blood. In this work three microfluidic concepts have been investigated. As the first two concepts based on microfluidic chips and on polymeric PVC tubing with an implemented cleaning procedure have proven inadequate, the utilization of the tubing as disposable with an additional constriction has lead to a reproducible method for coagulation time measurements. The fluidic concept is based on the analysis of an anticoagulated droplet (“plug“) of blood which moves actuated by pressure through the air filled tubing with an inner diameter of 0.5 mm. After activation with a thromboplastin reagent the plug volume reaches 3.5 μl. The plug is driven backwards and forwards through the constriction which has a ratio of surface to volume of about 38:1 mm−1 which is more than four times higher than in the open tubing. With advancing coagulation the driving pressure increases and the time corresponding to exceeding a predefined threshold is interpreted as prothrombin time. Times determined with this method at discrete heparin levels between 0.4 and 1.2 international units per milliliter whole blood show a linear correlation with times obtained with a laboratory coagulometer. A titration of blood with a high heparin concentration of 4 IU/ml with the heparin antagonist Polybrene has been demonstrated successfully. Impedance spectroscopy has been investigated as alternative measurement method of prothrombin times dependent on different levels of heparin in whole blood. A characteristic minimum has been found in the reactance signal correlating with reference coagulation times at a measurement frequency of 300 kHz. The adaption of this method to plug-based coagulation within the tube has been shown in principal using needle electrodes. The developed method of plug-based coagulation time measurement within inexpensive disposable PVC tubing fulfills the requirements of a coagulometer. Nevertheless the complexity of the setup has to be reduced and an interface for the automated drawing of blood samples off the extracorporeal circulation has to be developed before the system may be used as add-on.
Determination of Enzyme Kinetics in Micro Concentration Gradients
2013, Mathias Welsche
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK
KurzfassungAll functions of cells and even of whole organisms are regulated by many biochemical reactions, which influence each other in a complex and dynamic signalling network. Enzymes catalyse those reactions and thus are the basis of cellular signalling. To decipher the cellular signalling network, an all-embracing understanding of the underlying kinetics of its enzymatic reactions, and thus highly efficient analysis tools are essential. The idea of BIOSS Pathway Assembler is to enable a fast and efficient analysis of enzyme kinetics in signalling pathways by observing the interaction of signalling enzymes in overlaid micro concentration gradients, which represent continuous dilution series of enzymes. This concept has been proven in this thesis by injection of enzyme solutions (Src kinase and thrombin protease) into reaction confinements where the enzymes formed micro concentration gradients, by diffusion. Within these gradients the activity of Src and thrombin has been observed with a microscope by fluorescent reporter systems. These gradients starting at an upper concentration of 100 ng/μl Src and 1 μg/μl thrombin, respectively have been generated with a total enzyme consumption of 3 ng Src and 6 μg thrombin. Comparable state of the art experiments with discrete dilution steps in multiwell plates in contrast (20 μl reaction volume, 10 dilution steps 1:2) would require 4000 ng Src and 40 μg thrombin, respectively. There are two key requirements for the realisation of enzyme kinetics measurements in micro concentration gradients that have been treated in this thesis. First an adequate microfluidic dispensing unit to inject enzyme solutions into the reaction confinement is required. It has to be compatible and reliable with instable bioreagents, which are heterogeneous and poorly characterised regarding their physical properties like e.g. viscosity, surface tension, vapour pressure and diffusivity. Second biochemical reporter systems, to read reaction kinetics online and spatially resolved within microgradients are needed. Within this thesis, the microfluidic bubble jet dispenser Pico- Injector [1;2] has been characterised regarding its precision and compatibility to dispense biological reagents. The experiments have shown, that it is perfectly suited to generate micro concentration gradients with minimum consumption of precious biomolecules due to its small droplet volume (15 pl) and dead volume (< 1 μl). Two solutions of instable enzymes (Syk and Src kinase) dispensed with the Pico-Injector did not show decreased catalytic activity and thus, no degradation of enzymes seems to occur during dispensing process. But dispensing of solutions containing BSA (bovine serum albumin), glycerol or detergents increased its volume deviation and decreased the reliability of the dispensing process significantly. Solutions containing more than 10 μg/ml BSA could not be dispensed at all. The advantages of the BIOSS Pathway Assembler concept rely on spatially and temporally changing reagent concentrations in micro gradients. Thus, fluorescence based biochemical reporter systems that indicate the progress of reactions by a signal that is reversible and measurable online and spatially resolved are required. Such reporters have been developed and characterised for Syk kinase, a key player in B cell signalling, and thrombin, a protease involved in the blood clotting cascade. To read out the fluorescence signal a microscope has been used. The Syk reporter is based on the CHEF principle (chelation enhanced fluorescence) published by Imperiali et al. [3]. It was optimised for Syk kinase by mimicking the chemical properties of its natural substrate and modifications with lipopeptide and biotin residues have been introduced, to immobilise it on surfaces for a locallised read out. The modified Syk kinase reporter has shown to be reversible and online readable in solution, but lost its functionality completely when it was bound to surfaces. Thus, to finally proof the principle of measuring enzyme kinetics in micro concentration gradients, a simple thrombin reporter has been developed. The reporter is based on a fluorescently labelled peptide that is immobilised via micro contact printing. By thrombin activity the fluorescent label is cleaved wich could be measured online and spatially resolved with a microscope by a signal decrease down to ~ 50 % of the original fluorescence. However, the experiments have shown that the setup of micro concentration gradient based enzyme kinetic experiments is extremely complex. These systems require surface bound reporters, which are much more complicated than soluble reporter molecules due to effects like fluorescence quenching, sterical hindrance or depletion of reagents by adsorption at the surface. Additionally only the concentration of the reaction product can be measured directly via the reporter signal. The concentrations of all other involved enzymes and reagents have to be calculated by their diffusion coefficients. The determination of diffusion coefficients of enzymes however is extremely difficult. Most enzymes are precious and only available in minute amounts what precludes extensive pretests. Additionally their diffusivity is strongly dependend on environmental conditions, like pH, salt concentrations and interactions with other biomolecules. Thus, a quantitative analysis of enzyme kinetics in micro gradients is difficult and often impossible, since reagent concentrations cannot be determined with acceptable precision.
Fabrication of DNA microarrays by digital solid-phase PCR in a next generation sequencing chip
2013, Jochen Hoffmann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK
KurzfassungA process for the fabrication of DNA microarrays by copying a next-generation sequencing chip is presented in this thesis. DNA molecules of a DNA library are mixed with a biochemical reaction mix and loaded into hundreds of thousands wells of the commercial sequencing chip PicoTiterPlate (PTP, Roche). The DNA randomly distributes onto the wells so that statistically one well contains one single DNA molecule. All wells of the PTP are closed with a microscope slide and the DNA is amplified by polymerase chain reaction (PCR). Each single PCR product is attached as a DNA cluster in the PTP and on the corresponding position on the slide since both surfaces have been coated with PCR primers. The sequence of each DNA cluster can be obtained by a subsequent sequencing reaction in the PTP. This also reveals the sequences of each DNA cluster on the slide thus allowing the slide to be used as a standard DNA microarray. For achieving this aim, different methods were engineered. As a first milestone, a method for the immobilization of PCR primers onto microscope slides made of glass, cyclic olefin polymer (COP), polypropylene (PP), cyclic olefin copolymer (COC), and polydimethylsiloxane (PDMS) was successfully established. In a second step, a solid-phase PCR (SP-PCR) was performed on such slides. In SP-PCR, PCR products are attached to a surface via an immobilized primer and labeled fluorescently. Depending on the material, fluorescent signals of PCR primers increased by factors of 43.9 – 86.8, PCR-induced losses of control oligonucleotides were measured to 31.1 % - 55.6 %. As a second milestone, a DNA library was successfully amplified in 110,000 of 18.5 picoliter wells by the currently smallest on-chip PCR. By adaptation of the established methods for primer immobilization and SP-PCR to a PTP, generated products were successfully immobilized to the surfaces of both – PTP and slide. Surface-bound PCR products were fluorescently-labelled by unspecific and specific staining methods and subsequently detected. Template DNA in length of 100 bp, 346 bp, and 1,513 bp was amplified and immobilized in a PTP with comparable efficiency. As a third milestone, the combination of SP-PCR and digital PCR (dPCR) was performed in a PTP. By this unprecedented combination, PCR products were generated from truly single DNA molecules and immobilized as DNA clusters to PTP and corresponding positions on the slide. Such a PTP can be sequenced to identify the sequence of each DNA cluster. The corresponding slide is used as a master DNA microarray for a replication into further DNA microarrays. Since especially in dPCR carry-over DNA can lead to false-positive signals, two measures against carry-over DNA contaminations were examined. With a chemical decontaminant, DNA concentrations on surfaces were reduced by a factor of 106 in one hour. A biochemical decontaminant reduced false-positive signals from digital solidphase PCR in a PTP by a factor of 4,500. For the molecular replication of a master DNA microarray into a target DNA microarray a fundamental concept was described and verified. In experiments, a liquid polymer was applied onto a master array containing movable DNA molecules and subsequently sandwiched with a target array. During polymerization, the movable DNA molecules attached to the polymer and the polymer adhered to the target array. After separation of both arrays, up to 76.6 % of the movable DNA was detected on the target array, whereupon the master array remained unchanged. In the future, this process is to be applied to a microscope slide (analogue to a master DNA microarray) generated by digital solid-phase PCR in a PTP. The whole described process chain would allow the fabrication of DNA microarrays as molecular copies of a sequencing chip.
Highly Porous Platinum Electrodes for the Use in Potentially Implantable Glucose Fuel Cells
2013, Arne Kloke
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungImplantable glucose fuel cells (IGFC) are investigated as self-regenerative approach for the permanent power supply of medical implants, such as cardiac pacemakers. In contrast to batteries such fuel cells could continuously convert ambient energy stored in glucose and oxygen to electrical energy. This way surgical replacements of medical implants could be avoided which are required when their batteries are exhausted. Since both reactants are simultaneously present in body fluids, a concept for the spatial separation of the electrode reactions to anode and cathode is required to avoid mixed electrode potentials and thus drastic reductions in cell voltage. State of the art implantable fuel cells either use reaction-selective catalysts (enzymes), which suffer from low lifetimes or special design concepts for a physical separation of the simultaneously present reactants. Within this thesis, it is shown how variation of the specific surface area of platinum electrodes can be used to obtain a preference of one of the two electrode reactions and thus to reduce the influence of mixed electrode potentials. For investigation of the relationship between specific surface area and electrode potential a new process “Cyclic Electrodeposition of PtCu Alloys” has been established. This process allows for the fabrication of porous platinum electrodes with controllable specific surface area in a range of roughness factors from 20 to more than 4000. The high specific surface area is obtained by alternation of platinum-copper co-deposition and dissolution of copper from the deposited alloy performed during cyclic voltammetry in an electrolyte composed of diluted sulfuric acid and additions of hexachloroplatinic acid (H2PtCl6) and copper sulfate (CuSO4). The specific surface area can be adjusted by the number of applied deposition cycles. The resulting electrode structures consist out of mixed-phase PtCu nanocrystallites of 5-20 nm in diameter. The concentration of non-dissolved, residual copper decreases from bulk (3 - 8 %) to surface (8 - 20 %) indicating a core-shell structure. From technical point of view cyclic electrodeposition of PtCu alloy is advantageous to state of the art processes since it allows for online-control by tracking the evolution of the cyclic voltammograms, requires no templates, surfactants or elevated temperatures and can in principle be applied on every conductive substrate (e.g. pacemaker capsule). Load curve and continuous load experiments were performed to investigate the relationship between specific surface area and the electrode potentials of anode and cathode under conditions relevant for implantation (phosphate-buffered saline, pH = 7.4, 3 mmol l-1 glucose, 3.5 % - 21.0 % oxygen saturation). Oxygen cathodes were shown to best match the requirements of implantable glucose fuel cells when having a roughness factor in between of 100 to 300 depending on the present oxygen concentration. At this specific surface area, the electrode simultaneously features good accessibility of the entire catalyst surface for a high oxygen mass transport rate as well as a low polarization resistance. Best anode performance was obtained at maximum specific surface area, whereas no substantial change was observed for electrodes with roughness factors higher than 2500. A high specific surface area is beneficial to glucose anodes since a porous internal structure leads to long diffusion pathways and thus to substantial oxygen depletion in the deeper regions of the electrode. Hence glucose oxidation proceeds at mostly anoxic conditions enabling electrode open circuit potentials more negative than -350 mV vs. SCE, even in presence of 7.0 % oxygen saturation. With anodes and cathodes of corresponding specific surface area, no selective biocatalysts or fuel cell designs are required to spatially separate the two electrode reactions. This was clearly demonstrated by investigation of single layer glucose fuel cells which are assembled by simply placing anode and cathode side by side, instead of on top of each other as done in state of the art depletion design (oxygen consuming cathode is placed in front of the anode to enforce oxygen depletion): In single layer design, the same electrodes exhibited the same open circuit cell voltage as in depletion design. According to their simple assembly single layer fuel cells represent the easiest way to implement a surface coated fuel cell (e.g. on implant capsules). In terms of power density, the single layer design is disadvantageous compared to depletion design, since stacking of two electrode layers enables for twice the electrode area at the same geometric footprint. Fuel cells assembled in depletion design enabled power densities of up to 5.1 μW cm-2 which corresponds to an improvement in power density of 16 % compared to state of the art at simultaneously facilitated and technologically improved fabrication. Such a power density would be more than sufficient to power a cardiac pacemaker (5-10 μW) when implemented as surface coating on its capsule (30 - 40 cm2). Nonetheless, the decrease in power density observed during long-term investigations (0.036 μW cm-2 day-1, 90 day at continuous load of 10 μA cm-2) showed that further research on catalyst composition and fuel cell design (e.g.semi-permeable protection layers) is required to realize the concept of a regenerative, implantable long term energy source.
Lokale chemische Stimulation von Zelllinien durch Dispensieren von Tropfen mit dem StimuDrop
2013, Susanne Zibek
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Elke Guenther, NMI Reutlingen
Mediator Probe PCR: A novel assay principle for universal real-time detection of nucleic acid amplification
2013, Bernd Faltin
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: PD Dr. habil rer. nat. Manfred Weidmann , Georg-Augustus-Universität Göttingen
KurzfassungThis thesis describes the development and characterization of the mediator probe polymerase chain reaction (MP PCR), an innovative strategy for the detection of nucleic acids. The novel assay format combines analyte-specific reagents for target amplification with a standardized fluorescence reporter molecule circumventing the need for target-specific fluorogenic probes (e.g. hydrolysis probe, HP). The proprietary key elements of this assay technology are the bi-functional mediator probe (MP), consisting of a target-specific 3’ region (probe) and a generic 5’ region (mediator), and the universal fluorogenic reporter (UR). Upon target amplification, the MP is cleaved by the polymerase. The released mediator is captured by the UR and instantly induces a fluorescence signal. The striking feature of the MP PCR is the indirect detection of the target. Different targets can be detected employing only one design of the UR as the probe region of the MP is adapted to the specific target. In contrast, HPs have to be synthesized for each target with additional expenses. Consequently, the MP PCR benefits from economic probe design. The Mediator Design Software (M-Desire) was exclusively developed within the scope of this work. M-Desire generated random sequences of defined length and accounts for analysis of melting temperature (Tm), nucleotide composition, secondary structure formation and other properties of the mediator sequence. The software pipeline delivered several sets of random sequences without cross-interference or interactions with the target. In considering thermodynamic design, an optimal melting temperature (Tm) of the mediator in the range of the used primers (typically 60.0–64.0 °C) was determined. The Tm of the probe region is recommended to be between 69.0–73.0 °C and can be designed according to common guidelines for HPs. The UR is a self-contained fluorogenic signaling oligonucleotide with hairpin-like conformation due to an internal hybridization of its 5’ region. In this arrangement, a pair of fluorescent modifications is brought in close proximity. Four different designs of the UR were evaluated, with both in silico methods and intensive experimental studies. The high Tm of the internal hybridization sequence (71.4 °C) enabled stable hairpin formation under reaction conditions. The most appropriate design (UR-D) enables fluorescence resonance energy transfer (FRET) with high efficiency (Eq) (> 90.0 % (FAM-DABCYL) and 81.0 % (Cy5-BHQ-2), respectively). The obtained values are constant for each target and do not depend on the sequence and length of the probe molecule, as is the case for HPs. The unpaired 3’ region contains the mediator hybridization site (MHS). The annealing of the mediator is followed by polymerase dependent elongation facilitating conformational changes and disintegration of the UR, respectively. Both pathways suppress FRET and contribute to an arising fluorescence emission. The MP PCR was analyzed in terms of assay characteristics in comparison to a reference HP PCR. As model assay, the amplification of a dilution series of the human papilloma virus 18 Late 1 gene (HPV18 L1) was chosen. The focus was set on the determination of the limit of detection (78.3 copies/10 μl reaction mixture), intraassay precision (55.1–9.9 %) and accuracy (+21.6– -8.1 %), influence of the elongation time on the reaction efficiency (EPCR = 90 % (6 s)–79 % (50 s)), and the parallel amplification of two target sequences within one reaction. For all criteria, the performance was concordant between MP PCR and HP PCR. Furthermore, both amplification techniques were applied to the detection of HPV18, Escherichia coli, Homo sapiens, and Staphylococcus aureus. Amplification of respective DNA dilution series showed exceptional linearity when detected by novel mediator probes (r² = 0.991–0.999) and state-of-the-art HPs (r² = 0.975–0.993), respectively. In a qualitative screening, the MP PCR was evaluated using cervical specimens for the detection of HPV16 DNA. A comparative study of the HPV16 MP PCR with commercially available molecular testings, revealed a concordance of 79.8 % (Linear Array, Roche; n = 183) and 80.6 % (Real-time molecular beacon HPV, GenoID; n = 155), respectively. Further, cervical specimens from women who had precursor stage of cervical cancer as determined by histology, were analyzed. The state-of-the-art Hybrid Capture2 assay (HC2, Qiagen) was used as gold standard method. The negative predictive value and the sensitivity of the HPV16 MP PCR (both 41.1 %) was lower when compared to HC2 (80.0 % / 98.2 %); on the contrary, HPV16 MP PCR revealed a comparable positive predictive value and a significantly higher specificity (both 84.4 %) when compared to HC2 (76.7 % / 19.0 %). In terms of cost analysis, the MP PCR benefits from using a standardized signaling molecule in a multitude of assays. The analysis of numerous different target sequences with a low or medium number of individual reactions, as is typically applied in the development of PCR testings, expose the great potential of the MP PCR. In addition, the purchase of the UR in large quantity benefits from economies of scales and reduce the costs per testing.
Microfluidic Continuous Nucleic Acid Extraction
2013, Marc Karle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jens Ducrée, School of Physical Sciences, Dublin City University
KurzfassungCountless fields of application require continuous monitoring, particularly when monitored parameters underlie strong fluctuations, kinetics need to be determined or the parameter of interest requires frequent sampling and labour-intensive processing. Examples include monitoring of cell count in bioreactors, continuous recording of biomarkers during drug treatment of patients or biological safety applications. Continuous systems have been evaluated and/or commercialised for a number of applications. Continuous microfluidic DNA extraction which would allow for continuous analysis based on nucleic acids however, has not been developed so far. To close this gap, a microfluidic platform for continuous DNA extraction allowing for subsequent PCR analysis was developed within the scope of this work. For evaluation of the DNA extraction efficiency E. coli was chosen as model organism and a specific quantitative PCR based detection system was implemented by designing the respective primers and probe. The development of the microfluidic chip was split into the development of individual modules. For DNA extraction out of cell suspension, the lysis of cells had to be included upstream of the DNA extraction module. For this purpose, module 1 for a continuous cell lysis was developed providing a continuous stream of cell lysate to be fed into the DNA extraction module. Within the lysis module cell suspension and lysis buffer were combined in one microfluidic channel which provided for an incubation time of 305 s. For optimal lysis conditions, the channel was heated to 50 °C. Compared to batch-wise cell lysis in test tubes, the characterisation of the continuous cell lysis module yielded efficiencies of 77 % and 45% in two test runs. The combination of the cell lysis and DNA extraction modules required an additional microfluidic structure for DNA binding to magnetic beads. Therefore, module 2 was developed for DNA binding, in which lysate and magnetic beads suspended in binding buffer were combined in one microfluidic channel providing an incubation time of 169 s. In the binding structure, DNA was continuously bound to the beads with an efficiency of 57 % and 58 % compared to batch-wise references in test tubes. In the third and major part, the core module for continuous DNA extraction from crude cell lysate was developed which provided a continuous stream of pure and PCR-ready DNA. In this module, DNA bound to superparamagnetic beads was routed through microfluidic channels and several separation chambers by using one central rotating permanent magnet. The magnet facilitated the phase transfer of the DNA loaded beads across the phase interface of co-flowing laminar streams of different buffer solutions. With this approach, the extract-wash-elute process of DNA purification could be implemented on one microfluidic structure. The sample inlet flow rate was set to 0.75 μl·s-1 and the eluate flow rate to -0.35 μl·s-1 leading to a 2-fold enrichment of DNA. The characterisation of the continuous DNA extraction module yielded an efficiency of 147 ± 28 % compared to the same process performed batch-wise in test tubes. Additional alternative extensions for bead removal and increased DNA concentration in the eluate were successfully implemented on the novel platform. In a subsequent feasibility study, the combination with a downstream module for continuous microfluidic PCR was successfully shown. In a fourth and final step, all three modules were integrated monolithically on one microfluidic chip. The complete process of continuous DNA extraction directly from cell suspension was successfully shown with an efficiency of 106 ± 19 % compared to the same process performed batch-wise in test tubes. Now, for the first time continuous microfluidic DNA extraction was demonstrated to be feasible. The continuous DNA extraction module as well as the integrated microfluidic chip for continuous DNA extraction including cell lysis and DNA binding, clearly outperformed the off-chip batch-wise references in test tubes. In addition the chip was designed to be compatible with the injection compression moulding process to enable cost-effective mass production in DVD format.
Non-Contact Process Control Sensors for Nanoliter Dispensing Systems
2013, Andreas Ernst
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Leonahrd Reindl, IMTEK
KurzfassungThis thesis deals with the design, the development and the characterisation of non - contact process control sensors to monitor the droplet ejection process of drop-on-demand dispensing systems in the nanoliter range. The design of the sensors is focussed on a small mounting size to enable a smart integration to the dispensing device. The non - contact working method implements a contamination free and non - invasive measurement technique which does not affect the dispensing process. In contrast to the commonly applied process observation systems, which are mostly unable to provide online process information, the developed sensors serve with analogue signals which enable to realise a real-time process control. As the entire sensors are fabricated in printed circuit board technology, very cost effective competitors to the stateof- the-art systems can be placed on market. The implementation of the process control sensors is realised by an optical approach, based on the effects of geometrical optics induced by a droplet passing a measurement light beam and a capacitive measurement method, exploiting the interaction of a dispensed droplet with the electric field of an open capacitor. The volumes of the droplets which are measured by the sensors are in the range V = { 5 to 100 nl }. This volume range arises from the applied dispensing system, based on the PipeJetTM technology, and defines the minimum size of the individual measurement transducers. The application of the optical measurement method is based on the change of the intensity of a measurement light beam while a liquid droplet passes through it. The light beam is present in the flight path of the dispensed droplet and changes it’s intensity due to absorption, refraction and reflection effects. This enables to extract droplet dependent analogue signals which are used to analyse several parameters like droplet velocity, droplet shape, malfunctions in the dispensing process or to identify the reproducibility of successively ejected droplets by the correlation of so called droplet ’fingerprint’ signals. The smallest detectable droplet was identified to be Vmin= 1.5 nl, definig the resolution of the optical sensor. The determination of the droplet velocity could be implemented with an error in accuracy of Δuac= ±0.3 m/s. Additionally, the detection of free flying liquid jets was studied. It was found that the sensor can be applied for the determination of the volume of the liquid jets by the integral of the analogue sensor signals. This however requires to know the velocity of the liquid jet which can not be simply deduced like the velocity of single droplets. The gained results imply the feasibility to realise quantitative process control for micro liter dispensing systems like e.g. dispensing valves or jet ejecting pipettes. An application example is given which describes the successful improvement of the observation of batch process manufacturing of lateral flow immuno assay test stripes by the application of the optical sensor. The developed optical sensor provides a small and cost effective online process control unit which features functinallity far beyond simple droplet presence detection. The capacitive measurement method is based on the change of the capacity of an open plate capacitor induced by the presence of a droplet in between the electrodes. The change in capacity yields magnitudes in the region of ΔC = 0.7 to 3 fF for the considered volume range. The measurement capacitor is placed below the dispenser orifice and aligned to the droplet’s flight path to avoid the contact of the droplet to the electrodes. To study the occuring physical effects in detail, a multi disciplinary computational fluid dynamics (CFD) simulation was established. The correct physical system description was identified to be the effect of capacitive coupling, implying the specific signal characteristics featuring a leading negative signal dip. The target quantity of the simulation study was the change of the charge on the measurement electrode induced by a passing droplet. Based on the simulation results two different sensor electronic read out circuits were designed, differing in their front - end impedance and amplification circuits. The individual designs of the electronic circuits were improved by an established network simulation. The characterisation of both sensors led to a calibration function which was adapted to the individual sensor circuit performance by the adjustment of the calibration factors. The application of this function enabled to determine the volume of single dispensed droplets in the volume range V = 20 to 85 nl with an accuracy of ΔVsensor1 = ± 3 nl for sensor type 1 and ΔVsensor2 = ± 4 nl for sensor type 2 on the fly. The major difference of both sensor types was found in the volume sensitivity which was Sisensor1 = 5.3 mV/nl and Si-sensor2 = 77.2 mV/nl. The sensors resolutions were identified to be Vmin-sensor1= 1.5 nl and Vmin-sensor2= 6.5 nl. The sensors can also be applied to determine the velocity of the dispensed droplets with an accuracy of Δumax = ± 0.1 m/s. The influence of the misalignment of the droplet’s lateral position in between the capacitor electrodes could be quantified to falsify the determined droplet volume by an maximum error of ΔV = ± 12 nl. Also variations of the liquid’s relative permittivity were studied in detail by experiment. It turned out that the capacitive measurement principle is mostly independent to changes in the relative permittivity for values εr > 40. Therefore, an online process control sensor for the non - contact measurement of the volume of droplets in the nanoliter range, which is largely independend from the media properties, could be implemented.
Tomographic reconstruction of polymer electrolyte membrane fuel cell cathode catalyst layers
2013, Simon Thiele
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Margit Zacharias, IMTEK
KurzfassungPolymer electrolyte membrane fuel cells (PEMFCs) will make an important contribution to the energy challenge mankind has to face in the near future. In spite of the advances within the last decades, central technological and scientific challenges impeding the broad market introduction of PEMFCs remain. The oxygen reduction reaction (ORR) occurring in the nano porous cathode catalyst layer (CCL) is considered as the most performance limiting reaction in PEMFCs. This reaction is usually catalyzed by platinum nano particles. At a variety of operation conditions the reactants can only access a part of the catalytically active surface of the Pt. This severely limits the PEMFC performance and is not understood in detail until now. Before being able to improve performance it must be known how reactant transport limitations occur. To model reactant transport the nano porous geometry of the CCL must be known. Within this thesis, methods for the three‐dimensional reconstruction of the nano porous CCL have been developed. Techniques for the three‐dimensional imaging of the internal structure of porous media are called tomographic techniques. Two approaches for 3D reconstruction of PEMFC CCLs have been performed. In the first part of this work it was shown that a combination of focused Ion beam and scanning electron microscope can be used for PEMFC CCL tomography (SEMt). For the first time a three‐dimensional reconstruction of a PEMFC CCL was shown. It enabled to differentiate a solid phase and a pore phase in the nano porous CCL structure. With this approach morphological parameters such as porosity, connectivity, pore‐size‐distributions and grain‐size‐distributions were determined from the reconstruction. Additionally valuable information for valid modeling approaches of gas and liquid transport could be determined. One shortcoming of the approach described in the first part of this work is that position, size and surface area of the nanometer sized platinum particles cannot be determined using SEMt. Therefore in the second part of this work transmission electron microscopy tomography (TEMt) was combined with SEMt. From a TEMt reconstruction, morphological properties such as platinum particle surface area, volume and size distributions can be determined. For the first time an approach was shown to include this information into a SEMt reconstruction. Knowledge on both the positions of the active centers and the transport pathways of reactants is crucial information needed for a future optimization of the CCL structure. It was shown that the approaches developed in this work are very promising methods for future improvements of PEMFC CCLs. However, more research and development are necessary to propose improved CCL geometries. Even more research will be necessary to implement these propositions into manufacturing processes for PEMFC CCLs.
Towards a self-regenerating biofuel cell cathode
2013, Stefanie Rubenwolf
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungA rapidly growing number of distributed autonomous microsystems demand for a reliable long-lasting energy supply by “harvesting” and converting local ambient energy as chemical energy-driven biofuel cells. Enzymatic catalysts for biofuel cells are specific and get along with the typically unpurified fuels and the simultaneous presence of oxidants but the enzymes are deactivated rapidly within days to few weeks. Addressing the limited electrode lifetime, this thesis reports for the first time the compatibility of a mediatorless enzymatic biofuel cell electrode for selfregeneration. Self-regenerating enzymatic electrodes aim to decouple enzyme lifetime from system lifetime by refreshing the enzymes with help of integrated enzyme producing microorganisms, resulting in a theoretically unlimited lifetime. As shown by reviewing published strategies and concepts for enzymatically catalyzed electrodes with extended lifetimes, self-regeneration has so far only been introduced to mediated biofuel cell or indirect biosensor electrodes. For this proof-of-principle the oxygen reducing cathode enzyme laccase from Trametes versicolor was chosen, as it is naturally secreted and it adsorbs reversibly to carbon electrodes while directly transferring electrons. The choice of carbon material thus strongly influences enzyme load and direct electron transfer. This thesis reports the first systematic comparison of different carbon electrodes with adsorbed laccase, employing galvanostatic current density-cathode potential plots to characterize the oxygen reduction performance of half-cells. Multi-walled carbon nanotubes were identified as an efficient electrode material. Immobilization in form of paper-like mats (“buckypaper”) is a suitable method to improve surface availability, resulting in current densities of more than 450 μA cm-2 at 0.744 V vs. normal hydrogen electrode (NHE). To demonstrate the electrode’s compatibility with enzyme renewal, this thesis for the first time offers a quantitative description of time-dependent laccase deactivation and its adsorption behavior. After a deactivation-free grace period of 2 days, laccase activity decreases with a half-life of 7 days. Langmuir-type adsorption to buckypaper was observed with a mean resident time of 2 days. Assuming that adsorbed enzymes do not get inactivated faster than dissolved ones, exchanging the enzyme solution every second day is supposed to result in a stable electrode coverage with mostly active enzymes. In a long-term experiment at constant galvanostatic load, the exchange of the catholyte every second day resulted in a 2.5-times longer mean electrode lifetime (cathode potential above 0.744 V vs. NHE at 110 μA cm-2) of 19 ± 9 days compared to the control experiment. This clearly demonstrates the feasibility of the concept and is a first step towards the realization of a self-regenerating mediatorless enzymatic biofuel cell electrode. In next steps the continuous cultivation of an enzyme producing microorganism and an application based biofuel cell design can now be developed.
Unit operations for the integration of laboratory processes in the field of nucleic acid analysis based on centrifugal microfluidics
2013, Daniel Mark
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Ulrike Wallrabe, IMTEK
KurzfassungThe scope of this thesis is to advance the state of a centrifugal-microfluidic Lab-on-a- Chip platform. In general, Lab-on-a-Chip systems promise the miniaturization, automation and integration of biochemical assays. The centrifugal-microfluidic platform utilizes the pseudo-forces on a rotating test-carrier, such as the centrifugal force, Coriolis force and Euler force to control the movement of liquids. Additionally, capillary, magnetic and thermal effects can be utilized. In contrast to syringe-pump or air-pressure driven microfluidics, no connectors to the test-carrier except one sample inlet are required. This approach allows robust, user-friendly systems. In this thesis, the focus lies on microfluidic building blocks or “unit operations” for nucleic acid analysis. More specifically, unit operations for sample preparation and nucleic acid purification are presented. In the first part of the thesis, the framework is set by introducing basic physical principles which are required to design centrifugalmicrofluidic structures. Next, the state-of-the-art of microfluidic platforms in general and the centrifugal microfluidic platform in particular are critically reviewed. The second part describes unit operations and microfluidic systems which were developed during this thesis. Firstly and secondly described is a scalable, coatingless valve and aliquoting structure based the counter-pressure of entrapped air. Such aliquoting structures are an important component for multi-parameter analysis such as genotyping or testing for a panel of pathogens. The burst pressure of the valve can be tuned by the volume of the downstream chamber, the geometry of the valve and the surface tension of the liquid. Burst pressures in the range 2000 Pa to 20,000 Pa could be realized. With this valve, aliquoting structures for aliquoting into 8-16 volumes could be realized. This second unit operation proved robust and reliable for a number of manufacturing processes, including micro-milling, thermoforming and injection moulding. The CVs for the aliquoted volumes were in the range of 2-3%, corresponding to manufacturing tolerances. The aliquoting structure was successfully tested with water, including Tween 20 concentrations between 0.01% and 10%, and pure ethanol. The third unit operation that was developed is a waste containment structure based on an enclosed air volume for highly wetting liquids used as washing buffers in nucleic acid purification. With this structure and a previously published Coriolis switch (Haeberle et al.), 290 ng ± 80 ng DNA could be purified from 32 μL of lysed blood sample, compared to around 400 ng for the reference extraction. Fourthly, the encapsulation and pre-storage of liquids in centrifugal microfluidic systems is shown. Liquid reagent storage is an important aspect for user friendly and contamination-free disposables. The liquids are encapsulated in fused glass ampoules which can be easily crushed at the beginning of the assay. A typical volume of 100 μL was used to characterize the performance. For both water and ethanol, no loss of the encapsulated liquid could be measured within the measurement error after 300 days of storage. Fifthly, the generation of chitosan microbeads on the centrifugal microfluidic platform was shown in a side-project. While this is not relevant for nucleic acid analysis, it shows the versatility of the centrifugal liquid actuation. The fabrication relies on dispensing a 2 to 3% chitosan solution through a 127 μm nozzle into a polyphosphate solution as a gelation agent. This approach allowed the generation of chitosan beads in the range of 148 to 257 μm with CVs between 15% and 22%. In the next chapter, the integration of most of the previously described unit operations into a disk for automated DNA purification processed on a standard laboratory centrifuge is shown. This is noteworthy, since this approach could circumvent the necessity for the end-user to purchase specialized instrumentation to profit from microfluidic process integration and thus reduce market entry barriers. The structure features pre-storage of washing- and elution buffers and a microfluidic switch based on blocking of an air vent. The entire disposable does not require any local or global surface modifications and thus has the potential to be manufactured cheaply. The yield of extracted DNA was 24% in 10 minutes processing time. In the last chapter before the conclusions, the concept of “Microfluidic Apps” is presented. It is proposed to “upgrade” existing instruments with microfluidic disposable chips, instead of building custom instrumentation for microfluidics. This has the charm that the end-user does not have any initial investments for benefiting from microfluidic automation and a large base of existing instrumentation can be used. Besides the general concept, some examples of microfluidic Apps from various research groups are presented. Overall, several unit operations for nucleic acid analysis on the centrifugal microfluidic platform were developed and summarized in a disk for automated DNA purification on a standard laboratory centrifuge. Important concepts such as the coating-less aliquoting structure and switch as well as liquid reagent storage are an important step towards inexpensive, reliable and user-friendly centrifugal-microfluidic test carriers.

2012

Bubble-Jet Dispenser for Non- Contact and Leakage-Free pl Dispensing in Endoscopic Surgery
2012, Nicolai Wangler
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Non-contact Dispensing Technologies for Printing of Single Cells
2012, Azmi Bin Mohamed Yusof
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jan G. Korvink, IMTEK
KurzfassungKey issue in single cell analysis is the availability of tools to separate cells from larger cell population and isolate them individually into a retrievable confined space. Presently, the most commonly used techniques for isolation of individual cells on external substrate have been the passive immobilization on the treated surface and the cell entrapment into microwells. The limitations are that these methods are prone for cross contamination during transferring the cells and less flexible to handle various single cell patterns. Alternatively, non-contact dispensing technology has been employed to confine cells in a droplet and deposit/print them on external substrate. However, to date, the average efficiency to obtain printed droplet containing single cells follows the random sampling statistics in which, the number of cell distribution in the printed spot correlates with the cell concentration in the microdispenser. This thesis presents a different approach using a microdispenser to efficiently print single cells on external substrate surfaces in a controlled way. To enable drop-on-demand dispensers to print single cells according to the proposed method, three fundamental elements are required; 1) a sensor for detection of single cells, 2) a mechanism to establish a one-by-one cell flow past the sensor and 3) a mechanism to sort the droplets and deposit only those which contain single cells on the substrate. Two approaches have been investigated to evaluate the efficacy of adopting these three fundamental elements into a dispenser device. The first approach incorporates these elements into a commercialized on-demand dispenser assembly without altering the physical device features. By employing computer vision to analyze the region where the discrete flow of cell suspension exits the nozzle of the dispenser, the number of cell, to be expelled in each droplet, is predicted. This prediction requires a drop sorting mechanism to separates the droplets into void, single cell populated and multi-cell populated before deposit on the substrate surface. The second approach integrates all fundamental features into a single dispenser device, where the cell suspension is continuously screened in front of the sensor. It has been demonstrated that the presented approaches can be applied to a single cell applications within an automated single cell printing platform. The efficiency to obtain printed spots containing single cells improved significantly from 50% to 85%, by a factor of 1.7 compared with the technique reported in state of the art [110]. Printing single living cells has been shown within this work by using S. cerevisiae (average diameter 5 m -represents the smallest cell size) and endothelial HeLa cells (average diameter 10 m – represents common mammalian cells). Printing single cell in a 2 dimensional square array has been demonstrated on glass slides, standard petri-dishes and 96 well plates prefilled with growth media. This signifies a major advantage of using noncontact dispensing technology to print single cell that the cells can be collected one-by-one on basically any functional substrate surfaces. The average single cell printing efficiency for the S. cerevisiae was 75 ~ 84 % while the highest single cell printing efficiency for HeLa cells was more than 80%. The viability of the printed single cells was obtained in the average of 75%, exhibiting normal exponential growth trends throughout the cell culture evaluation. Besides exploring the fundamental cell separation and isolation using on-demand dispensing technology, also a single particle detection technique has been adopted using electrical impedance detection as alternative sensing approach. Innovation in process development has been introduced to resolve fabrication issues to realize integrated microdispenser featuring microchannel, metal electrodes and droplet generator. This cost efficient, wafer level fabrication has been successfully demonstrated using a combination of Silicon/dry-film photopolymer/Pyrex substrates. Characterization for the droplets dispensing performance showed comparatively similar with the dispenser’s developed using conventional Silicon/Pyrex technology. The embedded sensor consists of two sets of electrodes oriented in a parallel facing configuration. The sensor enables to explicitly determine the particle transit time which is required to precisely trigger the dispensing mode to expel the detected single particle within droplet in-flight. The differential impedance signal measured for polystyrene bead of size 10 m yield average amplitude of 0.39 mV, whilst for S.serevisiae cells with an average size of 5 m the amplitude is 0.12 mV. This result has essentially demonstrated the capability of the sensor system to perform particle/cell discrimination by size which is fundamental for future development of impedance based micro cell sorter.
PMP-NC² - A normally-closed, backpressure independent peristaltic micropump featuring a modular setup
2012, Fabian Trenkle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Peter Woias, IMTEK
KurzfassungThe presented thesis addresses a new concept for micropumps. The micropump PMP-NC² is based on a modular design with a re-usable actuator unit and a cost-efficient microfluidic chip, which is disposable and can easily be replaced after use or contamination. The micropump is driven in a peristaltic manner by three piezoelectric stack actuators. In the stand-by mode, meaning there is no electric voltage applied to the stack actuators, the pump is normally-closed. The normally-closed state prevents dosage errors. It is based on prestressed spring elements, which are implemented into the re-usable actor unit underneath the disposable fluidic chip. This construction enables a permanent deflection of the pump membrane towards the bottom of the pump chambers without electric energy consumption. The two orifices at the bottom of each pump chamber are sealed hereby and dosage errors are prevented even if the pressure difference between fluidic in- and outlet of the pump is varied. This was verified up to a maximum measured pressure difference of 150 kPa. The PMP-NC² was designed to enable precise pumping operations even if the backpressure varies. The stroke volume of one peristaltic movement is 0.64 μL with a maximum deflection of the peristaltic stack actuators of 40 μm. Up to a frequency of 5 Hz the flow rate is linearly dependent (R²=0.99) on the pump frequency and enables precise pumping operations in a flow rate range of 1 – 50 μL/min. For pumping, the immanent restoring forces of the three pump membranes are used, which are caused by the passive spring elements. This means, that the valve function of the normally-closed state in at least one of the three pump chambers is always active during the complete pumping cycle. This enables backpressure independent pumping up to 80 kPa. By reducing the maximum pump chamber height to 5 μm (instead of 50 μm) a reduced dead volume of the pump chamber (which includes the fluidic interconnection to the next pump chamber) of 1.25 μL was achieved. The reduced dead volume leads to a gas bubble tolerance. Air bubbles with different volumes (2 – 6 μL) were pumped with this modification of the fluidic chip and the flow rate returned to the initial value of 100 μL/min after the air bubble left the chip at the outlet. This demonstrates that gas bubbles can be transported through the whole fluidic chip without causing permanent impairment of the pumping operation. A simulation model with lumped elements was developed to achieve a better understanding of the new micropump concept. The new and characteristic factors of this pump concept are studied through different simulations. In particular the influence of the passive spring actuator underneath the microfluidic chip is discussed. As an alternative drive concept a laboratory prototype of a pump based on amplified piezoelectric stack actuators (APATM) is presented and characterized. The APATM technology allows to save the passive spring actuator underneath the fluidic chip because the metallic frame of the APATM undertakes this function. Although both actuator concepts include the same functionality concerning the normally-closed characteristics and the pumping operation, a comparison of the distinct measurements allows identifying the strengths and weaknesses of each actuator concept. On the one hand the APATM pump provides a reproducible pumping operation and higher efficiencies of 80% in comparison to 30% for the concept with classic piezoelectric stack actuators. On the other hand the functionality of the normally-closed state and the backpressure independency of the flow rate are better suited to the concept based on classical piezoelectric stack actuators. Regarding these different characteristics, design parameters for the further development of the micropump are discussed.

2011

Centrifugal Microfluidic Systems for Protein and Nucleic Acid Analysis
2011, Sascha Lutz
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungTwo centrifugal microfluidic systems for diagnostic applications are presented in this thesis. One system allows the processing of immunoassays for protein analysis while the other enables an automated detection of nucleic acids. The immunoassay system consists of a polymer cartridge featuring microfluidic channels and a device with a rotary drive and a detection unit. Each cartridge features four structures for the parallel processing of immunoassays. Six cartridges can be attached to a rotor of said device to allow the parallel processing of up to 24 assays. The microfluidic structures of each cartridge include a chamber for mixing and dissolution of reagents, a reaction chamber, integrated waste handling and valves for the control of highly wetting fluids. The fluidic layout is furthermore complemented with a blood plasma separation module for integrated sample preparation. A volume of 4 µL blood plasma can be separated from a sample of 10 - 15 µL whole blood with a CV of 6 %. To enable the control of highly wetting fluids a novel siphon valve including a hydrophobic patch is developed. The valve allows the repetitive control of surfactants containing fluids to perform serial washing operations in the immunoassay cartridge. An investigation regarding the valving process for various detergent containing fluids often employed in biochemical and diagnostic applications exhibits a drastically improved valving process employing the presented siphon valve. The hydrophobic patch is manufactured automatically with a dispenser and carbon black particles allow a quality control of the coating. Furthermore, a novel unidirectional shakemode protocol is developed within this thesis enabling long-term mixing and dissolving processes with highly wetting fluids. To demonstrate the systems ability to perform diagnostic applications two types of immunoassays are implemented. A sandwich assay for the quantification of IL8 with a sensitivity of 105 pg/mL is demonstrated with a time-to-result of 45 min. An investigation regarding the assays reproducibility reveals intra- and inter-cartridge coefficients of variation (CVs) of 3 - 9 % depending on the sample concentration. The inter-batch reproducibility for antigen containing samples is between 13.8 % and 27.5 % and 47.5 % for the negative control. Furthermore, a competitive assay for the quantitative measurement of estradiol with a sensitivity of 100 pg/mL is shown with a time to result of 45 min and the results are compared to a commercially available microtiterplate-based assay. The nucleic acid detection system for the first time reports on a fully integrated foilbased microfluidic cartridge for isothermal amplification of DNA exploiting the recombinase polymerase amplification (RPA). The foil cartridge is structured by thermoforming. The cartridge features pre-storage of all liquid and dry reagents for the reaction. The fluidic layout allows the parallel testing of up to 30 samples. All fluidic unit operations like mixing, dissolving, valving and aliquoting are investigated experimentally. The presented system features a reduction of consumed reagents by a factor of five and reduced handling steps for the user. The recombinase polymerase amplification in the foil disk allows the detection of the antibiotics resistance marker mecA with a high sensitivity of < 20 copies out of a sample with a time-to-result of 20 min. The system features compatibility to a commercially available real-time PCR device that is used for processing and detection.

2010

Abiotically catalyzed glucose fuel cells for powering energy-autonomous medical implants
2010, Sven Kerzenmacher
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungThe overall goal of this doctoral thesis is the development of implantable glucose fuel cells employing abiotic catalyst (e.g. platinum, activated carbon) as power supply for energy-autonomous medical implants. Originally envisioned in the 1960s, this newly re-discovered approach uses the body’s own glucose as un-exhaustible source of chemical energy that is continuously transformed into electricity by electrochemical reaction with oxygen. This way the present need for regular surgical replacement of spent batteries may be circumvented which would dramatically improve the life-quality of patients relying on active medical implants. In the first part of this thesis a fundamental framework for the advancement of abiotically catalyzed glucose fuel cells is provided. Thereto the state-of-the-art is critically reviewed, comparing the different embodiment concepts and historical achievements with special regard to their applicability as sustainable implant power supply. Motivated by the lack of comprehensive report on fuel cell fabrication and performance in the literature, a detailed manufacturing protocol for a fuel cell based on hydrogel-bound carbon particles is presented, together with the detailed analysis of its performance characteristics in neutral buffer containing glucose and oxygen. A novelty of this work is the characterization of fuel cell performance with individually resolved electrode potentials. Using this technique the major factors governing fuel cell power output have been identified as progressing deactivation of the anode catalyst, incomplete reactant separation, and a cathode reaction governed by oxygen mass-transfer under physiological conditions. Furthermore, a complete testing environment for the time-efficient simultaneous performance characterization of 24 biofuel cells is presented. It bridges the gap between the limited controllability of simple load resistors and the application of oversized (and thus costly) multi-channel potentiostat systems. In the second part of this thesis two novel electrode structures based on porous Raney-platinum films are introduced. For the anode a fabrication route using zinc as alloying partner was developed that overcomes the biocompatibility concerns associated with carcinogenic nickel used in previous works. At the cathode the use of Raney-platinum film electrodes for oxygen reduction has so far not been possible due to their high glucose-sensitivity. Here it is for the first time shown that sufficiently glucose-tolerant cathodes can be fabricated from sub μm-thin Raney-platinum films. This enables the realization of a glucose fuel cell that employs platinum as catalyst at both electrodes, and that at the same time can also be mounted on an impermeable surface such as the implant casing. In contrast to previous fuel cells based on hydrogel-bound particle electrodes, the novel fuel cell exhibits excellent stability against oxidative and hydrolytic attack. Furthermore, its power density is by approx. 30% increased, amounting to (4.4 ± 0.2) μW cm-2 in neutral buffer containing physiological amounts of glucose and oxygen. This results from the higher catalytic activity of platinum cathodes and in particular the increased oxygen tolerance of the Raney-platinum film anodes introduced in this work. In artificial tissue fluid containing endogenous amino acids, the Raneyplatinum film cathodes exhibit a potential-loss of approximately 120 mV, compared to operation in neutral buffer with only glucose and oxygen. By application of a protective Nafion coating this potential-loss can be diminished to 90 mV, which corresponds to less than 20% of the total fuel cell voltage under typical operation conditions. In contrast, the Raney-platinum anodes in their present form are prone to progressing electrode poisoning by adsorbed reaction products and in particular endogenous amino acids. Here the Raneyplatinum film concept is an excellent platform for further research and optimization. The abdication of polymeric hydrogel binders in electrode fabrication facilitates the optimization of their catalytic properties by versatile and promising methods such as ad-atom surface modification or the application of specifically designed protective polymers with controlled charge and mesh size.
Back-end Processing in Lab-on-a-Chip Fabrication
2010, Lutz Riegger
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungThis thesis presents back-end processing steps for polymer labs-on-a-chip which permit the realization of complex applications like biological assays onchip. Pre-treatment for surface cleaning, hydrophilization to promote capillary action, selective hydrophobization for flow control, dry reagent pre-storage to enable fully integrated chips as well as biocompatible sealing to ensure operation and biological activity after processing are covered. Most of these processing steps are developed on a sample lab-on-a-chip which aims for the on-chip amplification of mRNA by nucleic acid sequence based amplification (NASBA). In detail, pre-treatment of chips with hydrogen peroxide is introduced which effectively sterilizes polymer surfaces. As hydrophilic coating, PEtOx-BP, PDMAA-BP and PEG are evaluated in respect to hydrophilicity. All coatings render polymer surfaces strongly hydrophilic (water contact angle θ < 40°). It is further determined that only PEG exhibits full NASBA compatibility. A new method for the selective surface patterning of microfluidic chips with hydrophobic fluoropolymers is developed and demonstrated by the fabrication of hydrophobic valves via dispensing. It enables efficient optical quality control for the surface patterning due to incorporated dyes thus permitting low-cost production of highly reliable hydrophobic valves. Specifically, different dyes for fluoropolymers are investigated and two fluoropolymersolvent- dye solutions based on fluorescent quantum dots (QD) and carbon black (CB) are presented in detail. The latter creates superhydrophobic surfaces (typical layer thickness: 2 μm) on arbitrary substrates, e.g. chips made from cyclic olefin copolymer (COC, θ = 157.9°), provides very good visibility for the visual quality control in polymer labs-on-a-chip and increases burst pressures of hydrophobic valves by up to 35 %. On the sample lab-on-a-chip, the quality control in combination with Teflon-CB as coating reduces the burst pressure variability from 14.5 % down to 6.1 % compared with Teflon-coated valves. Two approaches for the pre-storage of bioreagents, i.e. spotting and drying as well as spotting and freeze-drying are presented and evaluated. Multiple 25 nL droplets of reagents are dispensed into sample reaction chambers while preventing any contact with the adjacent walls for the spotting and drying. The concept is validated for primers and probes by successfully conducting NASBA for mRNA of three different human papilloma virus (HPV) types, even after 2.5 months of storage. For spotting and freeze-drying, reagents are dispensed on frozen substrates whereas multiple droplets form small reagent pillars. These pillars are successfully freeze-dried on-chip using a custom developed protocol. Rehydration and positive NASBA amplification of susceptible enzymes is demonstrated after three weeks. Biocompatible sealing is realized via temperature diffusion bonding and adhesive bonding. The former process is optimized by the introduction of a transparent compound foil which allows for optical read-out techniques. With the adapted process, strongly bonded test chips are produced (delamination pressure > 3 bar). A potentially negative temperature impact of the sealing process on pre-stored reagents is further excluded for a minimum distance of 180 μm between interface and reagents. Finally, it is demonstrated that PEG as hydrophilic coating can interfere with temperature diffusion bonding as residual PEG on the chip surface promotes delamination. For adhesive bonding as versatile approach for the sealing of polymer microfluidic chips, the influence of process parameters is investigated. Specifically, a process chain comprising pre-processing, adhesive transfer as well as post-processing is presented and parameter recommendations are provided. As device for adhesive transfer, a modified laminator is utilized which transfers thin layers of adhesive onto only the chip surface via a silicone roll. Using this device and a high temperature compatible (Tg > 100°C) epoxy adhesive, adhesive layers in the range of 2 - 4 μm can be reproducibly transferred (CV < 4 %). For best bonding results, it is recommended to provide 2.5 μm thin layers of adhesive in combination with a subsequent evacuation step at 10 mbar for 3 hours. Further, it is proposed to integrate capture channels near large, featureless areas to compensate for variations in processing and thus prevent clogging of channels. With these recommendations, strongly bonded test chips (delamination pressure > 4 bar) can be reliably produced (yield > 80 %). Additionally, the process is inherently compatible with Vistex as hydrophilic coating due to similar coupling chemistry.
Dissipative particle dynamics as a simulation tool for capillary wetting and suspensions in microfluidic applications
2010, Claudio Giorgio Cupelli
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jan G. Korvink, IMTEK
Lab-on-a-Foil: Genotyping by real-time PCR in microthermoformed polymer foils on a centrifugal microfluidic platform
2010, Maximilian Focke
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungA novel Lab-on-a-Chip microsystem for analysis of desoxyribonucleic acids (DNA) was developed, validated and evaluated in the scope of this thesis. The microsystem is based on elaborate microfluidic networks that are integrated into a novel carrier substrate based on microthermoformed polymer foils. This thesis covers the full system integration from establishment of a novel conceptual framework over development and evaluation of a new fabrication process and microfluidic sample processing up to final implementation of the biochemical assays. Nowadays, decision making is frequently based on DNA analysis, for example in the fields of food or infection control, in clinical settings or forensic scenarios. The microbiological application of genotyping is frequently used to determine the existence of a specific DNA sequence in a sample which may for instance be associated with a particular subtype of pathogenic bacteria. At the same time, analytical tests must be fast, reliable and cost-efficient. This thesis aims at providing solutions to these demands by development of a disposable microfluidic Lab-on-a-Chip system comprising automated sample processing and highly sensitive genotyping capabilities even if the sample contains less than 10 copies of the DNA target sequence. The technological work was strongly inspired by industrial processes for packaging of consumable goods like food or pharmaceuticals into foil-based blister packages. Good packaging not only protects the inside from damage on its way from production to the point of use but also provides information about its content and makes it accessible as well as applicable for the user. Further, it must be very cost-efficient, too. Therefore, a novel conceptual framework was particularly established in this work. It perceives foilbased Lab-on-a-Chip systems (“Lab-on-a-Foil”) as functional packaging of microfluidics and biochemistry. A prime example for this concept is microthermoforming by soft lithography (μTSL) for fabrication of foil-based microfluidic devices. This process was developed, analysed and optimised by design of experiments (DOE) in the scope of this work. Fabrication of defect-free Lab-on-a-Foil substrates was achieved by systematic minimisation of deviation between critical geometries of a master tool and the respective geometries in the thermoformed foil substrates. The following process parameters were evaluated: evacuation and moulding temperature, pre-stretching pressure and duration as well as main pressure duration. All parameters have significant impact on the dimensional responses (probability of error p < 0.05), most considerably moulding temperature with > 40 % relative impact. Predicted results of the empirical process model established by DOE were in excellent agreement with the experiments typically exhibiting maximum deviations of less than 2 % to the master tool and replicate-to-replicate variations below 10 μm. The analysis was finally complemented by development of two moulding tools for characterisation of the capabilities and limitations of the process. This was eventually used to establish a set of design rules for robust fabrication of moulding tools. The μTSL process was further applied to fabricate Lab-on-a-Foil cartridges for a basic microfluidic genotyping assay for testing DNA samples in parallel. The microfluidic cartridges were designed for operation in a slightly modified commercial thermocycling instrument. The developed microfluidic protocol is controlled by capillary and centrifugal forces and divides the liquid sample in parallel into independent aliquots of 9.8 μl (CV 3.4 %, N = 32 wells). The genotyping assays were performed with prestored primers and probes for real-time polymerase chain reaction (PCR) showing a limit of detection well below 10 copies of DNA per reaction well (N = 24 wells in 3 independent disks). The system was evaluated by 44 genotyping assays comprising 22 DNA samples as duplicates in a total of 11 disks. The samples also contained clinical isolates of patients infected by methicilin-resistant Staphylococcus aureus (MRSA). The genotyping results were in excellent agreement with the reference assays performed in conventional microtubes. This basic design was eventually upgraded in order to allow primary and successive secondary PCR in a fully integrated and automated microfluidic cartridge. This implementation addresses shortcomings of conventional lab routines in which the lower limit of detection is drastically affected when a given DNA sample is aliquoted into several reaction tubes to be tested for a number of possible genetic sequences. In contrast, the final microfluidic system enabled real-time detection of specific DNA sequences in up to 14 reaction wells even if the sample contained less than 10 DNA copies. The implementation specifically pre-amplifies the DNA target sequences by a factor of ~1000 prior to aliquoting. PCR pre-amplification protocols are conventionally associated with severe risks of DNA contaminations due to handling and transfer of high-copy DNA samples. This problem was also successfully circumvented by the selfcontained microfluidic system design making additional handling steps obsolete. The system was verified by detection of genotypes of MRSA proving highest sensitivity by reliable detection of specific target sequences in samples containing less than 10 DNA copies. Each reaction well contained pre-stored dry reagents for specific secondary realtime PCR. The microfluidic protocol was extraordinary robust and worked in 52 of 52 cartridges including valving, mixing and aliquoting even under thermocycling conditions (where applicable). Further investigations will have to address implementation of sample preparation modules, integration of new passive valves to eliminate the need for local hydrophilic surface treatments for sufficient channel priming, assessment of long-term stability and shelf-life, integration of liquid reagent storage and upscaling scenarios for higher fabrication throughputs. Nevertheless, the fully integrated system provides prospect to convenient, automated and universal microfluidic genotyping assays that are implemented in microthermoformed foils. Furthermore, the demonstrated approach to implement microfluidic cartridges in (slightly modified) commercial lab instruments yields reduced barriers for a potential market entry.
Passive Fluid Management in Micro Direct Methanol Fuel Cells
2010, Christian Litterst
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Passive and self-regulating fuel supply in direct methanol fuel cells
2010, Nils Paust
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Passive components for the control of bubbles and droplets in microstructures
2010, Tobias Metz
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
KurzfassungIn this work components for the passive control of disturbing gas bubbles or liquid droplets in microfluidic structures are developed, studied and implemented in selected applications. In the first part of this work problems caused by gas bubbles in microfluidic liquid flows and problems caused by droplets in microfluidic gas flows are reviewed. It is found that in particular contact line and capillary pressure effects lead to resistant forces that often disturb the functionality of systems. Active and passive ways to deal with disturbing gas bubbles or droplets according to the state of the art are reported. Some information could not be taken from scientific literature as those problems may be considered often as merely of practical relevance. In contrast, advices to deal with those problems can sometimes be found in operation manuals of fluidic devices. While the active removal of the disturbing phase - gas bubble or droplet - requires mostly strong pumps and high manual efforts, here passive approaches to deal with gas bubbles and droplets in microstructures are considered. On the one hand passive approaches can directly perform the removal of the disturbing phase. On the other hand they may also just support the active removal by bringing the disturbing phase in advantageous positions or reduce resistant effects. As passive effects those effects are considered, that result completely from the interplay of the liquid and/or gaseous phase with special geometries or material properties. Using those effects for the control of a disturbing phase needs no additional energy to be brought into the system and no materials are perished. As application examples miniaturized fuel cells are considered during this work as both problems are known there: Gas bubbles appear in anode flow fields of direct methanol fuel cells (DMFC) and abundant water disturbs oxygen supply in cathode channels of any type of low temperature fuel cells. To understand the general concepts behind the components and applications that will be studied, the fundamentals of the related physics and chemistry are summarized first. Effects that can be considered for the passive transport of gas bubbles and droplets in microstructures are capillary forces, gravity and evaporation. In an own chapter different those effects in special geometries are summed up to make them easily available for the design and analysis of the microfluidic components in the following chapters. As a general approach for the description of capillary driven transport in microfluidic systems the method of modelling pressure characteristics was developed. It enables to simply predict the behaviour of a droplet or gas bubble in a complex microfluidic system at different positions. The heart of this work is the analysis of four microfluidic components for the passive control of gas bubbles and droplets in microstructures. First, a complete model for the prediction of bubble positions in the T-shaped channel is derived. In this channel gas bubbles are positioned by capillary forces in a way that a liquid can bypass the gas bubbles. T-shaped channels are therefore save from being totally clogged by gas bubbles. With the improved model, non clogging T-shaped channels can now be designed for any contact angle condition. The model is validated by comparison to experiments and computational fluid dynamics (CFD) simulations. The second component, the StarTube is an extension of the T-shaped channel and employs a star-shaped channel profile. As in the T-shaped channel, liquid and gas are separated in the StarTube by capillary forces. In experiments with the StarTube it was found that contact line forces, which normally hamper the gas bubbles mobility, are reduced by more than a factor of ten. This is performed by reducing the contact line perpendicular to the direction of movement. A model for the prediction of bubble configurations for arbitrary contact angles is derived for arbitrary geometric configurations of the StarTube as well. The third component, the tapered channel, is a microfluidic component that has already been used in previous works for the passive removal of gas bubbles from DMFC anodes. Here a model for the dynamics of movement of gas bubbles in this channel is derived and calibrated by experiments. The analytic model respects for viscous and contact line effects and can predict - in its validated range - the movement of a gas bubble in the tapered channel. The model is reasonable for the flow of methanol in channels with dimensions between 200 µm and 800 µm and half tapering angles up to 7°. The last component, the tapered channel profile ("keyhole" channel), is a transfer of the concept of bubble control in the tapered channel and the T-shaped channel towards the control of droplets. It is set up for the passive removal of abundant water droplets from the cathodes of miniaturized fuel cells. The profile is designed and characterized using CFD simulations and analytical models. The functionality was proven in a hydrogen driven PEM fuel cell where the water removal was applied to clear the cathode from abundant water. Finally two application examples are presented that make use of the developed components. The tapered channel profile is extended to a system for the passive water management in miniaturized fuel cells. Therefore additionally a capillary overflow valve is introduced that switches passively between water removal and water storage in dependence of the water content of the cell. The system was integrated in a test fuel cell and the stabilization of performance output could be found for strongly water producing as well as very dry conditions. In the last chapter the StarJet is shown, as an extended application example for the StarTube. Its star-shaped profile is used in combination with strongly non wetting surface properties to centre droplets in a dispenser nozzle. Using the low friction of droplets and the possibility of a bypassing gas flow in the nozzle, a self regulated, pneumatic dispenser was developed. The system was implemented for the dispensing of liquid metal (solder) and tested in experiments where droplets with volumes of 120 pl and 3,6 nl could be generated at natural frequencies between 30 Hz and 400 Hz.
Pico-Injector for the Discrete Chemical Stimulation of Individual Cells Featuring a High Temporal and Spatial Resolution
2010, Jürgen Steigert
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungThis thesis presents a new and flexible tool (Pico-Injector) for the chemical stimulation of individual cells in their physiological environment with a high temporal and spatial resolution by precisely controlled release of discrete biochemical agent solutions. Key component of the Pico-Injector is the developed dispensing unit based on the bubble-jet actuation principle enabling the pulsed (f  11 kHz) and reproducible (CV < 9%) release of discrete agent volumes in the lower picoliter (pL) range (Vd = 10 pL or 6 pL). The diameter of the released agent droplets (dd = 27 μm for Vd = 10 pL) matches the size of typical cells of interest (dcell  10-30 μm) enabling the chemical stimulation of a single cell within a confluent cell culture. The agents are released with a high temporal resolution (< 100 μs). The dispensing unit features two individually addressable actuation units allowing the synchronized release of two biochemical agents in parallel or in series to perform customized stimulation protocols. The two agents are focussed on a common release area of dra < 50 μm. The fabrication of the bubble-jet actuator is based on standard surface micromachining. For the fabrication of the fluidic layer, a novel and flexible fabrication process was developed to realize precisely embedded 3-dimensional microfluidic structures in biocompatible SU-8 photoresist. The full-wafer bond process based on a polyethylene terephthalate (PET) handling layer enhances previous low-temperature bonding technologies. A very high bond strength of 45 MPa, while requiring only small anchoring structures was achieved. Small channel structures with an aspect ratio > 2 as well as wide membranes with an aspect ratio < 0.02 were simultaneously bonded to realize precisely defined channel structures. Furthermore, the developed process features high yields (> 80%) and enables the integration of microelectronics. The fabricated dispensing units show an excellent lifetime of > 2·106 dispensing cycles and are thus compatible to commercially available bubble-jet actuators. For the bubble-jet actuation principle, a sufficient biocompatibility was proven via the release of functional biochemical agents for the chemical stimulation of cells. Besides ink, water and cell culture medium also liquids and buffers containing enzymes, DNA, neurotransmitters, proteins, peptides, growth hormones, antibodies or fluorescence markers are successfully dispensed. All materials the Pico-Injector consists of are successfully validated against cell cytotoxicity according to ISO 10993-5. The Pico-Injector system can be sterilized at 120°C within water vapor without influencing its performance. The whole Pico-Injector system is designed like a USB flash drive allowing a simple usability. The Pico-Injector is mechanically mounted and electrically connected by inserting the low-cost polymer housing into a USB-socket. The self-draining fluidic reservoirs (V = 10 μL), integrated into the housing, can be loaded by pipettes and prime the dispenser by capillary forces. The Pico-Injector system is compatible to standard optical (e.g. fluorescence microscopy) and electrical (e.g. patch-clamp or multielectrode arrays) read-out devices for cell investigations. For the precisely controlled transfer of defined agent concentrations at user defined local positions within aqueous liquid cell environments, while avoiding any diffusion-based agent leakage in the environment, a new diffusion barrier concept (phase-gap) was established. This is realized by a low-cost, disposable and biocompatible cap that can be placed on top of any pL-dispenser. It generates a phase-gap between dispensing agent and target liquid when the dispenser is dipped into the latter. Two different working modes were developed: (i) the standard mode enables an instant (<< 1 ms) injection of the droplet with a penetration depth of hdp = 100 μm and a spatial accuracy of CV < 15% in the liquid environment and (ii) the focus mode further increases the spatial resolution (e.g. from 100 μm to 50 μm) at the cost of slowing down the release time (200 ms). For the phase-gap we have proven an excellent long-term stability of more than 30 hours against capillary priming and maximum volume ejection rates of up to 220 nL/s. Therefore, the Pico-Injector can be dipped into the liquid cell environment and positioned at any site in a target liquid (e.g. next to an individual cell) to realize sharp and controlled concentration profiles which are predictable by an analytical diffusion model. The release of pL-volumes generates a defined drug concentration at the target cell while the concentration gradient in the vicinity decreases drastically in time due to the 3-dimensional diffusion and leads to remarkably low background signals. For example, dispensing individual droplets (Vd = 10 pL) of a c0 = 2 mmol/L Rhodamine B solution enables locally (c < 0.2 c0 outside a radius of r = 50 μm) and temporally (c < 0.2 c0 after 5 s at r = 0 μm) well defined concentration profiles. The Pico-Injector is compatible to established read-out methods and features the leakage-free delivery of biochemical agents in very precise and defined quantities within a physiological liquid cell environment at well defined locations with a high resolution to establish controllable concentration profiles in space and time. This was successfully demonstrated by highly resolved labeling of a single protoplast embedded in a thin alginate-gel layer as well as by labeling an individual animal cell (L-929) within a confluent cell culture in a physiological liquid cell environment. Furthermore, the gentle and fast single cell transfection by applying nanoemulsions of liposomes containing DNA in cell medium directly on a single mammalian cell within a confluent cell culture was shown.

2009

Simulation of Microfluidic Systems With Fluid Particle Methods on High Performance Computers
2009, Thomas Steiner
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jan G. Korvink, I
KurzfassungOne of the main objectives in simulating complex microfluidic phenomena in life science is the attempt to transfer fundamental research results to commercial applications and products. One of the fastest growing fields in this area is in particular the development of so called Lab-On-A 50 -Chip systems that contain various microfluidic processing steps for chemical reactions or transport of reagents. In the standard design processes for almost all microfluidic devices Computational Fluid Dynamics (CFD) is widely and successfully used but for an increasing number of advanced applications involving, for instance, the flow 55 behavior of small groups of beads, blood cells or biopolymers in microcapillaries, novel simulation techniques are called for. Representing moving boundaries or object shapes varying with time poses serious difficulties for traditional finite-element based CFD schemes. Meshless, particle based simulation approaches, such as Dissipative Particle Dynamics 60 (DPD) are suited for addressing these complicated flow problems with sufficient numerical efficiency. DPD was originally proposed in a heuristic manner, as an explicit model for solvents on mesoscopic scales in which fluids and soft matter are represented as dynamic ensembles of interacting point particles. Solid or soft extended objects can conveniently be represented as compound objects 65 embedded seamlessly within an explicit model for the solvent. However, the application of DPD and related methods to realistic problems, in particular the design of microfluidic systems, is not well developed in general. With this work it is demonstrated how this method appears when used in practice, in a process of designing and simulating a specific microfluidic device. But 70 not only the how is covered within this work but also what is necessary in terms of efficient simulation techniques to treat these types of problems. Thus this work is organized in two parts. Part I deals with the simulation approach of fluid particle methods, its hydrodynamic characteristics, the setting of boundary conditions and the application to a microfluidic problem with aggregating 75 microspheres. In part II the parallelization approach and its performance is presented, including new algorithms to handle efficiently large numbers of different species or to optimize the load balancing in heterogeneous simulation problems. In the introduction chapter 1 the difference between mesh-based and meshless simulation approaches is highlighted and how DPD is related 80 to other particle based techniques. As DPD and its extensions leave room for interpretations and many authors has adapted it in different contexts to simulate complex phenomena, a review is given that lists up relevant literature with a short description of the topic. 85 In Part I of this work a systematic approach is presented to treat a realistic microfluidic problem. Chapter 2 presents the theory behind DPD and introduces the basic equations that describe the dynamics of the fluid particles. Extensions to the original approach are explained that implement additional degrees of freedom to 90 further enhance the hydrodynamic behavior and to improve the description of shear rheology of fluids and colloidal suspensions. Then Multibody Dissipative Particle Dynamics (MDPD) is presented as one possibility to handle multiple phases in free surface simulation and to study capillary phenomena or the dispensing of fluids. Subsequently and as a central part of this work, 95 the Quaternion approach is introduced that allows to describe the dynamics of arbitrarily shaped objects in an elegant and numerically efficient manner. The last section of this chapter deals with the Velocity-Verlet based numerical integration scheme for the calculation of all previously presented force models. In chapter 3 fluid properties and hydrodynamic behavior is discussed together 100 with the application of setting appropriate boundary conditions. In the first section of this chapter the fluid viscosity is measured and how it is related to model parameter such as repulsion, density or the random force of the DPD approach. Then techniques, typically used in Molecular Dynamics (MD) simulations, are reviewed to setup Pressure Boundary Conditions in fluid particle 105 methods. In particular Reflecting Boundary and the Gravitational methods are validated in detail with respect to their controllability and numerical efficiency in DPD. The hydrodynamic behavior is then studied based on this investigation by comparing the analytical solution of velocity profiles in different channels with corresponding simulations. 110 In chapter 4 DPD is applied in a design process of a microfluidic chamber where microspheres are supposed to aggregate in as regular pattern as possible. In the first section it is presented how DPD can be used as a supplementary tool to CFD simulations to treat phenomena that can not be treated otherwise. Then the experimental situation is outlined in combination with necessary modifications of boundary conditions in the simulation setup. 115 Experiment and simulation are matched within this process by a set of relevant dimensionless number such as Reynolds number, Peclet number, Mach number as well as the Drag Coefficient for spherical objects. Further it is presented how the DPD fluid determines a specific unit system for length-mass-time and how it is mapped 120 within this work with the metric MKS (meter-kilogram-second) system of a real experiment. With respect to these parameters the hydrodynamics in confined geometries is validated with reference to the applied pressure boundary conditions. Further the drag forces exerted on dispersed beads are checked and whether the correct dynamics within a Reynolds number range of 0.5 to 10.0 125 is adequately recovered. This chapter ends with a side by side comparison of the experimental structure and the simulated situation with time and length scales matched to the real experiment. Part II contains all part of this work that deal with customizing the simulation approach for parallelized code design, new developed simulation techniques and 130 optimizations for high performance computing. In chapter 5 the applied parallelization strategy based on Domain Decomposition with the Message Passing Interface (MPI ) as well as the structure of the parallelized program is presented. Then essential routines necessary for the parallelization process are explained with reference to corresponding code seg135 ments in the appendix. In the next section a new approach is presented to encode material definition parameter and indices of different moving objects into one single value by applying a so called Multi-Species Identification scheme. The advantage of this method is that it simplifies the code design and allows for an efficient realization of managing material properties in multi-processor 140 environments independent of the complexity of the simulated system. Then the new concept of Master Objects is introduced that allows to handle any number of different shaped objects in parallelized multi-processor programs. The basic idea is to define so called master objects in the pre-processing, duplicate these objects during simulation and calculate only changes in orientation 145 with respect to the initial configuration. Finally the difference between the applied distributed memory and a shared memory parallelization is discussed. The last chapter 6 discusses the versatility of the developed code across different computer architectures and how the performance is kept at an optimum level. The first section presents the applied and well-established techniques of 150 Cell Indexing Arrays and Verlet Skins and their influence on the performance. In the following two sections a Particle Based and Time Dependent Load Balancing are introduced to further increase the performance of the parallelized code. In particular the time dependent load balancing with its diffusive character in relocating processor boundaries proofs that also in simulations with large density gradients the workload can be distributed 155 equally. Finally the performance of the code on different computer platforms is discussed with up to 64 processors, respectively 100 million fluid particles. The tested platforms ranging from a low cost Opteron cluster with 12 CPUs at the Laboratory for MEMS Application (Institute for Microsystem Technology - IMTEK) in 160 Freiburg up to a NEC Xeon cluster with 400 CPUs of the High Performance Computing Center (HLRS) in Stuttgart in comparison to a shared memory system of the Fraunhofer IWM in Freiburg.
TopSpot: Highly Parallel Nanoliter Dispensing - Operating Conditions and Design Rules
2009, Remigius Niekrawietz
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Hermann Sandmeier, IFF, Uni Stuttgart
KurzfassungThe development of microarray technology has revolutionized the field of biological research and is nowadays an established method in diagnostics and drug discovery. This powerful tool provides the ability of a parallel high throughput analysis of hundreds to thousands of samples on a single microarray slide. Due to the steadily increasing number of samples, which have to be measured in research, highly parallel and flexible dispensers are required for spotting of microarray slides. One of these microarrayers is the pressure driven TopSpot nanoliter dispenser, which uses the non-contact printing method for spotting microarrays. Core of the TopSpot dispensing technology is a re-usable micromachined printhead, which allows the parallel ejection of up to 96 different samples and its one to one format change from the microtiterplate ordered reservoir pattern to the 500 μm pitched microarray grid (see Fig. 8.1). The overall goal of this work is to improve the quality of the microarray spotting by characterizing and optimizing the TopSpot printing process. One part of the optimization is to determine technological limits for integration and fabrication of Topspot printheads and to derive design rules for an improved printhead performance. The attained design rules should allow the development of highly integrated printheads, which raise the number of simultaneous dispensed droplets up to 384. The first section introduces the present trend of microarray applications and the different manufacturing technologies. It further gives a short overview of the state-of-theart microarrayer systems compared to the TopSpot technology. In the second chapter the basic differential equations are presented, which are relevant for the capillary filling of the printhead, the droplet formation and the droplet impact on surfaces in the micro scale. In order to provide a fundamental understanding of the pneumatic actuation process for the droplet ejection of the TopSpot dispenser, an analytical model of the generated pressure pulse is presented. The time depending pressure pulse and the approximated resulting flow through the nozzle is further developed into an analytical model to determine the requirements for a successful droplet tear-off. The implementation of an integrated process control for the highly parallel non-contact dispenser, which is essential for the high-quality microarray production, is addressed in section three. A camera system mounted under a special tray is used as optical process control for the spotted microarray. This online monitoring gives precise information about the droplet diameter and the homogeneity of all ejected droplets in one evaluation step during the microarray production. Based on this technique an automatic adaptation of the optimal dispensing parameter for unknown printing media, the detection of missing spots, satellites, dirt particles on the slide and the position of the spots in the array is established. As a result multi-ejection of droplets per nozzle could be eliminated and a CV of 1.5 % for all 24 spot diameters is achieved. For achieving a higher pressure pulse for the droplet ejection a new sealing method was developed, which enabled a direct sealing on the silicon layer and eliminated the dead-volume in the actuation chamber. In comparison to the standard method a four fold increase in the printable viscosity range from 2.5 mPas to 10.8 mPas has been achieved. A further prerequisite to increase the microarray quality is monitoring the pressure pulse, which is necessary for the single droplet ejection process. Hence a pressure sensor was integrated into the actuation chamber to acquire the transient pressure pulse during the droplet ejection. This fundamental process parameter provides information of possible failure modes of the dispenser like an increased pressure amplitude in a flooded actuation chamber or a reduced pressure amplitude from an empty nozzle. Chapter four presents the significant influence parameters of the TopSpot dispensing method. These parameters are subdivided into those related to the print module, the highly integrated printheads and the operation conditions. Two separate fully three-dimensional numerical simulation models were set up using the Volume of Fluid method to study the influence of the parameters. The first model analyzed the droplet ejection and the droplet impact onto the slide. The deviations between simulation and experiment regarding the droplet volume and droplet velocity were less than 5 %. The influence of specific design parameters like the printhead nozzle dimensions, liquid properties of the printing media and the pressure pulse shape were analyzed. The capillary liquid transport for the first priming of the printhead was researched in the second model. The new passive capillary filling structure 1 B developed within this work reduced the evaporation of the dispensing liquid due to the sealed microchannels in the actuation chamber. It further enhanced the reproducibility and stable first priming of the nozzles with the highest successful self-filling rate of 99 % and the lowest CV (1 %). The design rules for highly integrated TopSpot printheads are the topic of chapter five. Compared to the standard printhead design the research of the nozzle length down to 50 μm enabled the reduction of the critical pressure by 20 % for the single droplet ejection. Different printhead designs enabled the size optimization of the nozzle expansion diameter. A value of 150 μm at the nozzle expansion diameter meets the demands of a minor increased pressure pulse (8 %) for a single droplet ejection process and the requirements for a highly integrated 384 nozzle printhead design. In addition the microchannel width reduction down to 20 μm allowed a reliable capillary self filling of the nozzles while providing more space for higher spatial integration. The ensured cross-talk free printing enabled the manufacturing of highly integrated 384 nozzle printheads with a pitch of 500 μm. Droplet diameters have been measured by a stroboscopic camera using the automated image processing software NeuroCheck®. For the standard operating conditions of the 384 nozzle printhead the characterization showed CVs lower than 1% per nozzle with a calculated droplet volume of 825 pl. The measured fluorochrome signal intensity of the spotted 384 array demonstrated a homogeneity better than 2.8 % depending on the used printing buffer. The newly developed multi-layer design overcomes the technological limitation of the small space inside the nozzle array with the help of additional silicon levels for the microchannels. This approach could even allow for the fabrication of printheads with 1536 nozzles in the future.

2008

Multiphase Microfluidics on a Centrifugal Platform
2008, Stefan Andreas Häberle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK
The Twin Epipoly Process: A Foundry Process Flow for a Micrfluidic Application Kit in Silicon/Glass
2008, Tjalf Pirk
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jürgen Wilde

2007

Droplet Generation From the Nanoliter to the Femtoliter Range
2007, Timo Lindemann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jan G. Korvink, IMTEK
KurzfassungThis thesis provides a general description of micro droplet generation at low and medium Weber numbers using analytical, numerical and experimental methods in an engineering manner of contemplation. Based on the presented results guidelines are deduced to support the designing and operating any type of micro droplet generator. In the first chapter the basic differential equations are presented which are relevant for the droplet formation in the micro scale. Based on these fundamentals analytical and numerical descriptions of the droplet formation process will be discussed throughout the thesis. Important dimensionless numbers like the Reynolds, the Weber and the Ohnesorge number are introduced to describe the fluid flow in micro dimensions and the droplet formation qualitatively. Simple analytical expressions are derived for fluidic components like fluidic resistance, fluidic inertance, fluidic capacitance, an outflow model of a nozzle, an inlet resistance and junction effects like a contraction or an expansion of the cross section. Such compact models can be applied to build equivalent fluidic networks for more complicated fluidic systems. In this work some of the considered droplet generators are described by such a network approach. The main part of the work is engaged with the fundamentals of droplet formation especially with the necessary criteria for a droplet ejection. Therefore the Weber number respectively the critical Weber number is used to derive sufficient critical parameters for the droplet formation like the critical velocity, the critical pressure, the critical time and the critical power. These parameters are introduced using an energetic approach based on the formulations stated in the previous chapter. These critical values are subsequently used to describe the sufficient boundary conditions for a successful droplet generation with a given setup. Moreover these critical values can also be used to depict the influence of the design and geometrical variations or parameter variations of the applied liquid. Since the energetic model is not capable to state the dynamic behavior of a given system it is complemented by a computational fluid dynamics (CFD) study concerning the dependence of the droplet formation process on the actuation dynamics. The role of the critical values is successfully validated using the CFD model. Based on this parameter maps for successful droplet generation in different droplet generation regimes like drop-on-demand, jet-on-demand or a jet ejection with subsequent Rayleigh breakup are presented. Finally a new, unique classification of the various droplet generation mechanisms known today is proposed. This classification is based on the underlying fluid dynamic working principle rather than on obvious design elements of the system or historical reasons. As a prominent example of a droplet generator a bubble jet printhead design provided by Olivetti I-Jet was studied intensively. This work was carried out within an European project supported by the Federal Ministry of Education and Research (BMBF), Germany (grant no. 16SV1607) within the EURIMUS program (IDEAL EM 42) applying the previously explained numerical and experimental methods. For a better understanding of the working principle the design and manufacturing of a bubble jet printhead and the characteristic values are presented. A fully three-dimensional CFD simulation model was set up to simulate the ejection process, the capillary filling and the thermal behavior of the printhead. Furthermore, the influence of specific design parameters and the optimization potential are explained in detail. The threedimensional simulation model of the thermal inkjet printhead developed in this work provides a valuable approach to optimize the printhead regarding droplet volume, droplet velocity, droplet quality and print frequency including 3D sensitive aspects. The correctness of the used pressure boundary condition and the simulation model in the three-dimensional case was verified by comparing simulations with gravimetrical and stroboscopic results. Thus, for the first time a fully 3D simulation of a bubble jet could be presented and validated. For the optimization or the design of a new printhead a variety of model parameters was investigated to study effects on, for example, geometry and ink properties. The detailed description of the bubble jet printhead is complemented by the examination of seven alternative droplet generators in a more general manner to verify the analytical approach and the criteria to predict the conditions for droplet formation derived in chapter 3. The single droplet generators are studied with respect to dosage technology, experimental results, theoretical description or numerical models. Conclusively the agreement with the considerations presented in chapter 3 is verified. The results presented in this chapter are partially based on work by other persons and are cited where appropriate to complete this thesis. The obtained findings confirm the analytic model approach in general. The derived critical parameters for droplet ejection are in good agreement for most of the systems. The critical parameters provide a sufficient condition to predict droplet breakup in general. In one special case (TopSpot dispenser) droplet breakup is even observed below the critical parameters. This observation is explained by the fact, that the necessary condition for droplet breakup in fact is different from the conditions given by the critical parameters of the model. To complete the thesis, guidelines for the systematic design of droplet generators are provided based on the presented results. Because the quest for a suitable droplet generator strongly depends on the main requirements and specifications of the system, it is distinguished therein between important main requirements along which the guidelines are developed. After the definition of these main requirements and the presentation of general design rules, specific design rules are recommended for typical applications. Using these recommendations an appropriate droplet generator can be designed from scratch or an existing droplet generator can be optimized.

2006

PCR-Slide: A modular and cascadable platform for DNA sample processing with integrated nanoliter dosage
2006, Rolf M. Kaak
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
KurzfassungSubject of this thesis was the conception, development and validation of a novel and modular platform for performance of DNA-related multistep reactions based on ther-mal cycling. Compared to the state of the art, the expenditure of laboratory equipment for DNA-based assays was reduced significantly. The platform that was developed within the scope of this thesis consists of three components: 1. a microfluidic chip (PCR-Slide) with the outer dimensions of a microscopic slide with up to 24 parallel reaction cavities 2. a dispensing station for the simultaneous and parallel extraction of nanoliter quan-tities from the reaction cavities 3. a thermal cycler for parallel thermal sample processing The PCR-Slide is a micro-structured chip made of polycarbonate, consisting merely of simple microfludic structures that can be produced cost-effectively. This means that the PCR-Slide can be realized as a low-cost disposable. Despite the simplicity of the structures, they evolve a high degree of functionality, enabling the parallel extrac-tion of reagents in nanoliter quantities with a high accuracy. For example, it could be shown that by simple pneumatic actuation aliquots of 60 nl could be extracted from the cavities with a standard deviation of 4 %. The structures of the PCR-Slide are arranged in a pitch corresponding to the pitch of the 384 well microtiter plates (4.5 mm). This allows the transfer of sample liquid into the wells of such a microtiter plate. As the reaction cavities of the PCR-Slide are also arranged in the same way, it is possible to dispense from one PCR-Slide into another one, allowing for the performance of several assay steps of a multistep assay on the PCR-Slide platform. For the actuation of the functional structures being integrated in the PCR-Slide two devices are needed, a thermal cycler and a dispensing station. The thermal cycler has two individually controllable plates, which can reach temperatures ranging from 4 °C to 120 °C. With the dispensing station the dispensing event is initiated by means of a pressure pulse. The applied dispensing principle is based on the Dispensing Well Plate technique, which allows to dispense liquid in the range of several nanoliters in a free jet and with high accuracy. Within the scope of this thesis it turned out that the dispensed volume of a Dispensing Well Plate dispenser is only independent from the actuation parame-ters for certain geometries of the dispenser's structure. By means of a developed mathematical model design rules for the layout of a dispensing geometry could be formulated. The functionality of the PCR-Slide was proofed by performing two different assays on the chip, a hybridization assay and a genotyping assay. The hybridization assay consisted of four different steps: 1. PCR 2. sample post-processing based on exonuclease digestion 3. sample liquid transfer by means of the inbuilt nanoliter dosage (transfer of 5 times 100 nl = 500 nl of the post-processed sample into 1.5 µl hybridization buffer in a microarray) 4. hybridization and detection The genotyping assay included several reaction steps, of which two were performed subsequently and successfully on the PCR-Slide. The genotype specific product was detected using capillary electrophoresis.
Polymer Fabrication and Microfluidic Unit Operations for Medical Diagnostics on a Rotating Disk
2006, Thilo Brenner
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungThis work is motivated by the vital trend in medical diagnostics towards decentralized testing and the potential of miniaturization to support this trend. Aim of this thesis is the development of a centrifugal microfluidic platform for medical diagnostics. Compared to the state-of-the-art, novel efficient techniques for polymer microfabrication and new microfluidic principles and operations are proposed. These operations are integrated in a microfluidic toolbox which contains all essential operation units for automated assay procedures on a rotating disk. An important prerequisite to the efficient development of microfluidic devices is a prototyping technology for the rapid and precise fabrication of polymer chips. The implemented processes comprise SU-8 lithography, polymer replication by soft embossing, surface modification and thermal sealing of channels. The outstanding feature is the rapid tooling of microstructured mold inserts made from silicone rubber and epoxy resins which reduce the fabrication time for a new design to less than four days. The mold inserts are used in both, hot embossing and injection molding and so enable a seamless transfer from the level of prototyping to pilot series production. Furthermore, a new experimental test stand is developed to resolve micron-scale hydrodynamics on fast spinning disks. Two novel microfluidic principles for plasma separation from whole blood are proposed. A decanting technique supplies 2 μl (CV 3%) plasma from 5 μl whole blood within 20 s at a rotational frequency of 40 Hz. The second technique centrifugally separates the plasma in a 2 μl whole blood sample at high rotational frequency of 100 Hz within 35 s and subsequently extracts 0.5 nl plasma (CV 1%) through a duct using capillary and centrifugal interaction. For both techniques, the residual red blood cell concentration in the extracted plasma amounts to less than 0.11%. For the first time, the outstanding role of the Coriolis force in centrifugal microfluidics is investigated by experiments and CFD simulations. Beyond a specific threshold frequency, the Coriolis force dominates the centrifugally driven radial flow and can be used to switch a flow between two symmetric outlets which are selected by the sense of rotation. The rotational threshold frequency has been measured to 55.7 Hz for a channel width of 360 μm and a depth of 125 μm. Furthermore, the inhomogeneous distribution of the velocity-dependent Coriolis force induces convective mixing in radial microchannels. By investigating the key impact parameters which are the geometry of the channels and the speed of rotation, it is shown that the contact surface between two laminar flows can be folded to shorten the mixing times by up to two orders of magnitudes. The good mixing homogeneity and the extraordinary high volume throughput (up to 1 ml/s) in these planar channel networks may lead to novel applications especially in fields of chemical synthesis. Flow sequencing on the platfrom can be achieved by hydrophobic valves which release a liquid sample to a specific channel segment when a critical burst frequency is exceeded. The working range of the implemented valves spans a frequency range from 25 Hz up to 45 Hz whereas the burst frequency can be adjusted at a precision of 1 Hz. The parallel assembly of valves allows portioning of one common sample into several aliquots of 250 nl (CV 6%). A structure for the dilution of blood samples with buffer is proposed. Furthermore, a novel capillary siphon structure is introduced as another simple and robust principle for flow valving and sequencing. The controlled coupling of molecules in a liquid sample to a solid surface is essential to any biochemical assay. The incubation time of sample in a flow cell which is packed with functionalized beads must be sufficiently long so that all potential molecules in the sample can bind to the bead surface. Techniques to stack beads into columns as well as elaborate liquid handling techniques to adjust flow rates between 3.5 nl/s and 75 nl/s by the rotational frequency are developed. The maximum flow rate where all molecules can still bind onto the bead surface is experimentally determined to 13 nl/s. This way, quantitative detection of mouse IgG down to a concentration of 1ng/ml on beads by a fluorescent assay is accomplished. The integration of several developed fluidic operations to a process chain is demonstrated by a disk-based alcohol assay with blood separation and a structure for complete sample preparation for immunoassays on whole blood.
Production of Custom Microarrays Using a Highly Parallel Pressure Driven Nanoliter Dispenser
2006, Oliver Gutmann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Ullrich Certa, F. Hoffmann La-Roche, Basel
KurzfassungThe microarray technology has revolutionized the fields of biological research. It is established in many fields of basic research, diagnostics and drug discovery. Custom made microarrays extend the researchers toolbox and enable flexible and fast layout changes of microarrays. The highly parallel pressure driven nanoliter dispenser Top- Spot can be used for non-contact printing of microarrays. The goal of this work was to enable the system to produce high-quality custom microarrays with various media. In Section A the optimization of the microfluidic TopSpot printheads is described. The droplet ejection of printheads is highly depending on the hydrophobic coating of the nozzle array. For re-coating three different methods were used for applying different hydrophobic silanes to the nozzle array. Best results were achieved with a new developed selective removal coating method, which takes only 5 minutes, is easy applicable and more reliable than other methods. Highly reproducible droplet volume, speed and spot position were achieved, even with sticky or wetting media. The automated filling of printheads was achieved by using a modified pipetting robot. With it the filling of a 96 nozzle printhead takes only 12 minutes. During filling the evaporation of printing media out of the printhead was avoided by a dew point controlled cooling stage. Filled printheads could be stored on the cooling stage and re-used in several spotting runs over one week. This procedure may dramatically reduce reagent consumption and setup times. Carry-over tests with fluorescencently labeled printing media (DNA, proteins) showed that after the standard washing procedure no residues of former fillings were detectable. This solves a prime critical point of other microarrayers. Furthermore a new modulary washing device was developed, enabling a semi-automated, parallel washing of multiple printheads. This lowered setup times dramatically. In extensive studies the correlation between satellite free droplet dispensing and liquid properties was investigated. Variation of pressure pulse amplitude and duration led to stable and reproducible dispensing of liquids in a range of 1.0 to 1.9 mPas of viscosity and a range of surface tensions of 26.0 to 72.8 mN/m. For liquids of different viscosities the duration of the pressure pulse was the predominant effect on satellite free droplet release compared to pressure amplitude. Stroboscopic evaluation of oligonucleotide and protein solution printing showed CVs of droplet diameter lower than 1 % per nozzle and lower than 1.5 % over all 24 nozzles of a printhead. The pitch of printhead nozzles was 500 μm and the deflection of printed spots on the slides was random and less than ±10 μm. Both, the high spot position accuracy and the spot diameter reproducibility enabled interlaced printing with a spot spacing of 250 μm that led to an increase of spot density by a factor of four. Quality control of microarray production is essential. For on-line control of the printing process a two-camera-system was established. A stroboscopic camera allowed recording of droplets during flight and withit the precise adjustment of the pressure pulse to the specific printing media. A second camera monitored spot presence and position on the slide. An integrated image processing software was used to categorize the printed microarray (A-Quality, B-Quality, Reject). After starting of quality control the yield of A-Quality slides per print run was increased to over 90%. However, not only the technological issues of TopSpot microarray printing were addressed during this thesis. In Section B the specific biochemical demands of Oligonucleotide, plasmid DNA, protein and living cell microarray productions are described. For oligonucleotide microarray production optimized printing buffers and protocols were established. Best results were achieved using 0.5M sodium-phosphate (pH 8.0) as printing buffer in combination with the UV cross-linking method. A scoring matrix was established for the evaluation of 15 different microarray slides. Corning Epoxy slides scored highest and are used for the production of custom oligonucleotide microarrays now. For the removal of unbound material a slide washing protocol was established. Whereby a newly developed dip-washing device prevented the smearing of spots during the slide washing procedure reliably and led to circular and homogeneously shaped microarray spots. The produced custom oligonucleotide microarrays were used successfully for gene expression profiling experiments in pharma research. In collaboration with the Swiss Tropical Institute (Basel, Switzerland) a plasmid microarray for the differential genomic analysis of Myobacterium ulcerans was produced. The microarray consists of 800 different plasmids, printed in duplicate on one slide. Only 2 μl of each plasmid solution were needed to produce 54 slides. The varying fluidic properties of plasmid solutions were addressed by using the stroboscopic camera to adjust the optimal pressure pulse. The hybridization efficiency of the plasmid array was unexpected low compared to an earlier prototype. It was assumed that repetitive freezing during storage led to degradation of plasmids. Since the DNA of M. ulcerans was sequenced a PCR based approach was tested as an alternative approach. Protein microarrays are the fastest growing field of microarray technology. For protein microarray production the immobilization efficiency was optimized by testing 144 different printing buffers for printing two different proteins onto seven different microarray slide types. Additionally, two improved immobilization strategies were developed and tested successfully. The commonly used EDC-NHS system was miniaturized by a drop-in-drop printing technique. This resulted in up to 30 times higher signals compared to the standard protocol. The very fast UV cross-linking was used with no detectable negative effect in performed microarray assays. Not only forward phase microarrays (e.g. antibody arrays), but also reverse phase cell lysate microarrays were produced in a highly parallel manner. With it a major limitation of to date reversephase microarrays may be overcome: a very time consuming production process. Although high amounts of surfactant are used in cell lysates optimal printing parameter were found and 1300 cell lysate arrays were produced. The reproducibility of printing was shown by the low CV of reference spots (~5%) in the performed ß-Tubulin assay. Cell microarrays enable multiplexing of biomedical assays. During this thesis elementary demands of living human cells assay on microarray printing technology were solved. With it highly reproducible living cell counts per spot on the microarray slides were achieved while keeping their vitality and function for analysis. It is expected that this methodology will greatly expand the scope of current microarray technology. In conclusion the TopSpot system fulfills the elementary demands of high quality custom microarray production. The system was optimized and it is now enabled to be used for fast and flexible production of above mentioned custom microarrays.
Readout of Diagnostic Assays on a Centrifugal Microfluidic Platform
2006, Markus Grumann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Ulrike Wallrabe, IMTEK
KurzfassungThis thesis presents the technical achievements towards establishing a novel centrifugal microfluidic platform for multiplexed immunoassays in the field of point-of-care applications. Multiplexing of immunoassays is here defined as the detection of a set of different antigen species from a given sample in a single channel. Mixing as one of the crucial microfluidic unit operation is drastically accelerated by two novel fluidic concepts. First, magnetic beads confined in a mixing chamber are periodically deflected by a set of permanent magnets aligned at spatially fixed positions. The resulting relative motion of the beads with respect to the liquid induces advection. In the second concept, without magnetic beads, the disk is spun with periodic changes in the sense of rotation (shake-mode) where inertia effects induce stirring of the liquids. As a result, both strategies speedup mixing from about 10 minutes for mere diffusion to the 1-second range. In several developed disk-based assays, shake-mode mixing could proof to be a robust and versatile microfluidic tool. Multiplexing of immunoassays on the centrifugal microfluidic platform is technically implemented in two formats: as bead-based, or microarray- based assays. Bead-tagging in the multiplexed immunoassay scheme is implemented by integrated quantum dots, or organic dyes. Here, a succesfully implemented Hepatitis-A assay demonstrates the performance of the approach with quantum dots as bead-tag. A theoretical modeling of the bead-aggregation process in microfluidic channels accompanied by experimental investigations resulted in a microfluidic disk design, which allowed the successful conduction of bead-based immunoassays. Microarrays as an alternative to bead-based multiplexed immunoassays are implemented and evaluated implementing a BSA assay on the centrifugal microfluidic platform in three ways: on a rigid polymer disk featuring microfluidic channels structures, on a PDMS lid, which completely covers a lab-on-a-disk, or on a slide-based microfluidic chip attached to a rotor (applied for patent). Beneficial in all cases are the reduced sample and reagent consumption, the accelerated processing, and the well controlled environmental settings along the complete procedure. The colorimetric absorbance readout as common scheme for metabolic assays are implemented on the microfluidic centrifual system whereas the optical path length through the detection cell as limiting factor is drastically extended by monolithically incorporated V-grooves (applied for patent). The performance of the simple and rugged concept is successfully demonstrated on a modular setup to measure the concentration of glucose (CV = 4%), hemoglobin (CV = 3%), and alcohol (CV = 4%) in human whole blood. The measurement of the hematocrit as an important marker for medical diagnostics is transferred to the centrifugal microfluidic platform. Here, a radially aligned dead end channel is first bubble-free primed with blood. Here, trapping of gas upon priming is reliably avoided by capillary wicking, which is solely promoted along predetermined edges of the channel (applied for patent). After a sedimentation step, the hematocrit is read out by visual inspection with a disk-imprinted scale. As a result, an overall CV of 5% over the whole physiological-pathological range is achieved. A desktop-sized actuation and readout device for metabolic assays on the centrifugal platform is conceptually designed; a demonstrator device is assembled. A designed and constructed test and development stand to accurately visualize the propagation of fluids and the flow patterning in micronchannels on fast spinning disks constitutes the experimental base for this work and related ones.
TopSpot Vario: A Novel Microarrayer System for Highly Parallel Picoliter Dispensing
2006, Chris Steinert
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jan G. Korvink, IMTEK
KurzfassungThis thesis reports about the conceptional design, development and characterization of a novel system for production of microarrays, a so called microarrayer system. In the recent years microarray technology had a high impact in basic research, diagnostics and drug discovery. Its success caused tremendous activity of different companies to provide the most suitable microarrayer system. The here presented microarrayer bases on the commercially available TopSpot technology and is called TopSpot Vario. The TopSpot technology is an alternative to the piezo tip and pin printers which are currently dominating the microarrayer market. Its main advantage is the parallel ejection of up to 96 different samples and its one to one format change from the microtiterplate ordered reservoir pattern to the microarray spot pattern. The nozzles of the system are fabricated very homogeneously and very precisely in parallel using silicon micromachining. This is a clear advantage compared to other systems using single part productions like piezo tips and pins with a wider spread of important geometries. The novel developed TopSpot Vario system improves the standard TopSpot technology by replacing the pneumatic actuation principle by a direct liquid displacement method. This was realized by a piezoelectrically driven deformation of elastomer material into filled cavities and nozzles placed in their bottom. Core of the TopSpot Vario technology is a silicon micromachined printhead as it is for the standard TopSpot technology. Up to 24 different samples can be filled into the reservoirs which are ordered in a standard 384 well plate pattern. The one to one format change from the 6 by 4 ordered reservoir pattern with a pitch of 4.5 mm to the 500 μm pitched microarray grid is provided by microchannels connecting each reservoir with the corresponding nozzle. During actuation a parallel ejection of small droplets, one out of each nozzle, takes place. Different printhead designs and concepts were pursued. All components of the printhead like nozzles, displacement chambers, microchannels, etc. were separately optimized by different test-structures. For example, the optimized geometries of a via structure which connects microchannels in different levels improved the successful self-filling rate of the standardly used geometries from 62 % to 96 % for the applied test procedure. All optimized geometries were integrated into a finally used TopSpot Vario printhead design which was extensively and systematically characterized by using statistical planning of experiments. It provides an elastomer placement into the printhead before the printhead is filled. This enables a reliable sealing of all microchannels from each other during filling and actuation. Hence a reliable exclusion of crosscontamination between the samples is guaranteed. Key of success for this approach was the invention of a new microchannel structure for bubble-free priming of blind channels. The actuation principle of TopSpot Vario allows a tunable droplet volume of aqueous solutions from 250 pl up to 1600 pl. Indeed, one major conclusion of this work was the recommendation to print multiple small droplets into each other to achieve larger spot diameters instead of ejecting only one large droplet. This was due to the measured decrease of array homogeneity with increasing droplet volume. Compared to the standard TopSpot system, TopSpot Vario allows a much more flexible tunability of droplet volume and dynamics, a smaller minimum droplet volume (TopSpot Vario: 250 pl; standard TopSpot: ~700 pl), a higher maximum printing frequency (TopSpot Vario: 400 Hz; standard TopSpot: 30 Hz) and dispensing of higher viscous media (TopSpot Vario: η < 11 mPa.s ; standard TopSpot: η < 2.4 mPa.s). Further advantage compared to standard TopSpot is that not all nozzles of a TopSpot Vario printhead have to be used in parallel, so that even arrays with a pitch higher than 500 μm are possible. On the other hand, a pitch of 250 μm is possible on nearly all substrate surfaces due to the small minimum droplet volumes. Finally the reliable exclusion of cross-contamination during actuation is a clear advantage compared to standard Top- Spot, where a blocked vent hole can result in a mixing of the samples in the actuation chamber. The finally used 24 channel printhead design can be easily enhanced to fabricate a 96 channel TopSpot Vario printhead which is already commercially available for the standard TopSpot system. The characterization of the TopSpot Vario system showed an intra nozzle reproducibility of droplet volume of 1.9 % and an inter nozzle reproducibility of all 24 droplet volumes in one array of 7.5 % at the recommended droplet volume of 270 pl. Reproducibility of average droplet volumes of one array from experiment to experiment was measured at 5 %. With these values the TopSpot Vario system can compete with all the other state-of-the-art microarrayers. Still to be optimized is the filling performance of the TopSpot Vario printheads. There, the main focus has to be set on minimization or even better an exclusion of the present risk of filling failures due to trapping of air bubbles in the displacement chambers. During 2040 performed fillings, 31 (1.5 %) displacement chambers with a trapped air bubble were observed. Due to the fact that even one fail filled displacement chamber results in the need of cleaning and re-filling of the entire 24 channel printhead, overall 11 % of the printhead fillings showed at least one of that filling malfunction. The current state of development of TopSpot Vario allows the use of the presented technology for a highly flexible microarray production. It is ready for beta testing where an occasional cleaning and re-filling of the printhead due to filling failures can be tolerated.

2000

Elektrostatisch angetriebenes 3/2-Wege-Mikroventil für pneumatische Anwendungen
2000, Stefan Messner
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. W. Menz, IMTEK
KurzfassungIn dieser Arbeit wird nun erstmals die Entwicklung und Herstellung eines 3/2?Wege-Mikroventils beschrieben, welches die Anforderungen der Industriepneumatik erfüllt. Ausgehend von einer Betrachtung des Marktpotentials für Mikroventile in der Pneumatik wird zunächst ein Überblick über die am Markt verfügbaren, konventionellen Miniaturventile sowie über die bereits entwickelten Mikroventile und deren Antriebsmechanismen gegeben. Darauf aufbauend wird ein theoretisches Modellsystem erarbeitet, mit dem das struktur- und strömungsmechanische Verhalten von Mikroventilen für Gase berechnet werden kann. Auf dieser Grundlage wird mit Hilfe von Simulationsmodellen die Auslegung des in der Arbeit realisierten, elektrostatisch angetriebenen 3/2-Wege-Mikroventils detailliert beschrieben. Allgemeine Betrachtungen zu möglichen Antriebsprinzipien sowie die ausführliche Darstellung des verwendeten elektrostatischen Antriebs beschließen den theoretischen Teil der Arbeit. Das umfassende Verständnis der sich gegenseitig beeinflussenden Ventilparameter wurde in der Realisierung des elektrostatisch angetriebenen 3/2-Wege-Mikroventils umgesetzt. Dazu werden überwiegend Standardverfahren der Silizium-Mikromechanik eingesetzt, wodurch die Voraussetzungen für eine industrielle Serienfertigung weitgehend gegeben sind. Der detaillierte Ablauf dieser Fertigungsverfahren wird ausführlich aufgezeigt. Ausgehend von den vereinzelten Silizium-Ventilchips mit einer Abmessung von etwa 6 x 6 x 1,5 mm³ werden die verwendete Gehäusekonstruktion und die eingesetzten Montageverfahren grob erläutert. Die meßtechnische Charakterisierung der Mikroventile zeigt, daß trotz des extrem geringen Ventilhubs von nur etwa 3 µm Durchflüsse bis 1 l/min erreicht werden können. Der Vergleich mit den simulierten Werten zeigt eine sehr gute Übereinstimmung.

Master-Arbeit

Jahre: 2024 | 2023 | 2022 | 2021 | 2020 | 2019 | 2018 | 2017 | 2016 | 2015 | 2014 | 2013 | 2012 | 2011 | 2010 | 2006

2024

Characterization of Termini-Influence on Nanopore Measurements Towards in-situ Digestion
2024, xx
Erstgutachter: apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Kurzfassung
Deep Learning Based Instance Segmentation Of Organoids For Real-Time Application
2024, xx
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung
Hybrid 3D-bioprinting towards an advanced bone healing model and in vivo testing
2024, xx
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Kurzfassung
Hybrid 3D-bioprinting towards an advanced bone healing model and in vivo testing
2024, xx
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Kurzfassung
Integrating Pick-and-Place Subsystem in Hybrid 3D Printing Systems for Process Automation of 3D Printed Electronic Assemblies
2024, xx
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung
Optical detection of multicellular spheroids in nanolitre sized free falling droplets
2024, xx
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: PD. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Kurzfassung
Optical detection of multicellular spheroids in nanolitre sized free falling droplets
2024, xx
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Kurzfassung

2023

Automation of hybrid capture based target enrichment method for whole exome sequencing by centrifugal microfluidics
2023, XX
Erstgutachter: PD Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK - Chemie & Physik von Grenzflächen
Kurzfassung
Design, Manufacturing and Evaluation of a Microfluidic Cartridge for Molecular Sample-to-Answer Analysis
2023, XX
Erstgutachter: Apl. Prof. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Development of a non-contact capacitive sensor for in-flow microparticle detection
2023, XX
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Jochen Kieninger, IMTEK - Elektrische Messtechnik und Eingebettete Systeme
Kurzfassung
Development of carbon supported Ag nanoparticles as catalyst for CO2 electrolysis
2023, XX
Erstgutachter: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung/Elektrochemische Energiesysteme
Zweitgutachter: Dr. Jochen Kieninger, IMTEK - Sensoren
Kurzfassung
Entwicklung eines auf Calciumphosphat-Zement (CPC) basierenden hybriden 3D-Bioprinting-Verfahrens zum Drucken von Knochenersatzprodukten
2023, XX
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Evaluation of cfMeDIP-qPCR for colorectal cancer detection in liquid biopsy samples
2023, xx
Erstgutachter: Prof. Dr. Bernd Kammerer, Organische Chemie, Universität Freiburg
Zweitgutachter: PD. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Kurzfassung
Fully functional hybrid 3D-printing of multi-layered electrical circuit
2023, XX
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Bastian Rapp, IMTEK - Prozesstechnologie
Kurzfassung
Integrated reference electrode for anion-exchange membrane water electrolysis
2023, XX
Erstgutachter: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Jochen Kieninger, IMTEK - Elektrische Messtechnik und Eingebettete Systeme
Kurzfassung
Konstruktion eines thermo-pneumatisch aktuierten Labormusters für die mobile Diagnostik
2023, XX
Erstgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Screening of protein biomarkers for hypomethylating agent resistance in acute myeloid leukemia using absolute quantification by protein interaction cou-pling
2023, XX
Erstgutachter: PD. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Kurzfassung
Vanadium redox flow battery performance of pretreated Aemion+™
2023, XX
Erstgutachter: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung/Elektrochemische Energiesysteme
Zweitgutachter: Dr. Jochen Kieninger, IMTEK - Sensoren
Kurzfassung

2022

A multi-cellular 3D bioprinting approach to monitor cell-cell interactions and to enhance understanding of vascularization processes
2022, Joshua Weygant
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Günter Finkenzeller, Uniklinik Freiburg
Kurzfassung
Development of a microfluidic channel structure to increase the interspacing between micro tumors in a fluid flow
2022, Trong Nguyen
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: PD. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Kurzfassung
Development of porous Bipolar Membranes for the Application in CO2-Electrolysers producing CO
2022, Stefan Ingenhoven
Erstgutachter: Dr. Severin Vierrath, EES-Group, IMTEK
Zweitgutachter: Dr. Jochen Kieninger, IMTEK - Elektrische Messtechnik und Eingebettete Systeme
Kurzfassung
Ein mikrofluidischer Chip für die automatische Sample-to-answer-Analyse am Beispiel eines MRSA Nachweises
2022, Benedict Martens
Erstgutachter: apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK - Sensoren
Kurzfassung
Increasing multiplexing degree of digital assays by monochrome multiplexing – Development of a 6-plex dMP – PCR for quantification of KRAS/BRAF point mutations
2022, Anja Markl
Erstgutachter: Prof. Dr. Winfried Römer, Centre for Biological Signalling studies - BIOSS, Uni Freiburg
Zweitgutachter: apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Kurzfassung
Normalization of DNA for Library Preparation for Next Generation Sequencing using Carboxylated COC Surfaces
2022, Moritz Huber
Erstgutachter: PD. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Optimization of the cathode catalyst layer composition of a zero-gap CO2 electrolyzer
2022, Luca Bohn
Erstgutachter: Dr. Severin Vierrath, EES-Group, IMTEK
Zweitgutachter: Dr. Jochen Kieninger, IMTEK - Elektrische Messtechnik und Eingebettete Systeme
Kurzfassung

2021

A novel microporous layer with integrated flow field structures in PEMFCs
2021, Neethu Philip Thombra
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Stefan Glunz, Fraunhofer ISE
Kurzfassung
Anode Development for Alkaline Direct Alcohol Fuel Cells
2021, Edgar Cruz Ortiz
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Hanemann, IMTEK - Werkstoffprozesstechnik
Kurzfassung
Automated generation of platelet poor plasma in a microfluidic cartridge
2021, Lena Karkossa
Erstgutachter: PD Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Ulrich Walker, Universitätsspital Basel / CH
Kurzfassung
Development and characterization of Pt/C catalysts for proton-exchange membrane fuel cells
2021, Miriam von Holst
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Hanemann, IMTEK - Werkstoffprozesstechnik
Kurzfassung
Entwicklung und Charakterisierung einer Multiplex-LAMP zur Differenzierung von Makrolid-Resistenzen bei Treponema pallidum subsp. Pertenue
2021, Helena Gmoser
Erstgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK - Sensoren
Kurzfassung
Improvement of the Water Management of Anion Exchange Membrane Fuel Cells
2021, Jakob Salewsky
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung
Machine-learning for enhanced single spheroid detection
2021, Sarah Eisenkolb
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Measuring the Volume of Liquid Jets with Computer Vision
2021, Guilherme Miotto
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Brox, IMTEK - Computer Science
Kurzfassung
Mechanisch verstärkte Kohlenwasserstoffmembran für die Polymerelektrolytbrennstoffzelle
2021, Clara Schare
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung
On the development of fluorine free membrane electrode assemblies towards scalable production of proton exchange membrane fuel cells
2021, Farmal Khan
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung
Paste Transfer Simulation in Stencil Printing
2021, Kiarash Karimi
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Lars Pastewka, IMTEK - Simulation
Kurzfassung
Thermal cycling by centrifugation-controlled convection for ultra-fast polymerase chain reaction
2021, Niklas Virks
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Wilfried Weber, Inst. Für Biologie II, Uni Freiburg
Kurzfassung

2020

Efficient solid phase extraction of short DNA fragments on carboxylated surfaces using centrifugal microfluidics
2020, Sophie Jenne
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Entwicklung und Validierung eines Augmented Reality Reportingtools
2020, Darko Ilić
Erstgutachter: Dr. Philipp M. Scholl, TF, Uni Freiburg - Rechnerarchitektur
Zweitgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Kurzfassung
Evaluation & Optimization of Inkjet Printing Process with UV Curable Hotmelt Hybrid Inks for the Production of a Selective Emitter PERC Solar Cell
2020, Malik Taimoor Elahi
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Roman Keding, Fraunhofer ISE
Kurzfassung
Evaluation of the production and opening dynamics of double chamber stickpacks
2020, Hedieh Ludwig
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Ulrike Wallrabe, IMTEK - Mikroaktorik
Kurzfassung
Herstellung von Reaktionsgefäßen für die Miniaturisierung von Einzelzell-Analyse-Workflows auf planaren Oberflächen
2020, Merit Sieben
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Materailien der Mikrosystemtechnik
Kurzfassung
Hybrid 3D Printing: A coalesced 3D printing of polymer and solder
2020, Zeba Khan
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Bastian Rapp, IMTEK - Prozesstechnologie
Kurzfassung
Hybrid bioprinting of thermoplastics and hydrogels for 3D tissue cultures
2020, Ole Thaden
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Influence on Break-in Process of PEM Fuel Cell Materials and Hot Press Production Conditions
2020, Irene Franzetti
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Michael H. Eikerling, Institute of Energy and Climate Research, Forschungszentrum Jülich
Kurzfassung
Study on the implementation of direct hybrid fuel cell/battery systems for different electrically assisted bicycle types
2020, Leonardo Ranck Zingano
Erstgutachter: Jun-Prof. Dr. Stefan Pauliuk, Nachhaltiges Energie- und Stoffstrommanagement, Uni Freiburg
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung

2019

Characterization of rheological properties of liquid during dispensing using deep learning
2019, Pranshul Sardana
Erstgutachter: Dr. Peter Koltay, IMTEK / Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Brox, Informatik / Pattern Recognition and Image Processing
Kurzfassung
Dependency of Polymer-Polymer Adhesion on Process Parameters in 3D-Printing of Polycaprolactone
2019, Stefan Conrad
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung
Development of a tubular flow sensor
2019, Mona Masouni Goudarzi
Erstgutachter: Prof. Dr. Hans Zappe, IMTEK / Mikrooptik
Zweitgutachter: Dr. Peter Koltay, IMTEK / Anwendungsentwicklung
Kurzfassung
Evaluation of StarJet temperature process window for maskless solder printing on ENIG
2019, Michael Fechtig
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jürgen Wilde, IMTEK - Aufbau- und Verbindungstechnik
Kurzfassung
Evaulation of a homogenous, digital single-cell transcriptomic assay
2019, Shadi Edalati
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Kurzfassung
LabSlide für die hochparalelle, LAMP-basierte Auswertung digitaler Immunoassays
2019, Julian Rüdiger
Erstgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK / Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jenny Kehrbusch, Hochschule Kaiserslautern
Kurzfassung
Lensiess imaging of prokaryiotic cells for single cell printing
2019, Kerstin Thiemann
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Kurzfassung
Microfluidic Perfusion Functional Lid for Organ-on-a-chip Applications
2019, Ahmad Itani
Erstgutachter: Dr. Peter Koltay, IMTEK / Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK / Simulation
Kurzfassung
Reinforced membranes for polymer electrolyte membrane water eiectrolyzers
2019, Dunja Abed el Hafez
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung

2018

An Optical Method for Quantitative Volume Determination of Liquid Jets
2018, Kevin Pfleghar
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung
Bioprinting of endothelial cells towards controlled vascularization structures in artificial tissues
2018, Kevin Tröndle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. rer. nat. Günter Finkenzeller, Klinik für Plastische und Handchirurgie Freiburg
Kurzfassung
Development of a tubular flow sensor
2018, Mona Masoumi Goudarzi
Erstgutachter: Dr. Peter Koltay, IMTEK - Uni Freiburg
Kurzfassung
Recognition of living and dead cells for automated cell sorting
2018, Mais Hanoon
Erstgutachter: Dr. Peter Koltay
Kurzfassung
Towards Direct Membrane Deposition and Noble Metal Free Cathodes for PEM Water Electrolysis
2018, Peter Holzapfel
Erstgutachter: Prof. Dr. Simon Thiele, Electrocatalytical Interface Engineering, Friedrich-Alexander University Erlangen-Nürnberg
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwedungsentwicklung
Kurzfassung
“Developing of a signal fixation strategy tor the proximity ligation assay“
2018, Uljana Botscherowa
Erstgutachter: Dr. Matthias Meier, IMTEK - Uni Freiburg
Zweitgutachter: Prof. Roland Zengerle, IMTEK - Uni Freiburg
Kurzfassung

2017

Automatisierte DNA-Extraktion durch den Einsatz von Membranen im LabTube für die Tuberkulosediagnostik
2017, Leonard Kost
Erstgutachter: Prof. Dr, Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Alexander Rohrbach, IMTEK
Kurzfassung
Effect of Coriolis Force and Flow Rate on Binding Efficiency of Biomolecules in Centrifugal Microfluidics
2017, Ahmad Saleem Akhtar
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Alexander Rohrbach, IMTEK - Bio- und Nanophotonik
Kurzfassung
Guidelines für das MRD-Monitoring mittels biplex Mediatorsonden-PCR bei akuter lymphoblastischer Leukämie
2017, Franziska Schlenker
Erstgutachter: PD Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: PD Dr. Cornelia Eckert, Campus Virchow-Klinikum (CVK), Charité Berlin
Kurzfassung
Segmentierung von makroporösen Materialien mittels Infiltration und maschinellem Lernen
2017, Arne Götze
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
Kurzfassung
The Influence of Lactate Concentration and Oxygen on the Current Production and Biofilm Formation of Shewanella oneidensis MR-1 on Electrospun Anodes
2017, Zachary A. Pinder
Erstgutachter: PD Dr. Sven Kerzenmacher, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Siegfried Fink, Forstbotanik, Uni Freiburg
Kurzfassung

2016

Analytische Proteinaffinitätschromatographie in der offenen Mikrofluidik
2016, Boris Hamouda
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Oliver Ambacher, Fraunhofer-Institut für Angewandte Festkörperphysik IAF
Kurzfassung
Artifcial 3-D cell culture on a microfluidic chip
2016, Kerstin Schlegel
Kurzfassung
Development and evaluation of a methane-producing microbial electrolysis cell
2016, Marielle Fink
Erstgutachter: Prof. Wilfried Weber , BIOSS und Institut für Biologie II, Uni Freiburg
Zweitgutachter: Prof. Matthias Boll, Institut für Biologie II, Uni Freiburg
Kurzfassung
Development of a pneumatic Coriolis mixer in centrifugal microfluidics
2016, Pawel Ranoszek
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK-Simulation
Kurzfassung
Development of a synthetic switch for measuring in situ dynamics of protein turnover rates
2016, Christoph Gäbelein
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung
Development of techniques for the in-line viscosity determination in a low-cost flow sensor
2016, Fritz Koch
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Entwicklung eines Begasungssystems für den Betrieb abiotischer Glukosebrennstoffzellen in simulierter Gewebeflüssigkeit und Serum
2016, Melanie Bühler
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
Kurzfassung
Erforschung stabiler Emulsionen auf mikrofluidischen Chips für eine Lab-on-a-Chip-Anwendung
2016, Anja Schachtschneider
Kurzfassung
Konzeptionierung, simulationsgestützte Auslegung und Charakterisierung von Fluidikstrukturen für schnelle Zonen-PCR auf der zentrifugalen LabDisk Plattform
2016, Peter Berli
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. P. Woias, IMTEK - Konstruktion von Mikrosystemen
Kurzfassung

2015

Development of a microfluidic disk for digital droplet PCR
2015, Marcel Geltman
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: PD Dr. Albrecht Brandenburg, Fraunhofer Institute for Physical Measurement Techniques (IPM)
Kurzfassung
Gasblasenbasiertes Mischen für beadbasierte Affinitätsreaktionen in der zentrifugalen Mikrofluidik
2015, Martin Schulz
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK-Sensoren
Kurzfassung
Herstellung eines Sensors zur ortsaufgelösten Messung des elektrischen Potentials in einer Brennstoffzellen-Katalysatorschicht
2015, Michaela Frase
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
Kurzfassung
Open subtrate microbatch protein crystallization using the PipeJet™ technology
2015, Jonathan Kottmeier
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Oliver Einsle, Lehrstuhl für Biochemie
Kurzfassung
Zentrifugo-thermopneumatisches Wachsventil auf der zentrifugalen mikrofluidischen LabDisk Plattform
2015, Anna Drzyzga
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Dr. Andreas Greiner, IMTEK
Kurzfassung

2014

Aerosolbedingte Kontaminationsanalyse Mediatorsonden-PCR basierter Geneslices
2014, Alexander Heyne
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Charakterisierung und Optimierung des blasengestützten Mischens in LabTube-Kartuschen
2014, Maximilian Grösche
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, I
Kurzfassung
Charakterisierung und Optimierung des Nukleinsäureassays für neonatale Sepsis auf einer zentrifugal-mikrofluidischen Foliendisk
2014, Thorsten Meßinger
Kurzfassung
Conceptual study of LabTube based automation of a specific protocol for bacterial DNA extraction from whole blood samples
2014, Ardavan Shabanian
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Peter Woias, IMTEK
Kurzfassung
Herstellung und Charakterisierung von Platin- Legierungskatalysatoren für den Einsatz in der implantierbaren Glukose-Brennstoffzelle
2014, Felix Wiedenmann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK, IMTEK
Kurzfassung
Kühlraten-Ventilaktuator auf der zentrifugalen LabDisk Jakob Schott
2014, Jakob Schott
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Hanemann, IMTEK
Kurzfassung

2013

Aufbau mehrlagiger Mikrofluidikstrukturen aus thermogeformten Foliensubstraten am Beispiel der SAXS-LabDisk
2013, Dirk Buselmeier
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Automatisierung magnetpartikelbasierter Immunoassays auf zentrifugal-mikrofluidischem Lab-on-a-Chip System
2013, Jing Jin
Erstgutachter: Prof. Dr. Michael Köhler, Technische Universität Ilmenau
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK
Kurzfassung
Cartridge development for automated immunoassays in standard laboratory centrifuges
2013, Vaskar Gnyavali
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Charakterisierung der Bead-basierten Nukleinsäure-Extraktion auf der zentrifugal-mikrofluidischen Foliendisk
2013, Sergej Keil
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Enhanced in-situ generation of polonies in picowell arrays by solid phase PCR for sequencing
2013, Sebastian Hin
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Entwicklung eines integrierten Verschlusses für Lab-on-a-Chip Systeme aus thermogeformten Folien
2013, Jan Smetana
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Gas bubble based mixing for DNA extraction on centrifugal microfluidic platforms
2013, Jens Liebeskind
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Imaging technology for volumetric characterization of non-contact micro-dosages
2013, Muniyogeshbabu Thanikhatla Govindaiah
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Alexander Rohrbach
Kurzfassung
Implementierung und Optimierung eines Mikrothermoformprozesses mit dem Einsatz starrer Formwerkzeuge
2013, Janika Bischoff
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Claas Müller
Kurzfassung
Large volume handling on LabDisk
2013, Johannes Wöhrle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung

2012

Characterization and Optimization of DNA extraction in the LabTube cartridge
2012, Shou Zhang
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Charakterisierung und Optimierung unabhängiger Prozessparameter für einen Mikrothermoformprozess Marion
2012, Marion Finkbeiner
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Contact replication of DNA microarrays
2012, Andreas Grabow
Erstgutachter: Prof. Dr. rer. nat. Christiane Zell, Hochschule Offenburg
Kurzfassung
Design and Elevation of Tempered PDMS Flow Cell for Protein Microarray Generation
2012, Suleman Shakil
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jürgen Wilde, IMTEK
Kurzfassung
Development of Pneumatic Pumping Unit Operations on Centrifugal Platforms and Comparative Study to a Network Model Approach
2012, Steffen Zehnle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Digital solid-phase PCR in picowell arrays to generate DNA colonies for sequencing
2012, Martin Trotter
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Electrophoretic Separation of DNA in Written Gel Lines on Planar Substrates
2012, Ludwig Gutzweiler
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Margit Zacharias, IMTEK
Kurzfassung
Kontaktfreies Drucken von Zellen und Hydrogelen für Tissue Engineering und Einzelzellanalyse
2012, Sonja Niekrawietz
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
Kurzfassung
Neukonzeptionierung und Charakterisierung einer Fluidik zur PCR-basierten DNA-Analytik
2012, Miriam Kräft
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Quantitative measurement system for noncontact micro dispensers based on QCM technology
2012, Jin Zhang
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK
Kurzfassung
Weiterentwicklung und Automatisierung eines kontaktfreien Dosierers für den sub-μL Bereich
2012, Imad Malki
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jürgen Wöllenstein, IMTEK

2011

Development of a Fluorescence detection system using a semi-conductive photomultiplier for centrifugal microfluidic platform
2011, Muthukaruppan Gnanadesigan
Erstgutachter: Prof. Dr. Bernhard Vondenbusch, Hochschule Furtwangen
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK
Kurzfassung
Development of an Active Micromixer Using an External Mechanical Actuator Array
2011, Yawar Abbas
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Development, fabrication and characterization of a low-cost electromagnetic dispensing valve for the sub-μl range
2011, Sabrina Kartmann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Dr. Andreas Greiner, IMTEK
High Throughput Cell Lysis Based on Integrated Bubble-Jet Actuators
2011, Vitaliy Kondrashov
Erstgutachter: Prof. Dr. Ulrich Mescheder, Hochschule Furtwangen University
Zweitgutachter: Prof. Dr. Dietrich Kühlke, Hochschule Furtwangen University
Kurzfassung
High Throughput Cell-Sorter based on Integrated Bubble-Jet Actuators
2011, Natalia Andreevna Bakhtina
Erstgutachter: Prof. Dr. Ulrich Mescheder, Hochschule Furtwangen University
Zweitgutachter: Prof. Dr. Dietrich Kühlke, Hochschule Furtwangen University
Kurzfassung
Imaging of Immobilization Processes of Molecules with iRIfS
2011, David Lämmle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Günter Gauglitz, Eberhard-Karls- Universität Tübingen
Mechanical Lysis on a Centrifugal Microfluidic Disk
2011, Stefan Schuhladen
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Network Simulation of a Solenoid Dispensing Valve
2011, Shivam Dwivedi
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jürgen Wilde, IMTEK
Kurzfassung
Nukleinsäure-Extraktion auf einer zentrifugal-mikrofluidischen Foliendisk mit Hilfe magnetischer Partikeln
2011, Bülent Kanat
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung

2010

Charakterisierung und Implementierung der Lyse von Bakterien auf einem zentrifugal mikrofluidischen System
2010, Nadine Berger
Erstgutachter: Prof. Dr. Hessling, Hochschule Ulm
Zweitgutachter: Prof. Dr. Walter, Hochschule Ulm

2006

Development of a Device for Integrated Cell Culturing and Stimulation
2006, Markus Wintermantel
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Kurzfassung
Development of a valve-less micropump for fuel cell applications
2006, Yasir Osman Mustafa Elnager
Kurzfassung

Bachelor-Arbeit

Jahre: 2024 | 2023 | 2022 | 2021 | 2020 | 2019 | 2018 | 2017 | 2016 | 2015 | 2014 | 2013 | 2012 | 2011 | 2010 | 2009 | 2008

2024

Casting thin membranes of Poly-((4-acetylpyridine)-co-(para-terphenyl)) for Vanadium Redox Flow Batteries – influence of molecular weight and temperature on morphology and performance
2024, xx
Erstgutachter: Dr. Severin Vierrath, IMTEK - Electrochemical Energy Systems
Kurzfassung
Development of an automated optical fill level detection in microfluidic channels under rotation using image recognition
2024, xx
Erstgutachter: Apl. Prof. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Kurzfassung
Evaluating the Impact of Different Factors on Organoid Functionality
2024, xx
Kurzfassung
Hybrid 3D-bioprinting towards an advanced bone healing model and in vivo testing
2024, xx
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung
Implementierung und Charakterisierung eines optischen Füllstandsensors für das automatisierte Packen von "Dual-Mode"-Chromatographiesäulen
2024, xx
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Kurzfassung
Untersuchung des Salzausfalls in einem zerogap CO2 Elektrolyseur bei gepulstem Betrieb
2024, xx
Erstgutachter: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung
Kurzfassung

2023

Conductivity evaluation of anion-exchange membranes for water electrolyzers
2023, XX
Erstgutachter: Dr. Severin Vierrath, EES-Group, IMTEK
Kurzfassung
Rissfreie Anodenkatalysatorschicht auf Kohlenwasserstoffmembran durch Reduzierung der Membranquellung
2023, xx
Erstgutachter: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung
Kurzfassung

2022

Platin auf neuartigem Kohlenstoffträger - Optimierung von Katalysatortinten für die Polymerelektrolytmembran-Brennstoffzelle
2022, Hassan Fadlullah
Erstgutachter: Dr. Severin Vierrath, EES-Group, IMTEK
Zweitgutachter: Prof. Dr. Anna Fischer, Institut für Anorganische und Analytische Chemie, Uni Freiburg
Kurzfassung

2021

Charakterisierung der UV-induzierten Freisetzung von Hydrogel-Bead gekoppelten Primern für eine digitale PCR mittels eines neuronalen Netzes zur Tropfenklassifizierung
2021, Daniela Rassler
Erstgutachter: PD Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Charakterisierung eines dampfdruckunterstützten thermopneumatischen Pumpmechanismus
2021, Maren Kirste
Erstgutachter: PD Dr. Nils Paust, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Charakterisierung von Alginathydrogelen zur Herstellung einer Referenz-Biotinte für die Standardisierung von DoD-Bioprinting-Verfahren
2021, Lea Zausch
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Entwicklung neuer Wasserstoff-Brennstoffzellen mit fluorfreien Ionomeren und reduzierter Platin-Katalysatorbeladung
2021, Andreas Fischbach
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Etablierung und Optimierung einer 4-plex Mediatorsonden-PCR zur simultanen Detektion von DNA und RNA
2021, Larissa Graner
Erstgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: PD Dr. rer. nat. Stefanie Eyerich, Helmholtz Zentrum München
Kurzfassung
Kostengünstige funktionalisierte Elektroden für den elektrochemischen Nachweis elektroaktiv gelabelter DNA Sonden
2021, Dalina Dler Boya
Erstgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK - Chemie und Physik von Grenzflächen
Kurzfassung

2020

Druckkontrolliertes Direktvolumen - Verdrängersystem zur kontaktfreien Dosierung für Nano und Mikrolitertropfen
2020, Florian Lux
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Properties of solder metal bumps printed by StarJet technology on ENIG surfaces for backside contacting
2020, Caoting Li
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Lars Pastewka, IMTEK - Simulation
Kurzfassung
Untersuchung der Lagerstabilität von fluorogenen Reportermolekülen
2020, Sebastian Dominik Huber
Erstgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Lisa Becherer, IMTEK - Anwendungsentwicklung
Kurzfassung
Verbesserung eines Ultraschall-Sprühprozesses zur Herstellung von PEM Brennstoffzellen mit optimierter Leistung
2020, Souaad Saoud
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Patrick Ruther, IMTEK - Materailien der Mikrosystemtechnik
Kurzfassung

2019

Bakterien-Detektion mittels Dunkelfeldmikroskopie für den Einzelzelldruck
2019, Raphael Dammann
Erstgutachter: Dr. Peter Koltay, Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, Simulation
Kurzfassung
Nanostrukturierte Keramiken für elektrochemische Energieanwendungen
2019, Luca Bohn
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
Zweitgutachter: Dr. Patrick Ruther, IMTEK / Materialien für Mikrosystemtechnik
Kurzfassung
Trocknungspuffer für partikelgekoppelte C-reaktive Proteine im Point-of-Care
2019, Lena Karkossa
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK / Simulation
Kurzfassung

2018

CHARAKTERISIERUNG VON INDUSTRIELLEN AL-PASTEN ZUR EVALUATION DER Dispenstechnologie für die Rückseitenmetallisierung von bifacialen PERC Solarzellen
2018, Marcel Klemens
Erstgutachter: Prof. Dr. Roland Zengerel , IMTEK - Uni Freiburg
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Uni Freiburg
Kurzfassung
Charakterisierung von Hydrogelen für die Herstellung von 3D-Strukturen mittels Extrusion
2018, Bushra Banu Ansari
Erstgutachter: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK - Simulation
Kurzfassung
Charakterisierung von Stützmaterialien aus bioresorbierbarem Kunststoff für das 3D-Bioprinting
2018, Nicolas Bignani
Erstgutachter: Prof.Dr. Roland Zengerel , IMTEK - Uni Freiburg
Kurzfassung
Hochdynamische Multianalyt-Assays –Gleichzeitige Detektion von Proteinen und Nukleinsäuren
2018, Angela Schade
Erstgutachter: Prof. Dr. Dieter Stoll , Hochschule Albstadt-Sigmaringen
Zweitgutachter: Apl. Prof. Dr. Felix von Stetten, IMTEK, Uni Freiburg
Kurzfassung
Validierung der Fixierung von diagnostischen Reagenzien in Stickpacks
2018, Michael Eisele
Kurzfassung

2017

Microbial Electrolysis Cells for the Treatment of Industrial Wastewater: Evaluating the Effect of Different Separators on Reactor Performance
2017, Pia Kolb
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr, Sabine Sané, University College Freiburg
Kurzfassung
Verifikation eines Simulationsmodells zur Folienausdehnung auf mikrofluidischen Testträgern
2017, Valentin Dimitrov
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung

2016

Charakterisierung zentrifugal mikrofluidischer Testträger zur DNA-Extraktion von Bakterien aus Wasserproben
2016, Michael Zehnle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK-Simulation
Kurzfassung
Einzelzelldruck für die Analyse zirkulierender Tumorzellen
2016, Kevin Pfleghar
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK-Simulation
Kurzfassung
Elektrochemischer Hybridisierungsnachweis mit Mediatorsonden auf Inkjetgedruckten Strukturen für die Anwendung als E-DNA Sensor
2016, Monika Farina
Erstgutachter: PD Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK - Sensorik
Kurzfassung
Increasing current production in microbial electrolysis cells by adding granular activated carbon to the anode
2016, Veronika Vazhnik
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Kurzfassung
Influence of different separators on the performance of microbial electrolysis cells
2016, Dunia Abed el Hafez
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Dr. Andreas Greiner, IMTEK-Simulation
Kurzfassung

2015

Aufbau eines Prüfstandes zur Untersuchung der Präzision bei der kontaktfreien Tropfenpositionierung im Nano- und Pikoliterbereich
2015, Matthias Körner
Erstgutachter: Prof. Dr.-Ing. Volker Hirsch
Zweitgutachter: Prof. Dr.-Ing. habil. Catherina Burghart
Kurzfassung
Digital droplet loop-mediated isothermal amplification (ddLAMP) on standard laboratory devices
2015, Clara Siber
Erstgutachter: PD Dr. Felix von Stetten
Zweitgutachter: Prof. Dr. Gerald Urban
Kurzfassung
Membran-basierter Probeneinlass für pathogenes Vollblut auf einer zentrifugalen mikrofluidischen LabDisk Plattform
2015, Manuel Loskyll
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Oliver Paul, Materialien der Mikrosystemtechnik, IMTEK-Sensoren
Kurzfassung
Nachweis von respiratorischer Pathogen DNA auf einer zentrifugal-mikrofluidischen LabDisk Plattform
2015, Franziska Schlenker
Erstgutachter: PD Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
Zweitgutachter: PD Dr. A. Brandenburg, Fraunhofer Institute for Physical Measurement Techniques (IPM)
Kurzfassung
Voruntersuchungen zur Behandlung von synthetischem Brauereiabwasser mit der filtrierenden mikrobiellen Brennstoffzelle
2015, Jonas Rapp
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
Kurzfassung

2014

Charakterisierung des Ausdehnungsverhaltens einer flexiblen Messzelle unter Druckbeaufschlagung
2014, Malena Kellermann
Kurzfassung
Charakterisierung unterschiedlicher Elektrodengeometrien eines kapazitiven Einweg-Drucksensors
2014, Julian Rüdiger
Kurzfassung
Development of Detection Methods for Mediator Probe Cleavage during Recombinase Polymerase Amplification
2014, Laura Leonhardt
Kurzfassung
Grundlegende Untersuchungen zur Machbarkeit der filtrierenden mikrobiellen Brennstoffzelle
2014, Arne Götze
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
Kurzfassung

2013

Electrospinning of anode materials for microbial fuel cell applications
2013, Alexander Sido
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
Kurzfassung
Geometrische Multiplex Mediator-Sonden PCR in GeneSlices
2013, Sandra Cindric
Erstgutachter: Prof.Dr. Ulrike Salat, Hochschule Furtwangen University
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK
Kurzfassung
Mikrofluidische Integration und Charakterisierung einer Phagenselektion
2013, Tatiana Serr
Erstgutachter: Prof.Dr. Ulrike Salat, Hochschule Furtwangen University
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK
Kurzfassung

2012

Entwicklung von luftatmenden Kathoden für die mikrobielle Brennstoffzelle
2012, David Thissen
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
Kurzfassung
Etablierung eines Referenzprotokolls zur Pestiziddetektion mittels eines enzymatischen Biosensors
2012, Martin Schulz
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Untersuchung des Einflusses von Aminosäuren auf die Glukose-Oxidation an hochporösen Platin-Katalysatoren
2012, Lena Bleck
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
Kurzfassung

2011

Anodenmaterialien für die hocheffiziente Mikrobielle Brennstoffzelle
2011, Julia Koch
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
Kurzfassung
Beschichtungen für Lab-on-a-Chip-Systeme
2011, Dirk Rappenecker
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Charakterisierung eines beadbasierten Immunassays auf Foliendisk
2011, Dominik Feldmeier
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Dr. Andreas Greiner, IMTEK
Kurzfassung
Die Anwendung von Verbundfolien zur Reagenzienvorlagerung in LoaC-Systemen
2011, Yannick Barb
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Dr. Andreas Greiner, IMTEK
Kurzfassung
Experimentelle Charakterisierung und Parameteroptimierung eines Dosiersystems für den sub μl-Bereich
2011, Johannes Aleker
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Herstellung von hochporösen Platin-Elektroden mittels gepulster galvanischer Abscheidung als Anode für die implantierbare Glukose- Brennstoffzelle
2011, Anna Drzyzga
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
Kurzfassung
Langzeitstabilität Aquacer behandelter Oberflächen & Optimierung einer mikrofluidischen Blutplasma-Separation
2011, Johannes Fries
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Mikrofluidische Gradientenerzeugung
2011, Robert Bergmann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Kurzfassung
Rekombinase Polymerase Amplifikation im kontinuierlichen, mikrofluidichen Fluss
2011, Alexander Heyne
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Universeller DNA-Nachweis mittels Mediator-Sonden-Technologie
2011, Lucas Dreesen
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK
Kurzfassung
Verschließbare Einfüllöffnungen für Lab-on-a-Foil-Systeme
2011, Sebastian Reiter
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung

2010

Charakterisierung und Verbesserung der PipeJet™ eines Dispensers
2010, Ktaich Rabih
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Entwicklung eines Braille - Display betriebenen, mikrofluidischen PDMS - Pump- und Ventilsystems
2010, Henning Meier
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Immobilisierung von Primern auf Poly(-dimethylsiloxan) (PDMS) für Primer-Verlängerungsreaktionen
2010, Sebastian Hin
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Dr. Oswald Prucker, IMTEK
Kurzfassung
On-Chip Lyse von E. coli im kontinuierlichen Fluss
2010, Johannes Wöhrle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Reagenzienvorlagerung in delaminierbaren Folienbeuteln
2010, Jan Smetana
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Ulrike Wallrabe, IMTEK
Kurzfassung
Thermisches Deckeln von Foliendisks
2010, Dirk Buselmeier
Erstgutachter: Prof. Dr. Claas Müller, IMTEK
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK
Kurzfassung

2009

Detection of Bacteria with Magnetic Beads on a Centrifugal Microfluidic Cartridge
2009, Dureid Michael Qazzazie
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Entwicklung eines Teststands und einer Testfluidik für die Manipulation magnetischer Partikel auf einer zentrifugalmikrofluidischen Plattform
2009, Alexander Emplerle
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Claas Müller, IMTEK
Kurzfassung
Fluoreszenzdetektionsstrategien für folienbasierte Lab-on-a-Chip-Systeme
2009, Patrick Reith
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Leonhard Reindl, IMTEK
Kurzfassung
High resoluted chemical Cell Transfection in confluent Cell Cultures
2009, Moritz Thielen
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Implementierung eines kompetitiven und Sandwich Immunoassays in ein zentrifugal-mikrofluidisches System
2009, Imad Malki
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Kontrolliertes Einbringen hoch benetzender Flüssigkeiten in Lab-on-a-Foil-Systeme
2009, Roland Feuerstein
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Polymere Schutzschicht für implantierbare Glukose Brennstoffzellen
2009, Stanislav Sherman
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
Kurzfassung
Prozessentwicklung für mikrofluidische Chips auf Basis von vorstrukturierten Thermoplasten und Trockenresisten
2009, Ludwig Gutzweiler
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Selektive Oberflächenmodifizierung und verbesserte Prozessdynamik für die TopSpot Technologie
2009, Michael Bleile
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Specific Chemical Stimulation of Cells for Patch Clamp Assays
2009, Olga Sobolev
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung
Untersuchung eines neuen mikrofluidischen Ventils für hochbenetzende Flüssigkeiten
2009, Richard Jahner
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung

2008

Processing Optimization of the μ-Active NASBA Chip
2008, Marius Clad
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
Kurzfassung

Diplomarbeit

Jahre: 2015 | 2011 | 2010 | 2009 | 2008 | 2007 | 2006 | 2005 | 2004 | 2003 | 2002 | 2001

2015

RNAswitch auf einer mikrofluidischen Plattform
2015, Lukas Gladis
KurzfassungAptamere sind DNA- oder RNA-Oligonukleotide, die sich zu einer dreidimensionalen Struktur falten und wichtige Prozesse in Zellen steuern können. Durch ihre leichte Modifizierung und einer geringen bis gar keinen Immunogenität konkurrieren sie dabei immer mehr mit klassischen Antikörpern in der medizinischen Anwendung. Die Verwendung eines mikrofluidischen Mehrkammersystems erlaubt es dabei mehrere hundert Aptamer-Mutanten unter gleichen Reaktionsbedingungen zu untersuchen. In dieser Arbeit wurde ein Aptamer (Cap-Aptamer) auf einem MITOMI-Chip untersucht, das eine Bindungsaffinität für eine Cap-Struktur besitzt. Die Cap-Struktur befindet sich am 5´-Ende der mRNA und enthält ein methyliertes Guanin-Nukleotid (m7-GTP). In eukaryotischen Zellen besitzt die Cap-Struktur dabei wichtige Funktionen, wie beispielsweise die Initiation der Translation. Für die Untersuchung des Cap-Aptamers auf dem MITOMI-Chip wurde die Sequenz des Aptamers modifiziert. Hierfür wurde ein Cap-Aptamer entwickelt, genannt Cap-Aptamer (I), das mittels einer Anker-Sequenz auf dem Chip immobilisiert werden konnte. Das Cap-Aptamer (I) zeigte jedoch keine Bindungsaffinität für m7-GTP. Ferner wurde ein zweites Aptamer entworfen, dass sogenannte Cap-Aptamer Flush (F), welches nach Bindung an m7-GTP mit einer DNA-Sonde detektiert werden sollte. Die Messdaten zeigten jedoch auch hier keine nachweisbare Bindungsaffinität des Aptamers für m7-GTP. Der Einsatz des Cap-Aptamers in der moderen Forschung könnte dabei vielversprechende Einsatzmöglichkeiten bieten. Die Untersuchung von Cap-abhängigen Prozessen wie die Inhibition der Translation wären möglich. Ferner wurden in dieser Arbeit zwei RNA-basierte Glycin-Sensoren entwickelt (Glycin-Sensor (p) und Glycin-Sensor (n)), die bei An- bzw. Abwesenheit von Glycin durch ihre fluoreszierende Aktivität detektiert werden sollten. Der Glycin-Sensor ist dabei durch eine Kopplung des Glycin-Riboswitches und einem Baby-Spinach-Aptamer entwickelt worden. Der hier entwickelte Glycin-Sensor (n) konnte dabei eine Sensitivität gegenüber Glycin zeigen, die umgekehrt proportional zur Glycin-Konzentration war. Der Glycin-Sensor (p), welcher auf die Anwesenheit von Glycin ansprechen sollte, zeigte dabei keine messbare Fluoreszenzemission. Glycin spielt eine wichtige Rolle bei der Proliferation von Krebszellen. Durch den Einsatz des Glycin-Sensors, könnten Krebszellen dabei in vivo detektiert werden.

2011

DNA basierter Nachweis von Lebensmittelpathogenen auf einer zentrifugal-mikrofluidischen Disk
2011, Nico Marquart
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungThis thesis describes the development of a centrifugal microfluidic structure for simultaneous detection of 6 common food pathogen using real-time PCR. To accomplish this blow moulded COP foil disks are mounted and processed in a modified thermocycling system comercially available. Conventional real-time PCR requires preparation of a separate mastermix with primers and probes for each bacterium. This step is replaced by integrated fluid processing. Therefore the fluidic layout is supposed to aliquot PCR ingredients for a total reaction volume of 10 μl in two consecutive steps. Since this layout is designed as part of a Lab-on-a-Chip system additional restrictions are caused by limited physical disk space with diameter of 130 mm and a maximum rotation frequency of 25 Hz. The development concept includes combination of centrifugal microfluidic unit operations (valves, siphons and aliquoting structures) into a functional layout and validation of functionality by experiments. Rapid prototyping technology enables simple layout transfer from milled COC prototypes to blow moulded COP foil disks. A custom centrifugal Bio-Disk test system has been used to perform the following fluid processing steps resulting in an improved foil disk layout finally enabling the implementation of the on-disk real-time PCR assay. First step of the fluid processing is splitting 100 μl PCR buffer into 2 x 50 μl and aliquoting each part into 7 cavities with an aliquot volume of 5,8 μl (CV 5,7 %). Then 100 μl elution buffer is split into portions with 50 μl each and gated into sample chambers by capillary siphons within 7 minutes. The frequency protocoll pauses at this step to enable the addition of a DNA sample to one part of the elution buffer. The elution buffer with and without DNA sample is then aliquoted into volumes of 6,1 μl (CV 12,8 %) and mixed with the PCR puffer. Pre stored primers and probes for real-time PCR are rehydrated in order to complete a PCR mix in every reaction chamber. The analysis includes a non template control and positive controls with pre stored 10 pg bacterial DNA. The setup enabled the measurement of sample DNA concentrations with a precision of ±1 PCR cycle. Further experiments demonstrated the detection of 0,1 pg (equals 17 to 56 DNA copies depending on organism) aliquoted sample DNA per reaction. At the same time the on-disk NTCs allow non specific detection of contamination of less then 10 DNA copies per reaction. A unique concept different to similar foil disks offering real-time PCR performance is the modular design that allows the combination of the whole layout with other modules for cell lysis and DNA extraction. Therefore the developed layout fullfills the requirements for integration into a so called sample-in-answer-out plattform for detection of bacteria in a pre enriched food sample.
Geometrische Multiplex-RPA auf einer zentrifugalen Foliendisk
2011, Dominik Bär
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungDriven by the need for fast, automatic nucleic acid diagnostics, especially concerning threats with biological weapons or the occurance of pandemies as a result of increasing population density and mobility, the masterproject SONDE aims to develop a fully integrated and portable diagnostic platforms. As a part of the masterproject, the goal of this thesis is the development of a centrifugal microfluidic foil cartridge for the geometric multiplex recombinase polymerase amplification (RPA) of various biological warefare agents. In the past, isothermal amplification on microfluidics was mainly realized by NASBA, only supporting the amplification of RNA. However, sample preparation was not integrated in those devices [1]. Isothermal amplification by RPA was already performed on a centrifugal microfluidic foil-cartridge [2] but multiplexing was not possible since the specific primers and probes for amplification were part of the mastermix. In this thesis we demonstrate a novel approach for the parallel detection of up to 11 different RNA and DNA targets from one initial portion of eluat by geometric multiplex. The newly developed fluidic structure features chambers to prepare an initial mastermix of rehydration buffer, elution buffer with DNA/RNA and lyophilized RPA polymerase with a total volume of up to 140 μL. This mastermix can then be split into 11 aliquots with final volume of 10 μl with a CV of 3.7 % what is reduction of 80 % compared to the standard volume of 50 μL. Primers and probes for specific amplifications are prestored in the amplification chambers. Xanthan (0.1 M) and Trehalose (5mM) were identified as suitable stabilizers for prestorage of dried primers and probes over an extended time period. The longterm storage tests for up to 4 weeks were conducted in real-time at room temperature and accelerated at 70°C and no visible denaturation was observed compared to fresh primers and probes. DNA and RNA amplification can be conducted both with the Twist DX RT package enabling for an easy combination of DNA and RNA diagnostics on one cartridge. [3]. Furthermore the fluidics geometry is designed for upstream integration of lysis and extraction structure. Geometric multiplexing was demonstrated with prestored primers and probes in the amplification-cavities by successfull amplification of purified RNA and DNA from Bacillus anthracis, Rift Valley Fever virus, Marburg Virus and Francissela tularensis. While executing the fluidics on a centrifugal test set-up (approx. 10 minutes), amplification and readout (approx. 3 min) were performed in a standard real time PCR thermocycler (Rotorgene 2000). The lower limit of detection was determined to 104 to 105 DNA / RNA copies per amplification chamber cavity. In future, the lower limit of detection may be further reduced by adjusting the concentrations of polymerase. Presumably, very low reaction volumes (10 μL) may require higher polymerase concentrations to compensate for adsorption effects. For the first time, a connection of the herein developed module for amplification and detection to upstream sample preparation (lysis and extraction) would make a completely integrated sample-to-answer system available.
Quantifizierung von Cholesterol in einer mikrofluidischen Cartridge
2011, Viktoria Reitenbach
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungIn this diploma thesis a microfluidic centrifugal cartridge is successfully developed within the scope of the HDL-c project for quantitative measurement of cholesterol out of 40 μL whole blood sample. The microfluidic layout is comprised of five microstructures for unit operations: sample uptake, plasma separation, aliquoting unit and reagents-chambers for dissolving of dried reagents and protein precipitation and cuvettes for detection of cholesterol out of blood plasma. In this thesis the unit operations are developed in separate on the basis of general conditions in product concept catalogue. Fluids in microfluidic structures are controlled by passive valves like capillary siphon and hydrophobic restriction in a channel. The biochemistry for cholesterol detection is going to be evolved by project partner EKFdiagnostic GmbH, which will be located in reagents-chambers as dry reagents. In this diploma thesis they are not applied, yet. Hereinafter, a summary of results is following to each unit operation. The sample uptake unit consists of a 30 mm long heparin-coated end-to-end capillary from Vitrex. Three different methods are tested to integrate the capillary into the microfluidic cartridge. Those are: a) integration of capillary in radial position in the disc plane, b) rolling in capillary into a sail-structure, c) plug in capillary in an angle on the cartridge. The sample intake takes place by filling the capillary with a blood sample from fingertips, earlobes or from a droplet deposed on a flat surface. As agreed upon with EKF-diagnostic GmbH the choice for an appropriate sample uptake system is made for the integrated capillary in radial position in the disc plane. With this system an untrained user need to make only two working steps to achieve analysis results for cholesterol. There is no risk of contamination for the device. This system is compatible to established production and packaging technologies. There are two systems for plasma separation. The first one consists of a decanting structure, where plasma extraction is controlled by a siphon. The plasma separation in the second system takes place in a sedimentation chamber that had been developed in the Zentrilab project [1]. After two minutes separation time the decanting method extracts 9 μL (CV: 55 %), the sedimentation chamber gets 8,7 μL (CV: 14 %) plasma out of 35 μL blood sample. For the detection of cholesterol parameters the first three of four cuvettes should be filled with 2 μL plasma at least. The fourth cuvette functions as process control. Extracted plasma has to be portioned into volumes that are also sufficient for biochemical reactions in the reagentschambers. Therefore extracted plasma is divided into four droplet-shaped aliquoting fingers in the aliquoting unit, which is dimensioned for 1 x 5 μL, 2 x 2,5 μL and for waste 1 x 4 μL. A sample of 13 μL plasma can be aliquoted into average volumes of 5,4 μL (CV: 3 %) in the first aliquoting finger and 3 μL (CV: 12 %) in the next two aliquoting fingers, in about 1,35 minutes. Two different variations of integrated microfluidic are combined out of successful developed operation units. The succession of the first integrated microfluidic is sample uptake, plasmaseparation with decanting structure, aliquoting unit, biochemical chambers and cuvettes. To rehash 40 μL blood sample for the cholesterol detection this integrated microfluidic takes 7,25 minutes (CV: 18 %). The average yielded plasma volume in the first three cuvettes is 3,2 μL (CV: 18 %). Heretofore all developed microstructures have been hydrophilized by Vistex coating. To accelerate the processing time and save place on the cartridge an alternative integration microfluidic system with a hydrophilic sucrose surface coating is investigated on one microfluidic cartridge. The peculiarity of this integrated microfluidic is that the blood sample of 40 μL first divides into four aliquoting fingers, that are dimensioned for 1 x 15 μL, 2 x 8,75 μL and for waste 1 x 7 μL. The separation of plasma takes place in small portions in separate sedimentation chambers. Extracted plasma is channeled first to the biochemical chambers, then to the cuvettes. For all operations, excluded biochemistry in the reagents-chambers, the processing time takes 2,57 minutes. Average plasma volume in cuvettes is 4,8 μL (CV: 21 %).

2010

DNA Elution on the Phase-Transfer Magnetophoresis platform
2010, Gregor Czilwik
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungMicrofluidic platforms represent a promising technology to perform system-integrated biochemical assays for complete automation of sample analysis. Numerous applications have been reported, that perform diagnostics without additional laboratory support. Yet, although highly relevant for areas such as process control of pharmaceutical fermentation, no application has been established, capable of continuous DNA extraction. Therefore, until now, nucleic acid based monitoring of biological samples, cannot be realized. To provide permanent sample pre-treatment, a microfluidic polymer chip for continuous flow-through DNA purification is thus being developed, using “Phase-Transfer Magnetophoresis” (PTM). In this diploma thesis, the so called PTM platform is effectively optimized in functionality. For the first time, reproducible and permanent extraction of nucleic acids from cell lysate is achieved. The purity of the eluate product is greatly increased, without any sign of inhibition in subsequent quantitative PCR analysis. The chip uses buffers of the commercially available AJ Innuscreen DNA extraction kit to implement microfluidic purification. Working principle is to transfer DNA, bound to superparamagnetic particles, through adjacently flowing, laminar buffer streams, thus performing three essential purification steps: separation, washing and elution. A rotating magnet generates a time-varying magnetic field for redirection of the magnetic beads, which move in a rolling motion of chain-like filaments through the microfluidic channels. Starting from a proof-of-principle stage, the PTM system is improved significantly. Ethanol concentration in the eluate is decreased from an initial value of 3.94 ± 2.30 vol.%. to 0.22 ± 0.10 vol.%, giving rise to entirely circumvent inhibition of PCR. High quality eluates enable optimization of the extraction process. Applying an advanced surface coating method with PEG 8000 and by constructing a new set-up station, the reproducibility of the system is increased. Deviations of extraction yields are brought below 26.50 % (discarding extremely low starting DNA amounts). Only limited by saturation of accessible binding area on the bead silica surface, the platform is further developed to recover nucleic acid with high linearity (R2 = 0.999), starting from low DNA (~ 5 fgμl1) to high DNA concentrations (~ 0.5 ngμl-1). As this is the first continuous DNA extraction on a microfluidic chip, the performance is compared to batch-wise extraction systems in test tubes, to determine the extraction efficiency. The process of enhancing recovery rates of the PTM platform led to significant results: In a DNA dilution series experiment over 7 orders of magnitude, the chip outperforms the tube extraction at all dilution factors with relative extraction efficiencies of 150 ± 50 %. Here, the exact same chemistries are used, indicating large benefits of microfluidic processing. Concerning on-line processing, on-chip eluate specimens are successfully processed with a microfluidic PCR chip, paving the way for following system integration with amplification modules. Increasing the ratio of sample inlet flow rate to eluate outlet flow rate, DNA is concentrated by a factor of 2.62 ± 0.32 in the eluate. Yet, possible concentration factors ( > 50) of the reference extraction system cannot be matched. Furthermore, for the first time, experiments are successfully conducted to extract DNA over 120 minutes, with the sample syringe volume as the only limiting factor. Here, a constant DNA recovery over time is observed. Thus the principle of the flow-through PTM platform finally affirms its ability for permanent and reproducible DNA extraction. In summary, the PTM platform is brought to the final stage of closing the gap between sample preparation (cell lysis) and analysis with available flow-through PCR chips for continuous monitoring of biological samples.
Entwicklung eines IR-Thermocyclers für PCR auf zentrifugal mikrofluidischen Plattformen
2010, André Groß
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Leonhard Reindl, IMTEK
KurzfassungWithin the scope of this diploma thesis an IR-thermocycler for centrifugal microfluidic platforms was developed. Fundamentally the temperature measurement technique for rotating platforms and heating technique on basis of IR-radiators were realised. The system was especially designed for real-time-PCR in an already existing PCR-cartridge. The particular challenge during the development process of the measurement technique was on one hand the adaption of the system to the microfluidic structures on the PCR-cartridge, on the other hand the transmission of the measured data during rotation. Special interest was obtained to the direct measurement of the PCR-solutions temperature. This would allow getting detailed information about the thermodynamic activities inside the solution. In actual systems this was so far not possible. Numerous contact-based und non-contact-based measurement techniques were evaluated. A non-contact-technique fitting our requirements could not be found. From the range of contact-based techniques micro-thermocouples and micro-thermistors were chosen. Successful fundamental tests inside the PCR-cartridge could be accomplished. As final temperature sensor for the measurement system a SMD micro-thermistor was selected. To transmit the temperature measurement data during rotation, the chair of electrical instrumentation at the IMTEK developed a radio transmitter module with inductive energycoupling. The sender unit was connected to the micro-thermistor and fixed in the centre of the PCR-Disk. The stationary receiver unit converted the received radio signal directly into an input signal for the heating controller. For integration of an IR-heater successful fundamental experiments with infrared glowlamps and ring-radiators were done. The focus of these tests was on achievable heating rates and controlling behaviour of the IR-sources. In the run-up of these experiments the required heating rates and the estimated thermodynamic characteristics were simulated. As simulation model a thermal-electrical network was developed and used. It allowed a fast and sufficient valuation of the system. Changes in geometry of the PCR-cartridge and its materials could be implemented simply and fast into the model and their impacts could be estimated. During this thesis a system was developed which is able to measure temperature on centrifugal microfluidic platforms in PCR-cartridges under rotation. Due to the wireless data transmission the IR-heater can be controlled in real-time. The systems possibility to run a complete thermocycling process was shown. The gained average heating rates with IR-radiators are with 4.5 K/s clearly above the requirements the cooling rates are with 2.5 K/s likewise very good.
Implementierung und Validierung fluidischer Strukturen zur Blutgruppengenotypisierung in Folientechnologie
2010, Julian Raum
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungWithin this thesis a microfluidic structure for blood genotyping was implemented on foil. The system is based on centrifugal microfluidics to accomplish the needed fluidic operations. This implies the lysis from whole blood, the DNA-extraction from lysed blood using a silica membrane and a following aliquoting into 16 cavities. The realization of these three operational units was aim of the thesis. Therefore the whole fluidic was splitted into these units and each unit was optimized. Fabrication of the microfluidic system was done by structuring of a COP-foil using microthermoforming. The used reagents and the silica membrane were taken from the QIAamp DNA Blood Mini Kit from Qiagen. To guarantee the functionality of the siphons used in the microfluidic system a hydrophilic coating with Vistex was added. The lysis was implemented successfully. The DNA-yield was 923 ng in average out of 32 μl whole blood. A comparable reference lysis yielded 1437 ng in average. This results in an effiency of the on-disk lysis of 64 %. The difference is due to a missing heating step to 56 °C on the foildisk during incubation. The whole process takes about 15 minutes. The structure for DNA-extraction was successfully implemented on foil. The washing steps and the binding of the DNA to a silica membrane were realized. Using a vent control switch within this structure an early routing of fluid into the eluate chamber could be prevented. In about 50 % of the cases an elution without a contact between eluate and the fluid in the waste chamber was achieved. Using 170 μl elution buffer an eluate volume of ~100 μl was obtained which is sufficient for the subsequent aliquoting. Another 55 μl of the 170 μl were used as switch fluid in the vent control switch. The ethanol concentration in the eluate was below 2.5 % which is the upper limit for athanol not to inhibit a PCR. The duration of the whole process is 9 minutes. The aliquoting structure was also transferred on foil and the functionality was shown. A volume of 120 μl elution buffer was distributed into 16 cavities. Due to some differences between the designed and the milled volumes the use of 120 μl elution buffer was needed to prove the functionality. In the final application 100 μl will be used. The aliquoting process takes 6 minutes. The integrated system was produced on foil and tested. The functionality of the lysis and aliquoting structure was shown. In two of six cases enough eluate was achieved with the DNA-extraction for a subsequent aliquoting. The evaluation of the aliquoted eluate showed an average of 3,4 ± 0,9 ng/μl and 8,6 ± 3,3 ng/μl DNA within each of the cavities. The whole process time is around 30 minutes. With this microfluidic structure the base for blood genotyping on foil was created. In combination with a device which can perform a PCR and fluorescence readout, this system may lead to an integration of the moleculargenetic blood genotyping into the everyday transfusional medicine.
Investigation of Electric Field Characteristics With Respect to Capacitive Droplet Detection
2010, Klaus Mutschler
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jan G. Korvink, IMTEK
KurzfassungThis thesis was prepared within the framework of the „drop detect project“, funded by the Ministry of Science, Research and Art of the Federal State of Baden-Württemberg (Kap. 1499 Tit. Gr. 97). The aim of the project was the development of a contact free measurement system for the in-situ characterization of dispensed droplets in the nanolitre scale. For this, a capacitive approach was used. The change in charge of an electric capacitor was monitored while the droplet passes through in order to derive droplet characteristics such as volume, velocity and point of droplet tear-off. To model the droplet generation and dynamics and the impact of such flying droplets on the electric field of a measurement capacitor, the computational fluid dynamic (CFD) software package CFD-ACE+ providing a multi physics environment was used. Considering the passage of a spherical droplet through a plate capacitor, basically a symmetric change of the capacity can be expected with respect to the droplet entering and leaving the capacitor. However, the experimentally measured signal is non-symmetric and exhibits a negative dip when the droplet enters the capacitor. One major objective of this thesis was the investigation of the effect of capacitive coupling as the physical explanation for this negative dip by numerical simulations. To study this effect, a 2D simulation model was set up, which resulted in a good qualitative correlation of the simulation to the measured signals. However, the simulated droplets showed an unexpected wobbling behaviour, which was caused by nonrealistic 2D simulation artefacts. This was the reason to step forward to a 3D simulation model. The verification of the 3D model by comparison with an analytical model and experimental results did yield a good consistency. The ab-initio simulation based on idealmaterial parameters and dimensions of the capacitor, resulted in deviations of the calculated capacity smaller 30% - including numerical errors - which can be considered as a good quantitative result for this type of CFD simulation. Especially in view of the very small capacities that had to be considered here, which were in the range of 0.2 - 2.8 fF. Typical droplets in the volume range of V = 5 to 100 nl led to changes in charge in the range of = 2 - 28 fC. Based on the validated simulation model a parameter study was performed to optimise the design of the sensor. Apart from the influence of droplet volume and droplet velocity also different geometries of the measurement capacitor with variable dimensions have been studied. As final result it turned out that a half-shell shaped capacitor with a symmetric trench exhibits the best significance regarding the droplet measurements.
Methoden zur Dekoration hochporöser Platin-Anoden und Untersuchung des Vergiftungsverhaltens für den Einsatz in implantierbaren Glukose-Brennstoffzellen
2010, Christian Köhler
Erstgutachter: Prof. Dr. Harald Hillebrecht, Uni Freiburg
Zweitgutachter: Prof. Dr. Ingo Krossing, Uni Freiburg
KurzfassungImplantable glucose fuel cells based on platinum show high catalyst poisoning in consequence of product adsorption and interfering substances. The aim of this work is to decorate the currently used ultra porous platinum catalyst (Pt-Cu) with gold or bismuth and to investigate if the decoration can reduce the catalyst poisoning. Therefore this work entails selecting and investigating suitable methods for the decoration of porous platinum catalysts. The decoration with bismuth was undertaken by underpotential deposition of bismuth (UPD), in which a limited amount of bismuth is deposited on the electrode electrochemically. The decoration with gold took place via copper-monolayerreplacement, where the copper atoms of a UPD deposited monolayer of copper are replaced galvanically through immersion in a gold solution. Precipition-reduction-deposition (FRA) is a method that potentially can be used for the decoration with both metals and is carried out in two steps: Evaporation of a metal salt solution on a platinum electrode and reduction of the salts at high temperature under hydrogen gas. The rating of the decoration methods was based on general conditions with regard to homogeneous coverage, stability of coverage, process limitation and adjustability of the composition. The FRA was unsuitable to decorate ultra porous structures. The problem of the method results from formation of big, inhomogeneous crystallites during the evaporation step. The decoration with bismuth UPD achieved a maximal coverage of 40 %. The gold decoration via copper-monolayer-replacement yielded a gold coverage of 1.5 %. The material characterization of the decorated catalysts by scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDX) and X-ray diffractrometry (XRD) suggests that via UPD of bismuth a bi-metallic catalyst was built up on the surface. The analysis of the gold decorated electrodes exhibits an accumulation of a layer of gold spheres over the platinum catalyst. Therefore the formation of a bi-metallic catalyst cannot be assumed. To investigate electrode performance load curve experiments were carried out at 37 °C in a phosphate buffered saline (PBS) + glucose solution. At 0 % oxygen saturation the bismuth decorated anodes showed a significantly more negative anode potential compared to the Pt-Cu electrodes. Under physiological oxygen concentration (7 %) the gold and bismuth decorated electrodes indicate a similar behavior to the Pt-Cu catalyst. Nevertheless the gold decorated anodes run with a reduced polarization loss compared to the Pt-Cu and bismuth decorated anodes. In order to analyse the poisoning behavior, the electrodes were placed for nine days at a constant galvanostatic load in a neutral buffered solution under physiological concentrations of glucose and oxygen. The resulting potential loss of the bismuth decorated catalyst exhibits no significant improvement compared to the Pt-Cu catalyst. The gold decorated catalyst shows a reduced potential loss, however at a clearly worsened initial anode potential (180 mV). Due to the possibility of poisoning of the electrode potentials from the presence of chloride ions the chloride susceptibility of the electrodes was tested. From this, a sensitivity to chloride ions of all investigated catalysts was determined. This implies poisoning of the decorated platinum catalysts is based on chloride susceptibility and continuous product adsorption. The sensitivity of the electrodes against interfering substances was tested in a simulated tissue fluid. The presence of interfering substances such as amino acids lead to a factor 9 higher fall-off of the anode potential at all used electrodes when compared to working in PBS + glucose. Therefore the results show that the bismuth UPD is capable of creating a homogenous repoducable decoration, however, the decorated electrodes show no improvement of the poisoning behavior. The decoration of gold via copper-monolayer-replacement creates no homogeneous bi-metalic catalyst, although the decorated electrodes show a significant slowing of the potential decrease.
Optimierung und Charakterisierung des Blasformprozesses mit DOE
2010, Dylan Al-Bamerni Azad
Erstgutachter: Prof. Dr. Claas Müller, IMTEK
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK
KurzfassungIn the scope of this thesis, a blow molding process for microstructuring of polymer foils is optimised by statistical design of experiments (DOE). The basic process is used for fabrication of microfluidic structures for biochemical assays. So far, the present blow moulding process partly delivers insufficient moulding results that eventually can lead to microfluidic malfunctions. Apart from that, the blow moulding process has not been examined in detail so far. After definition of relevant input factors, the following targets for process optimisation were defined: • optimisation of moulding quality by maximisation of mould fidelity • minimisation of “webbing” between elevated adjacent structures • minimisation of process duration A moulding tool made of PDMS with typical microfluidic sample structures was designed and critical measuring points for analysis of target values were defined. In order to ensure equal processing conditions, preliminary experiments were performed. These experiments exhibited shrinkage of the PDMS moulding tool by 2.6 % during the first six process iterations until no further dimensional change occured. However, the moulded foils were not significantly affected by the shrinkage. In the first part of the process optimisation, screening experiments were conducted (N = 30 runs) in order to identify dominant input factors and to define a suitable parameter setup for the subsequent optimisation phase, the so-called response surface design. The screening already showed that the webbing was only little affected by any of the input factors. The response surface design (N= 36 runs) delivered an optimised parameter set that allows sufficient moulding of all critical structures. All experiments were analysed with the software JMP® 8. All measuring points could be moulded with a maximum deviation of less than 1 % or less than 5 μm compared to the results that were predicted by the software. This implies very high accordance between the statistical model and the experimental results. The optimised process was finally characterised with two specific moulding tools that were particulary designed in the scope of this thesis. In order to ensure sufficient mouldability of microstructures, all moulding results that were obtained from the characterisation tools were analysed and led to definition of the following design rules: • Structures with rectangular base area require a minimum distance of at least the height of adjacent structures. • Structures with round base area require a minimum distance of at least a quarter of the height of adjacent structures. • Distance between typical microfluidic channels must be at least 300 μm.

2009

DNA Extraktion mit mikrofluidischer Flüssigkeitssteuerung und Reagenzienvorlagerung auf einer Laborzentrifuge
2009, Martina Müller
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungIn this diploma thesis a microfluidic liquid-control for DNA extraction from lysed blood on a centrifugal microfluidic system is developed. Thereby, several components are functionally combined in a complete system. These components are the following: 1. A microfluidic switch for the separation of extracted DNA from unwanted liquids 2. Glass capillaries for pre-storage of liquid washing buffers 3. Burstable seals with time-controlled release for an automated process after adding elution buffer and lysed blood. This microfluidic system can be put into a standard laboratory centrifuge for DNA extraction so that the development of a special centrifugal processing device is not necessary. The DNA extraction is based on binding of DNA to a silica membrane from the QIAamp DNA Blood Mini Kit which also delivers the appropriate buffers. 1. In this diploma thesis a micofluidic switch is used, which blocks the venting of air during the elution step out of a chamber for waste liquids by a liquid plug. The liquid plug is separated directly from the elution buffer. Therefore, no additional switching liquid has to be pre-stored. By using 190 μl elution buffer, an average of 131 ± 38 μl flows into the elution chamber. Thereby an average of 18 μl is lost by flowing into the waste chamber. The rest of the liquid is used to block the vent. 2. The pre-storage of the highly wetting ethanol-containing washing buffers was realized by glass capillaries. Two different washing buffers in a volume of 100 μl each are pre-stored. During the release process, an average of 14 % of the total liquid is lost. 3. The burstable seals with time-controlled release were realized by delaminated lids during fast rotation. By varying the width of bars, releasing times between 80 ± 34 s and 532 ± 221 s could be achieved. The release times depended heavily on the properties of the used liquids (alcohol-based or water-based). The complete system with time-control and pre-storage of reagents was implemented as a proof-of-principle concept with 2 successful tests. The average processing time was 20 min (time of reference extraction: 20 min). Thereby 30.9 ± 3.8 ng DNA were extracted out of 32 μl blood (8.54 % of the reference extraction). The release of the elution buffer lead to a decreased elution volume due to the last burstable seal and the capillary filling of the siphon and thus probably resulted in the observed decreased elution yield.
Endoskopischer Piko-Injektor
2009, Armin Träger
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungSince the end of the last century, medical science has implemented endoscopes more and more in the field of diagnostical, operative or therapeutical surgical interventions on the human body. With the help of such minimal invasive surgery, the post operative traumata for patients are reduced to an absolute minimum. During such an endoscopic intervention the intra corporeal dispensing of fluid, e.g. the specific colouring of tissue or the procedure of a local antineoplastic Chemotherapy, using the current state of technology, is not precise enough and only possible with a considerable amount of fluid volume. The Endoscope Piko-Injektor (EPI) which was developed in this Diploma Thesis can be inserted through the working channel of an endoscope and makes it possible, for the first time in the use of flexible endoscopes on humans, the dispensing of highly potent fluids with a volume range from 10 pl to 4 μl, at a highly precise temporal resolution and localization. The EPI features 4 separate independent channels with 20 μm wide nozzle openings, which allows dispensing of up to four different fluids. The outside dimensions of the assembled EPI is designed to fit the working channel diameter of a commercial endoscope with D = 2.2 mm. The activator Bubble-Jet has been further developed from earlier dispenser generations and allows the discrete dispensing of Vdrop ≤ 10 pl size individual drops. With an integrated reservoir volume of V = 1 μl per dispenser channel, each actuator can dispense up to 100000 individual drops. To avoid diffusive leakages during the application of the EPI through contact with surrounding target fluids, the EPI has a diffusive barrier, the so-called Integrated Phase-Gap (IPG), which guarantees a defined separation of the fluids within and around the dispenser. Using geometry and surface coating, a captured air bubble inside the IPG has an estimated pressure resistance of Δp = 296.15 Pa, which is equal to a submersion depth in water of h = 30.19 mm. With a much simpler technical solution for a modular diffusion barrier, a possible submersion depth of h = 5.76 mm was successfully proven, which equals a pressure difference of Δp = 56.52 Pa. Permanent resist TMMF S 2000 was chosen and appropriate process parameters were evaluated, for the fabrication and capping of the fluid channels. A long time swelling test of 160 days was made because of a reagent exteriorisation inside the EPI, whereby a longer contact of TMMF with an aqueous medium can be considered uncritical. With the first assembled dispensers, individual droplets as well as a continuous flow of droplets, with an actuator frequency of fshot = 10 kHz, was successfully proven in the air and during submersion in water. The Endoscope Piko-Injektor paves the way for a wide spectrum of innovative possibilities for Endoscope medical intervention. It offers a highly precise dispensing of 4 various fluids and is therefore an extremely capable tool for use in an isolated therapy of carcinoma tissue with highly concentrated, cytostatic or cytotoxic fluids.
Entwurf und Entwicklung eines Thermocyclers für PCR auf zentrifugal mikrofluidischen Plattformen
2009, Florian Thoma
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Leonhard Reindl, IMTEK
KurzfassungIn the course of this diploma thesis a thermocycler has been planned and developed that can execute PCR on centrifugal, microfluidic discs. First, potentiel thermocycling methods were evaluated by means of defined requirements and selection criteria in order to find the most adequate method. Based on this thermocycling method, the whole system was generated. Thermocycling methods based on Peltier-elements, convection with air and IR-radiation were analyzed. IR-radiation features high heating rates, but the temperature measurement in the PCR-chambers is technically difficult. The use of Peltier-elements would require detailed analysis of both the thermal interface and the prevention of shear forces. Therefore, the thermocycling method of convection with air was chosen. The heating of air with convection needs aggregates to heat and guide the air. Also, streaming action and the housing material must meet high requirements. Because the thermocycling of separate parts of the disc needs to be locally flexible, the housing was constructed with inlets for the airflow. The concept of the air channel is composed of two structures with different tasks. Initially, air is blown through two inlets into the first structure where it is homogeneously distributed by applicable air channel. After that, air is lead into the second structure in which it is guided to the PCR-chambers by means of airflowstructures. Then it is blown out of the housing. In order to optimize the airflow through the housing, simulations have been developed. These simulations show that a homogeneous distribution of air can be achieved. Also, the airflow to the PCR-chambers has been improved by optimizing the admission. When constructing the thermocycler, the compatibility to the Zentrilab basic device had to be considered. The basic device consists of a rotary axis and a readout system for the results of real time PCR. For thermocycling, a blower of hot air by Leiser was chosen, which is connected to the housing by a pneumatic tube. In addition, a measuring device was generated that allowed the temperature measurement in the PCR-chambers during rotation. The results of the measurements prove that the concept principally works. The required temperatures from 40 °C to 95 °C could be reached. To reach the necessary heating rates, the heat capacity of the system should be reduced though. At the outlet of the Leister bower, a 12 °C/s heating rate can be reached compared to only 7° C/s at the outlet of the pneumatic tube. The heating rates in the PCR-chambers are 0.6 °C/s and the temperature differences between the PCR-chambers are 0.3° C at most.
Isothermal Amplification of DNA on a Centrifugal Microfluidic Platform
2009, Patrick Weber
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungFast and mobile screening of pathogens or specific markers for epidemics is of great importance in emergency diagnostics, transplantation medicine and the food industry. Therefore the related market also known as Point of Care (PoC) has shown a constant growth over the past years. The estimated revenue of $ 5.05 billion in 2005 and 13.8 % average annual growth until 2010 leads to sales of $ 9.65 billion [1]. PoC testing requires costefficient, fast and easy-to-handle systems [2]. Two topics have gained great importance in PoC Life Science applications: Nucleic acid based diagnostics, the central tool of modern molecular biology, and Lab-on-a-Chip (LoaC) systems based on microtechnology [3,4]. Labon- a-Chips are microstructured reaction platforms on which the necessary fluid handling steps of the test assays are performed [5]. LoaC systems simplify and accelerate complicated tests due to their high level of automation. The state-of-the-art method for nucleic-acid-based diagnostics is the polymerase chain reaction (PCR) which is mainly performed in central laboratories. PCR has not found its way into the field of PoC yet because of the need for rapid and accurate thermocycling in order to achieve high efficiency and fast results. This however does not meet the easy-to-use and low energy requirements of PoC devices. In this thesis we validate a completely new approach for nucleic-acid-based diagnostics on the LoaC platform. Within this thesis the development of a centrifugal microfluidic disk for isothermal amplification of DNA is presented. The production of a cost-efficient disk platform is shown as well as the complete fluidic integration of the assay including reagent prestorage. The disposable disk substrate itself serves as a blister packaging for all reagents which are needed for the assay including glass capillaries for liquid reagents and lyophilisates which are prestored directly on the substrate [8]. The polymer foil substrate is structured in a blow moulding process and subsequently sealed using a commercial available microtiter plate sealing foil. The employed reaction mechanism is the Recombinase Polymerase Amplification (RPA) developed by TwistDx in 2006 [6,7]. It is a fast qualitative assay which shows high sensitivity with a detection limit of several copies of sample DNA and a short time-to-result of only 12 minutes. The advantage of this assay is its constant operating temperature of 37 °C. Prime system features are automatically performed assay preparation steps on the disk with an outstanding level of parallelisation which results in only one pipetting step per sample. The assay for 40 reactions can be prepared in less than 8 minutes including multiple mixing steps, aliquoting and metering of 10 μL volumes with a CV of 3.5 %. When the disk is equipped with a DNA sample, the user has to activate the platform and mount it into the readout device. In this context, it is shown that a commercial device (RotorGene2000, Corbett Research) can be equipped with the disk to perform the fully automated assay including real time readout. To demonstrate the screening for a highly relevant DNA target, a test assay for the detection of methicillin resistant Staphylococcus aureus (MRSA) is integrated on the disk. MRSA is a major safety hazard and economic threat for institutions such as hospitals or retirement homes [9,10]. The assay shows a high biological functionality on the disk while samples containing less than 10 copies of DNA can be successfully amplified with a time-to-result of only 12 minutes. The absence of cross-contamination on the disk is demonstrated by running several negative control reactions next to the compartments containing high DNA concentrations. The unique investigations made in this thesis highlight the enormous potential of RPA when performed on centrifugal LoaC platforms. Real-time readout under rotation enables new prospects in the monitoring of 40 or even more simultaneously started RPA reactions in multisample screening experiments. This establishes the basis for developments such as Nested- RPA or Multiplex-RPA. The readout device is kept at a constant temperature of 37 °C during the reaction which emphasises the low price and energy demands of an on-site readout device that could potentially be developed for the platform.
Microfluidic Immunoassay in a blow moulded Foil
2009, Gregor Welte
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungThis diploma thesis evaluated the implementation of an immunoassay on a blow moulded centrifugal Lab-on-Chip platform as a cost efficient fabrication method [1]. The ability to perform assays on miniaturized (Lab-on-Chip) devices is of high interest due to the reduction of volumes together with low material consumption. The unique behaviour of fluids at the microscale allows better control of molecular interactions [2,3]. The main market drivers in diagnostics are cost efficiency, reproducibility and short time to result [4]. Lab-on-Chip Point of care (PoC) testing revenue reached an estimated $5 billion in 2005 and is expected to expand with an average annual grow rate of 13.8 % through 2010, yielding sales of nearly $9.6 billion [5]. Recently the usage of a blow moulded microfluidic platform was successfully demonstrated in our group. The isothermal DNA amplification [6] was implemented in an Lab-on-Foil system with a complete fluidic processing and a real-time readout [7]. Therefore the main goals of this thesis were to characterize and evaluate the microfluidic design (transferred onto the blow moulded foil), to implement and to perform an immunoassay. Heterogeneous immunoassays require a capture antibody immobilized on a solid phase. Therefore immobilization of capture antibodies on a blow moulded polymer surface was established (DHB entry: Antibody-Coating of Polymer Surfaces (200902019)). Two assays were evaluated, a Follicle Stimulating Hormone (FSH) and an Interleukin-8 (IL-8) assay. The FSH assay was characterized in detail and established in a microtiter plate. This assay was used for an investigation concerning the feasibility of an antibody immobilization on a blow moulded polymer surface. To corroborate the reliability of this polymer surface as a viable platform for carrying out heterogeneous immunoassays, an assay against Interleukin-8 was furthermore successfully implemented. The efficiency of antibody immobilization was determined to 50 % compared to the standard high binding rate microtiter plate (NUNC Maxisorp). However both assays showed the same lower limit of detection in comparison to the microtiter plate approach. The fluidic test platform accommodates six structures where every structure is used to carry out one assay. The fluidic layout is designed to get a 50 % reduction of volumes regarding necessary reagents compared to a microtiter plate. Implemented unit operations provide sample preparation, mixing and waste handling. Up to 20 assay steps with a volume of 50 μL per step can be performed including washing steps resulting in a total assay volume of 1 mL. Capillary siphons are valving the reagents within the disc. A blood plasma separation was investigated as initial process of the assay followed by the fluidic protocols to perform an immunoassay on disc. Each valving step was tested subsequently. Due to ongoing development of the blow moulding process resulting in low yields concerning the fluidic, a complete assay on foil couldn’t be provided, but each single step was analyzed in detail. An experimental setup to read out absorptions was tested. Carrying out immunoassays based on absorption requires a device which is capable of measuring absorption through the polymer disc. This readout device is still under development but settings concerning positions of the cavities to measure were determined. In total all steps which are necessary to implement an immunoassay on a blow moulded foil were shown, especially the antibody immobilization. Therefore an immunoassay on foil is feasible.
Mikrofluidische Flüssigkeitssteuerung zur DNA Extraktion auf einer Laborzentrifuge
2009, Markus Rombach
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungThis thesis comprises the development of a microfluidic liquid-controlling for the automated extraction of genomic DNA from whole blood samples on a commercially available standard laboratory centrifuge. The DNA purification is based on the solid-phase extraction with a silica membrane in combination with other well optimised components of the QIAgen DNA Blood Mini Kit, which are to be integrated into the fluidic network. The liquid-control is realised on a centrifugal microfluidic platform. The boundary conditions of a laboratory centrifuge are the limiting factors of the liquid-control, in particular the non-adjustable acceleration and the unidirectional sense of rotation. The development of a novel switching concept is required to separate unwanted sample ingredients and washing buffers. For the applied extraction method, the correct switching of four liquid 100 μL volumes is necessary. The purpose is to separate the purified DNA properly during the final step from the three preceding liquids (elution step). In the process of switch development, three approaches are tested on a centrifugal analyzer. In the first approach, the switching is frequency-controlled. Depending on the rotational frequency, the vent of a chamber will be opened or blocked by a water column. At high frequencies, the vent is unblocked which enables the liquid flow into this chamber. By decreasing the rotational speed, the vent is blocked by the water column and thus the liquid flow into the chamber is hindered (elution step). For switch validation, varying channel and chamber geometries are tested. The switching of up to 98 % of the elution buffer volume is observed, but leads to insufficient results in the reproducibility test. The vent control is easily affected by thickness variation of the COC substrates of partly ±100 μm. The second approach is controlled by the liquid volume. Thus, the first 3 liquid volumes are routed into the waste reservoir under high rotational frequencies, while the low liquid volume in the elution step is collected in a reservoir and capillarily transported into the extraction chamber. However, the dimensioning of the switch structure is cancelled after the third iteration due to filling problems of the capillary siphon with the watery elution buffer. Finally, a third approach is developed on the basis of the first approach, utilising a controllable instability of the liquid–air interface. The instability occurs at high rotational frequencies, which enables a liquid flow into the waste reservoir. At lower frequencies, the interface is stabilised by the air pressure resulting from the unvented waste reservoir, which is used to switch the liquid in the elution step. The obtained results show reproducible switching procedures with an average liquid volume of 88 % flowing into the extraction chamber. The third approach is used for DNA extraction on the laboratory centrifuge, performing 7 comparable extractions. An average DNA yield of 42 % compared to the reference extraction is achieved. A maximum DNA mass of 171 ng out of 32 μl blood sample is measured. A subsequently successfully performed real–time PCR shows a shift of the Ct value of +1.83 cycles to the comparable DNA template of the reference extraction.
Mikrofluidische Plattform für hochsensitive DNA-Analytik mittels Vor- und Hauptamplifikation
2009, Fabian Stumpf
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungIn the scope of this thesis a microfluidic platform was developed, which allows highly sensitive nucleic acid analysis by pre- and main-amplification polymerase chain reaction (PCR). The platform is based on centrifugally actuated microfluidics, which was implemented in a commercially available, slightly modified thermocycler (Rotor-Gene 2000, Corbett Research, Australia). The main-amplification is obtained by real-time PCR. In the future, the developed platform will be used for genotyping. Genotyping by PCR is a standard method of molecular biology to identify subtypes of certain pathogenic strains (e.g. methicillin-resistant staphylococcus aureus, MRSA). Usually, a DNA sample is pre-amplified with a few thermocycles at first, in order to significantly increase the number of DNA copies. Subsequently, the pre-amplified sample is diluted and aliquoted. Then a second PCR, the main-amplification, takes place with an additional set of primers and probes, which allows a very specific identification of the respective subtypes. Within this thesis, the advantages of the so-called lab-on-a-chip technology are combined with the possibilities of a pre- and main-amplification of DNA samples. The lab-on-a-chip technology allows processing of very small sample volumes in an operator-independent, semi-automated and parallelized manner. As a result of the full system integration, laboratory contamination risk during handling of the pre-amplified sample is drastically reduced compared to manual handling. The system consists of the here developed, polymer-foil-based microfluidic cartridge and a commercially available, slightly modified thermocycler. By rotation of the foil cartridge and the thereby induced centrifugal forces all liquids can be actuated in the thermocycler. Microfluidic valving is accomplished by capillary siphon valves (yield = 99.3 %, N = 288 siphons). After the pre-amplification, which will increase the number of DNA samples by a factor of 1000, the PCR mix (20 μl) is diluted 1:9 and divided into 14 aliquots (accuracy 9.8 μl ± 3.4 %, N = 32 reaction cavities). Detection and optimisation of the biochemical analysis was carried out solely with the Exfoliative Toxin A gene of the MRSA-strain. A PCR-buffer (RealMasterMix, 5 Prime GmbH, Hamburg) with contact angle > 50° was chosen that proved feasible for all fluidic and biological requirements. Specific primer and probes are pre-stored in the respective reaction cavities and rehydrated prior main-amplification. The addition of bovine serum albumin (BSA) and (sacrificial) salmon sperm DNA results in more homogenous and more sensitive fluorescence signals during real-time detection. The lower limit of detection is less than 10 DNA copies for the fully integrated system. There is no cross-contamination detectable between the 14 reaction cavities. Two samples can be analysed simultaneous and independently in the foil cartridge. The duration from pipetting of the sample to the final result takes approximately 150 minutes. The developed system represents the starting point for future genotyping analysis, which can be even more efficient by a still-to-implement multiplex-pre-amplification and the prestorage of additional primers and probes for main-amplification.
Monolithic Biofuel Cell Using Microspheres as Insulator
2009, Arne Simon Dannenberg
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungThis thesis presents a monolithic assembly method for thin film glucose fuel cells that uses self-assembled microsphere layers as micro-perforated, electrically insulating support for a vacuum-metalized cathode. This is intended for the coating of medical implants, e.g. cardiac pacemakers, with fuel cells as sustainable energy supply. In the developed process, the microsphere insulator can be directly deposited on the anode. Its micro-perforation allows glucose diffusion through the cathode and the insulating layer to the anode. In contrast to photolithography, micro-structuring by polymer spheres can also be used on reflective, rough substrates. Besides the thinness of the layers, the key advantage of this method is the monolithical deposition of insulator and cathode. Manually clamping of anode, membrane and cathode, which is often used to assemble glucose fuel cells, is thus rendered unnecessary. The key step in the process chain is the self-assembly of polymeric microspheres on the anode in a thin (< 60 μm), ordered oligo-layer. The latter serves as insulating support (< 60 nS/cm2) and micro-structuring element for the cathode, which is deposited by evaporation of platinum in high vacuum after partial sintering of the microsphere layer. For this purpose, a new technique had to be developed that even works without surfactants. The anode samples (0.25 cm2) were successfully coated with one to three layers of 19.67 μm spheres. Polystyrene crosslinked with 2% of divinylbenzene proved to be feasible with respect to desired partial sintering to ensure layer adherence whilst avoiding layer closure at sterilization temperatures. The latter caused no layer delamination of the complete device. The process was successfully carried out on ultraporous, Raney-type platinum-zinc anodes. The deposited cathode achieved an open circuit potential (OCP) of (+227 ± 15) mV vs. SCE at physiologic concentrations of glucose (3 mmol/l) and oxygen (7% air sat.). Refining the cathode by electrodeposition of Pt-black lead to a polarization behavior similar to state-ofthe- art Pt-black cathodes and an OCP (+266 mV) comparable to Pt-Al cathodes (+270 mV). Despite the fact that the anode was indeed supplied with glucose through the other layers, its performance was low due to its loss in surface roughness of (43±7)% caused by a tempering step in the fabrication. In a side project, preparatory work addressed the enhancement of electrodeposited Pt-Cu anodes by microsphere templating. The Pt-Cu anode was thereto deposited within a multilayer of polymeric 2.55 μm spheres. Subsequent dissolution of the polymer produced a micro-porous, mechanically stable, 37 μm thick structure. As-produced micropores might improve accessibility of fuel to nanopores of the Pt-Cu and increase active surface area compared to non-templated Pt-Cu anodes. The results indicate the opportunity that higher surface roughness might indeed be obtainable. Since this is easily realizable on implant capsules, it might complete the monolithic coating process of implants with biofuel cells.
Re-design and validation of a centrifugal microfluidic lysis, switching and aliquoting structure
2009, Sidney Hakenberg
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Leonhard Reindl, IMTEK
KurzfassungThis thesis is embedded in the advancing progress of the ZentriLab genotyping project. The goal of this project is the implementation of a deoxyribonucleic acid (DNA) based automated blood group test with a disposable test carrier which is processed on a centrifugal analyzer. The target application is a point of care testing system capable of identifying the exact blood group genotype including “rare blood groups” that can be crucial in transfusion medicine. This diploma thesis builds up on an existing concept for the centrifugal microfluidic structure and provides solutions for its main shortcomings. These shortcomings are 1. malfunction of a valve for retention of highly wetting blood lysis buffer, 2. irreproducible switching performances of a microfluidic switch based on the Coriolis force used for separating washing buffers from downstream applications, 3. inaccurate metering within an aliquoting structure which splits a 110 μl solution into 16 aliquots for parallel determination of all relevant gene sequences. These microfluidic structures are re-designed on Cyclic Olefin Copolymer (COC)-cartridges and validated on a centrifugal setup following all quoted ZentriLab design rules and using reagents closest to what is used in the application. The lysis valve is realised in the form of a restriction channel as a hydrodynamic resistance element. Complete blood lysis sequences are performed using different channel cross sections of the valve restriction channel. The resistance Rhd of 8400 Pa·s·mm-³ gives reproducible performance, capable of retaining the liquid during the lysis procedure while ensuring on-demand liquid emptying in less than 50 seconds. Total liquid losses during lysis are kept below 1 μl which is tolerable for the downstream applications. The Coriolis switch is re-designed based on two different approaches and tested for switching reproducibility within a sequence of 4 reagent switching steps. The first approach is a modification of the state-of-the-art Coriolis switch. In order to improve its switching reproducibility, parameter changes are made, namely surface modification by applying different Teflon concentrations and geometrical variations of the switching chamber. Within the new second approach a Polydimethylsiloxane (PDMS) inlay is integrated in order to redirect droplets away from the lid surface thus switching contactlessly. In both realisations flawless switching sequences are observed in some experiments. However, the results are not reproducible. CVs of switching a total amount of fluid into the designated chamber are 1.3 % (Teflon based) and 3.3 % (PDMS based). Reasons for switching errors are probably irreproducible frictional forces upon the Teflon coated lid surface, COC milling residuals at droplet release orifices and inhomogeneities in thickness across the substrates. With the re-designed aliquoting structure 110 μl of elution buffer is successfully aliquoted into uniform volumes in 16 metering chambers with no loss by adding 0.1 % of the surfactant TWEEN 20. This is achieved in four out of four complete structures resulting in a successful test of 64 out of 64 metering structures. Furthermore the burst frequency characteristics of the centrifugo-pneumatic valves incorporated into the aliquoting structure are determined under varying geometries and different surfactant concentrations. The resulting large set of data thoroughly characterises the centrifugo-pneumatic valve. All results are documented in a laboratory internal design handbook for future activities on this project.
Single molecule analysis of feature size influences on mass transport limitations
2009, Phillip Kuhn
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungCommon immunoassays and microarrays are severely restricted by mass-transport limitations. It is assumed that these restrictions can be reduced by a miniaturization of spot size, thus only the reaction kinetics would be limiting the assay speed. The time-to-result could be reduced, which naturally leads to cost reduction. The general assumption is that smaller spots yield a higher signal density and reach the chemical equilibrium more quickly than larger spots. The aim of this thesis is to investigate and validate these assumptions which have been influencing the design of microarrays for the last 30 years. The binding on microspots smaller than 100 μm is analyzed with spatially-resolved single molecule detection. The fabrication of micrometer-sized spots with different diameters has been successfully established by microcontact printing. Two variations of microcontact printing, the conventional and an inverse technique, were investigated and used for the printing of capture proteins onto a solid support. The binding was detected at the single-molecule level with conjugated nanogold in combination with SEM imaging. Antibodies coated on the surface of nanogold particles bind specifically to the immobilized capture proteins. Single molecule detection was performed with high sensitivity, specificity and spatial resolution below 50 nm. The spot size dependance of nanogold particle density was only observed for stand-alone spots. If spots interfere with each other, no spot-size dependance could be observed. It is important that spots are not influencing each other, i.e. the depleted volume should be confined for each spot. The experimental observations were supported by numerical simulation. The simulation predicts a higher signal yield for smaller spots, compared to larger spots. It was shown, experimentally and by simulation, that edges of spots saturate more quickly compared to the center of the according spot. A statically firm validation of the simulations could not be provided because only a small dataset could be obtained in the given time. It is shown that with the given setup and more experiments a more reliable validation can be achieved. Additionally to the mass-transport investigations of microspots, electroless deposition of silver was studied which enlarges the gold particles to a size which is optically detectable. It was shown that read-out of enlarged nanogold can be performed either with a microscope or a laser from a CD-drive. This will enable quantitative protein detection on lab-on-a-disc systems in future applications.

2008

Binder-less Platinum Cathodes for Oxygen Reduction in Potentially Implantable Glucose Fuel Cells
2008, Ulli Kräling
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungImplantable abiotically catalyzed glucose fuel cells are a promising approach towards the realization of a sustainable power supply for medical implants. Situated in the body tissue, these devices generate electricity from the electrochemical reaction of glucose and oxygen from body fluids. Due to chemical instability of the state of the art polymer-bound activated carbon cathode and the difficult modification of such electrodes with protective polymers and adatoms a binder-less cathode design is developed and implemented in this work. In a first step, 500nm thin platinum layers, deposited onto a 300 μm thick silicon substrate, are porosified in a Raney-type process by alloy formation with aluminum, zinc, or bismuth. Upon annealing and subsequent dissolution of the non-noble metal a catalytically active structure with a high surface roughness has been obtained. The oxygen reduction performance of samples prepared using different annealing times and temperatures has been characterized at 37 °C in sterile phosphate buffered saline under physiological concentrations of glucose and oxygen. For more realistic conditions, a porous filter membrane with diffusion characteristics comparable to the tissue capsule forming around medical implants was placed in front of the cathode. The experimental results show that this additional diffusion barrier leads to oxygen mass-transfer related losses and limits the electrode performance at higher current densities. Under these conditions the best cathode performance has been obtained with electrodes fabricated from platinum-aluminum bi-layers, annealed at 300 °C for 1 h. In a second step different feedhole geometries were evaluated by numerical simulation of reactant diffusion. Evenly distributed trapezoid feedholes, fabricated in silicon by anisotropic KOH etching have been identified as the most suitable feedhole design for the cathode. This geometry ensures a sufficient glucose flux trough the cathode substrate and homogenous availability of glucose at the anode. Finally, cathodes with feedhole size of 1818 μm by 50 μm or 200 μm (corresponding to a reduction of the geometric cathode surface by 7.5 % and 22.9 %, respectively) have been fabricated and characterized as a complete binder-less fuel cell together with the already established binder-less platinum-zinc anodes. Fuel cells with 50 μm wide feedholes in the cathode showed the best performance. Their maximum power density amounts to (3.9 ± 0.2) μW/cm2 at 8.9 μA/cm2 and 65 mbar pO2. This value is significantly higher than the maximum power density of ~ 2.6 μW/cm2 reported for the state of the art fuel cell operated at the same oxygen partial pressure but without a diffusion barrier accounting for tissue capsule formation. Besides exhibiting a higher performance than the conventional polymer-bound glucose fuel cells, the novel glucose fuel cell also offers higher chemical stability. Furthermore, the binder-less fuel cell electrodes open up an easy possibility for further modifications, e.g. with protective polymers and adatoms to further increase their performance and poisoning resistance. Although constructed from potentially non-toxic materials, especially the application of aluminum mandates dedicated biocompatibility investigations if future implantation of the device is desired. Alternatively a careful optimization of the presented less problematic alloying partners zinc and bismuth towards higher oxygen reduction performance appears feasible.
Comparison of Laccase-Catalysed Biofuel Cell Cathodes Fabricated from Carbon Based Macro- and Nanomaterials
2008, Oliver Strohmeier
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Markus Biesalski
KurzfassungEnzymatically catalysed biofuel cells represent a promising future technology when it is up to supply low-power remote applications with energy derived from the environment. This approach of energy generation, denoted micro energy harvesting, establishes the basis for a sustainable long-lasting power supply. As a further advantage, especially in terms of environmental protection, batteries become unnecessary. However, all state-of-the-art set-ups are designed different and usually investigate complete fuel cells only. Thus an independent assessment of the single electrode performance from literature data is not possible. The test set-up used in the present work therefore was designed to overcome this problem since all influences of the counter electrode are excluded. The goal of this work was to characterise different carbon based macro- and nanomaterials as cathodematerial, independently from the anode. As cathode catalyst, the enzyme laccase from the white rod fungus Trametes versicolor is used since it shows direct electron transfer and adsorption to carbon based electrodes. From different powder-like nanomaterials as well as 2-D and 3-D solid carbons, cathodes were manufactured and implemented in the fuel cell. For each electrode, volume dependent potential-current characteristics were measured allowing a comparison of the materials. For the 2-D and 3-D solid materials, the performance correlates with the surface available for enzyme adsorption. However, it was revealed that this relation is not given for the nanomaterials anymore. Multi-walled nanotubes with a diameter of 10 nm were identified to be the best material to build cathodes although their surface to mass ratio is less than half of that of single-walled tubes (173.5 m²/g vs. 382 m²/g). This effect is explained by the fact that each enzyme molecule can adsorb to more than just one tube. With decreasing diameter more tubes per enzyme molecule adsorb, what compensates the advantage derived from the huge surface. Furthermore, the efficiency of the direct electron transfer to all nanomaterials was investigated. It could be proven that direct electron transfer to the amorphous surface of carbon nanofibers (1.5 μA/μg @ 0.4 V) is about three times more efficient than to the highly ordered surfaces of double-walled nanotubes (0.4 μA/μg @ 0.4 V). Concerning the volume dependent current density, the efficient electron transfer makes carbon nanofiber electrodes (12.3 m²/g) competitive to those of multi-walled nanotubes with a diameter of 60 to 100 nm (55.2 m²/g), although the surface to mass ratio is less than a forth. Besides improving the cathode performance, the implementation of carbon nanofibers (2.30 €/g) or multi-walled tubes (4.50 €/g) instead of single-walled tubes (86.00 €/g) reduces the costs to build a single cathode tremendously. Moreover, several modifications of materials were tested. Firstly, surface hydrophilisation revealed to have a negative influence on the electron transfer. Secondly, a modification of graphite felt with gold nanoparticles was tried and showed a downgraded cathode performance compared to the untreated. With the knowledge of the best cathode material, further challenges can be approached. To overcome the handling problem of the powder-like nanomaterials, suitable concepts for their immobilisation need to be developed. One possible solution to overcome this problem was pre-investigated. An immobilisation of nanotubes with ionic liquid was shown to be a very promising approach. As a vision, nanotubes should be grown on graphite felts combining the excellent performance of a nanomaterial with the easy handling of a solid.
Design und Herstellung des Zellstimulationswerkzeuges Piko-Injektor – 2. Generation –
2008, Michael Laufer
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungThe discrete chemical stimulation of single cells based on the release of precisely defined biochemical drugs gains more and more importance in the field of neurotechnology, biology and pharmacy. The Pico-Injector is a new user-friendly cell stimulation tool. This thesis focuses on the re-design of the Pico-Injectors's dispensing unit, which features two individually addressable fluid channels, as well as their integration into a low-cost housing for simple handling. Due to the reduction of the dispenser tip by 20 % to 340 μm in width, the re-designed dispensing unit features a more needlelike design, which leads to an easier positioning under the microscope. Moreover, the nozzle distance is reduced by 25 % to 90 μm for a higher spatial resolved drug release. The manufactured and characterized dispensing units resulted in a droplet volume of Vt = 9,3 pl with a reproducibility of CV = 13,8% (20 μm nozzle, 25 dispensing cycles each with 1000 droplets) as well as a maximum actuation frequency up to 11 kHz. To realize the Pico-Injector as a disposable device, a low-cost housing is required, which also implements macroscopical interfaces to the microscopical one of the dispensing unit. The housing has been created as USB-Stick to achieve at one hand the mechanically stabilization as well as the electrical connection and on the other hand the simple handling. The fluidic connection was realized with self-draining reservoirs (V = 10 μl) which were integrated into the housing. For the fabrication of the housing, consisting of lid and case, the polymer Cycloolefin Copolymer (COC Topas 5013) and Epo-Tek 353ND have been used. This combination proved most suitable in a systematical investigation of different materials. The fabrication process of the assembling of the Pico-Injector is based on three steps. The first step, gluing of the lid into the case, is to realize integrated fluidic structures. These fluidic structures withstand a pressure of up to pmax > 5 bar and a yield of Y1.step = 90 % is achieved. The second step, soldering the dispensing unit onto the flexible printed circuit (flexcable), achieved a yield of Y2.step = 100 %, as well as a suitable stability of the soldering connection, which withstand a force up to 35 N. The third and last step, gluing the dispensing unit with the flexcable into the case resulted in a yield of Y3.step = 50 %. The following characterization showed that the glued dispensing units withstand a pressure up to pmax > 5 bar without any leakage. The overall yield of Yoverall = 45 % as a result of the three single steps turned out rather low due to the third step. However, this was caused by the aging of the SU-8 resist, which has been used to build up the new dispensing unit. Preliminary tests showed the potential to gain an overall yield above 80 %. The necessary high spatial resolution (< 50 μm) for single cell stimulation as well as the possibility to stimulate single cells itself by the Pico-Injector could be demonstrated. Therefore, a single plant cell (protoplast) with a diameter of Ø = 30 μm was labeled with a fluorescent dye using the Pico-Injector for the discrete dispensing of indivdual droplets with a diameter of Ø < 25 μm.
Development and Integration of an Optical Process-Control Sensor for the PipeJet™-Dispenser
2008, Julien Maksymowiez
Erstgutachter: Prof. Dr. Bernhard Vondenbusch
KurzfassungThe importance of microfluidic dispensing systems for the nanoliter range in the chemical, biological and pharmaceutical industry has enormously increased in the past. Dosing liquids reliably and precisely is the main task of dispensing systems. Therefore, also the demand for a precise online-process-control has increased with the growing importance of dispensing systems in Life Science applications. In this thesis a non-contact online process-control-system for dispensing systems in the nanoliter range is developed. It is based on an optical detection of single droplets in flight. A droplet, which passes through the light beam of the optical transducer leads to a dilution of the detected light intensity and enables for the extraction of time dependent signals. The system is adapted and validated by the use of a PipeJet™-P9-dispensing system. The process-control delivers several levels of information. A first level provides the information about the presence and absence of a droplet and counts the number of dispensed droplets. This allows for the re-dispense of missing droplets, that a required target volume can be reached. The smallest detectable droplets have a radius of only ~70 μm, which corresponds to a volume of ~1.5 nl. The application of the developed sensor on dispensing robots, which provides to dispense single droplets to defined geometrical positions on a target substrate, allows for the determination of the destined position of the absent droplets. Re-dispensing of missing liquid quantities prohibits a further processing of defective substrate material and the waste of expensive sample liquid. A further control-level is based on the so called „fingerprint signals“, which depend on several droplet properties. The shape, the velocity, the size and the medium of the dispensed liquid define the extracted signal characteristics. The high reproducibility of the sensor signals for constant causative conditions enables to evaluate small deviations in the characteristics of the detected bodies. A stable dispensing process generates high reproducible droplets, thus small changes in one of the mentioned droplet parameters can be detected as a deviation in the sensor signal. Due to the normally high reproducible dispensing process of the PipeJet™ dispenser, signal deviations of ~5 % indicate for a unstable process. Even small changes in the droplet’s volume of ~2 nl can be detected as a change in the signals amplitude. The velocity of the detected droplet can be related to the signal length. Typical signal periods lie in a range of 1 to 1.5 ms, which indicates for droplet velocities from 0.5 to 1 m/s. Furthermore, malfunctions in a dispensing process can easily be detected by evaluating the detected signal. The sampling rate of the developed process control system is determined to be 44 kHz, thus allows for a satisfying resolution of the digitised sensor signals. The fabrication in a standard printed circuit board (PCB) process enables a easy adaption of the sensor to different kinds of contactless dispensers and implements a very cost efficient realisation.
Development of a Solid Phase Detection and Amplification Method for Nucleic Acids
2008, Simon Wadle
Erstgutachter: Prof. Dr. Ralf Reski, Biologie, Uni Freiburg
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
KurzfassungDifferent nucleic-acid-based assays have been developed for rapid detection of specific gene sequences. Fast and sensitive identification of genetic loci is at least required for the analysis of clinical samples. State-of-the-art genotyping assays can meet this demand and therefore in vitro diagnostics has become the major field of their application. Current studies estimate a worth of assays based on (real-time) polymerase chain reaction (PCR) and DNA microarrays between $ 6-7 billion, therefore they have a toehold in the molecular diagnostic market and further growth of their market share is expected [1, 2]. However their market potential, the mentioned methods most of genotyping assays are based on bare limitations. They are restricted concerning the multiplexing degree of real-time monitoring and the microarray sequence layout and fabrication. Facing these limitations a novel genotyping assay, the Mediator Probe Assay, is being developed. The Mediator Probe Assay allows for parallel detection of several genetic loci in one sample. This is facilitated by combination of target amplification by PCR and a detection reaction on a (low-density) DNA microarray. During liquid phase amplification of a gene of interest, a Mediator is activated by cleavage of a Mediator Probe specific for this gene. The Mediator can hybridise at an immobilised oligonucleotide with the specific Mediator binding site. After hybridisation, the Mediator triggers a reaction that enables signal generation. Goal of this thesis is the development of a solid phase detection method for the activated Mediator. Two different detection strategies were investigated: Triggering of direct fluorescence activation by the Mediator and triggering of the activation of an immobilised primer. The activated primer can initiate solid phase amplification which is accompanied by signal generation. After systematic liquid and solid phase characterisations, a proof of concept for a detection method based on the latter principle was performed. Reaction efficiencies were low using this detection method, but a significant signal was generated in samples containing the gene of interest. Two systems were evaluated for solid phase amplification as a means of space resolved signal generation. Amplification products could be detected in case of both solid phase systems. A glass-slide based format provided higher singal-to-noise ratios compared to a microwell-based solid phase amplification system. Reaction tubes and cartridges are usually fabricated of polymers. If reaction vessels are meant to be used for PCR they must comprise compatibility regarding the reaction conditions and components. As a supplemental task of this thesis, a PCR compatibility test for polymers was developed and preselected polymers were tested for PCR inhibition. Based on this test setup, a PCR compatible polymer for an intended Mediator Probe Assay reaction cartridge could be defined.
Integrierte Qualitätskontrolle für Dispensiersysteme mittels thermischer Strömungssensoren
2008, Norbert Schmitt
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Ulrike Wallrabe, IMTEK
KurzfassungThe application of fully automated pipetting systems has gained in interest enormously in the past years [1]. Especially the fields of pharmaceutical research and combinatory chemistry are in the need of dosing small amounts of liquids in the range from nano- to microliters. To ensure the accuracy and the reliability of pipetting processes, accurate sensor systems are required to observe certain parameters of the liquid handling progress. The topic of this diploma thesis focusses on the automated detection of the surface of sample liquids with an air driven pipetting system from Stratec Biomedical Systems. A thermal flow sensor monitors the fluidic characteristics in the pneumatic system of the pipettor, thus it enables the detection of liquid surfaces - liquid level detection (LLD), allows for the identification of malfunctions in the pipetting process and facilitates a quantitative volume evaluation of the handled liquid. To detect the contact of the pipette tip to the assay's surface, the sensor systems in the applied pipetting system "Integrated Pipettor" are based on the change in capacity of a measurement capacitance or the alteration of the pressure ratio inside the pneumatic system. To realise a capacitive method, special conductive tips are required which implicates enormous additional costs. The pressure based method was developed by Stratec Biomedical Systems to avoid the use of these expensive tips. While an open tip touches the surface of a sample liquid, a pressure peak appears which indicates for the LLD. Both of the explained methods could be improved in reproducibility, significance and response time by the integration of the thermal flow sensor. The standard sensor systems allow only for the single detection of a liquid surface with empty pipette tips. However, the here developed method allows for a LLD after the aspiration of even three liquid plugs into a pipette tip. A mechanical vibration generates an oscillation in the aspirated liquid. Further, this oscillation is transfused to the air column at the thermal flow sensor which finally can be detected as an analog sensor signal. By touching the liquid surface, the pipetting tip becomes clogged which leads to a damping of the oscillation in the liquid- air system inside the pipettor. The consequential change in the analog signals enables the indication of a stop criteria for the motion of the pipette robot. Even after the aspiration of three liquid plugs into the tip, the response time of the LLD allows for an immerse depth of the tip into the assay of less than 1mm. Further, the use of the calibrated flow sensor enables a real time volume detection of the motion of the pneumatic pipetting system. By integrating the analogue sensor signals, a volume detection of the pump movement with a deviation of only 2 % could be evaluated. Additionally, the control of very small volumes in the range of only a few nano liters was investigated by the use of the PipeJetTM dispensing system [6]. The air which flows towards a liquid reservoir was measured by the thermal flow sensor after the ejection of single droplets.
Liquid Reagent Storage for the Centrifugal Microfluidic Platform
2008, Jochen Hoffmann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungMEMS based microfluidic platforms represent a promising technology for rapid in-vitro diagnostics. Such systems performing bio-chemical assays on miniaturized cartridges by precisely handling minute amounts of liquids are often referred to as Lab-on-a-Chip systems. Advantages of theses systems are a fast time-to-result at reasonable costs and the potential for largely automated analysis. A frequent shortcoming of current platforms is the necessity to manually introduce liquid reagents before cartridge processing. The solution is to pre-store the liquids, thus enabling fully automated diagnosis, allowing save operation, and reducing human handling errors. Until now, only few approaches have been published in literature. Aim of this thesis is to investigate concepts for storage and release of liquid reagents on centrifugally operated Lab-on-a-Chip systems. Three concepts have been designed and tested. In the first approach, microencapsulated liquid reagents were considered for storage and release. Industrially manufactured paraffin spheres containing water were embedded into reservoirs on a plastic disk. By global heating of the rotating cartridge, the paraffin shells were molten and the enclosed liquid volumes were released. As a result, it was no possible to discharge defined and pure liquid amounts into the fluidic network. A fundamental problem is the low ratio of 1:7 of encapsulated liquid volume and encapsulating material. In the second concept, liquid reagents were directly introduced into cavities on disk. A burstable seal (weak-bonded interface) separates the liquid volume from the rest of the fluidic network. Liquids were released by a centrifugally induced delamination of the sealing foil at the predetermined area. By geometrical variations of the weak-bonded interface, its radial position, and the rotational frequency, release times have been adjusted between 31 and 321 seconds showing CVs between 1.9 and 68.8 %. Liquid volumes of 69 μL from a 100 μL prestored volume were released with a CV of 5.7 %. Shortcomings are the vapour transmission rate and the insufficient chemical inertness of the used sealing foil. For the third approach, liquids were encapsulated within glass tubes. Liquid containing tubes were enclosed by fusing or by a wax plug. Release concepts are shivering of the glass walls or melting of the wax plug. The released liquid content, excluding glass shivers, was transferred into the fluidic periphery by the centrifugal force. The concept was characterized for the following parameters: mean crushing force (9.0 N, CV = 32.0 %), glass deformation at the breaking point (102 μm, CV = 14 %), released content (98.7 μL of 100 μL, CV = 2.5 %), and vapour transmission rate (< 0.1 % liquid loss after 72 days). A disadvantage of this concept in the need for an external energy source required for liquid release. The applicability and usability of the last approach was demonstrated in two applications. First application: Required buffers for a DNA extraction on disk were encapsulated within glass, embedded into a microfluidic cartridge, and sequentially released by manual crushing. Reagents could be successfully released and transferred into the fluidic system. A DNA yield of only 4.1 % was caused by insufficient operation of the fluidic structure. Second application: Glass encapsulated reagents were incorporated into the fluidic cartridge for Isothermal Amplification of DNA. Real-time testing of the structures with pre-stored reagents has revealed no difference in the amplification performance compared to the “classically” tested structures, proving the suitability of the used method for pre-storage.
Modelling a Laccase-catalysed Biofuel Cell Cathode with experimental Quantification of Key-Parameters
2008, Johannes Kestel
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Markus Biesalski, Chair of Molecular Biology, TU Darmstadt
KurzfassungThe protein Laccase from the white-rod fungus Trametes versicolor is a powerful biocatalyst that is able to enhance the four electron reaction of molecular oxygen and four protons to water. This catalytical ability can be used to set up a biofuel cell cathode with an open circuit voltage of 580 mV to a saturated calomel reference electrode. When laccase is adsorbed on graphite, it is capable of taking up electrons for the reaction by direct electron transfer from the electrode. This allows a setup that is not dependent on the long-term stability of a mediator. The main challenge for such a biofuel cell cathode is the long-term stability of the biocatalyst laccase. The state of the art solution to meet this problem is to immobilise laccase on the electrode in order to stabilise the tertiary structure of the molecules. Here the demands on a novel system for achieving unlimited stability of a direct electron transfer laccase catalysed cathode are investigated. The key parameters that determine the long-time stability are the time dependent inactivation and the exchange of adsorbed laccase on the electrode. For the measurement of these parameters a measured setup that excludes diffusion limitations on adsorption and desorption was developed. The laccase preparation in use was characterised by a SDS-Polyacrylamide gel electrophoresis. The main of the four detected fractions has a molecular mass of 70.7 ± 4.2 kDa. The other fractions have a negligible amount. The amount of proteins in the preparation was determined by photo-spectroscopy to 37.39 ± 0.0031% of the weighted mass. The exchange on the electrode can be described by a time constant τ in which 63% of the adsorbed molecules are exchanged. This constant was calculated to τ = 105.69 ± 2.45 s from the desorption constant that was found by a two parameter fit to the measured Langmuir adsorption kinetic. For the adsorption- and desorption constant values of 0.094 ± 0.00015 ml/s/mg and 0.0065 ±0.00022 1/s were found. The Equilibrium coverage of the molecules in the lowest layer that contribute to the current was determined to 40.88%. This shows that the main principle for an unlimited operation of the cathode is possible: The adsorbed laccase can be substituted before it is inactivated. Based on the experiments a model in Matlab/Simulink was set up that can be used to calculate the ratio of active to inactive laccase molecules on the electrode as a measure for the long-term stability and performance of a self regenerative cathode. The model was validated by measured data. Based on this model a design rule for a self-regenerative biofuel cell cathode was given for the necessary generation and consumption rates in dependency of the demanded ratio of active to inactive molecules on the electrode and the number of adsorption sites.
Oxygen Depletion in Abiotically Catalyzed Glucose Fuel Cells
2008, Bruno Biller
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungAdvances in microelectronics and MEMS technology have led to a significant decrease of power requirement in medical implants, leading to a new interest in self-sustaining power sources. One technology that allows to supply low power implants, in close proximity or even on the surface of an implant, is the abiotically catalyzed glucose fuel cell. There an electrochemical reaction of the endogenous substances glucose and oxygen releases electrical energy. Simultaneous availability of glucose and oxygen poses a big challenge on abiotically catalyzed glucose fuel cells. This is due to the fact that there are no catalysts available that promote glucose oxidation in the presence of oxygen, leading to a decrease in overall cell voltage. To overcome this challenge, reactant separation has to be effectuated. A strategy reported in literature is to mount the cathode in front of the anode, so that oxygen is consumed in the cathodic reaction, resulting in a depletion of the oxygen concentration at the anode. The aim of this thesis is to introduce a method to quantitatively investigate the oxygen management in abiotically catalyzed glucose fuel cells for the first time. Therefore the two electrodes of an abiotically catalyzed glucose fuel cell are examined separately. On one hand, influences of oxygen depletion towards the anode potential are investigated, by exposing the electrodes to different oxygen saturations. On the other hand oxygen depletion itself is analyzed, which is induced by the cathodic reaction, thus the oxygen saturation is measured in the bulk liquid and behind the cathode. For each of these two tasks a setup is constructed, which allows to simulate physiological conditions. Implemented materials are carefully chosen, giving the possibility of autoclaving, thus ensuring a sterile working environment. Together they form a powerful tool to characterize the oxygen management in glucose fuel cells. Suitable working conditions within the physiological range ( ~ 4.5…7 % oxygen-saturation; ~ 3…5 mmol/l glucose concentration) are determined to allow for an efficient operating state of the art platinum-zinc alloy anodes. It is shown that glucose concentration has a strong effect on the stability of anode potentials, when they are exposed to oxygen. The influence of increasing oxygen saturations can be kept within reasonable limits at glucose concentrations not below 2.5 mmol/l. Platinum cathodes are investigated with respect to their applicability to reduce oxygen concentrations. Oxygen saturations behind the cathode were observed to be depleted up to 40 % and 55 % compared to oxygen saturations in the bulk liquid of 3.5 % and 7 %, respectively.

2007

Biocompatible Sealing of Microfluidic Substrates
2007, Carolin Henze
KurzfassungThe sealing of microfluidic chips is an integral part in the fabrication of lab-on-a-chip systems. Within this diploma thesis, two different sealing technologies, namely the temperature diffusion bonding and the adhesive bonding, are established and optimized with respect to functionality and biocompatibility. The microfluidic chip consists of cyclic olefin copolymer (COC) and features pre-stored bioreagents for the amplification reaction of HPV-mRNA which is read out by fluorometry. Accordingly, suitable sealing materials are selected and implemented in the processes, i.e. a cyclic olefin polymer (COP) as cover foil material and an epoxy resin for the adhesive bonding, both featuring USP class VI approval, high transparency and low autofluorescence. For the temperature diffusion bonding, the bioreagents which are pre-stored in a channel of 200 μm depth are exposed to a temperature of 61.1°C which is even below the acceptable temperature of 65°C. However, the process exhibits incompatibility to the hydrophilic coating and a time-consuming fabrication of the applied compound foil. In the adhesive bonding process, the preparation of the bonding materials is time-efficient and the process is compatible with the hydrophilic coating. Further, as the epoxy resin is transferred by a roll system as a thin layer (~ 5 μm) onto the chip surface instead of the cover foil, channel contaminations are prevented. With the evaluated parameters for both sealing processes, a high bond strength of 2.9 bar for the temperature diffusion bonding and 3.9 bar for the adhesive bonding for a contact surface of only 500 μm between channels is achieved. To conclude, the adhesive bonding is selected as suitable sealing method for the amplification chip since it fully complies with the boundary conditions and features some advantages over the temperature diffusion bonding. Additionally, concepts for the parallelization of both sealing technologies are presented which enable a medium-size batch production of the sealed microfluidic chips.
Design und Fabrikation der Pikoinjektor Dosiereinheit
2007, Olivia Brett
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungThe development of chemical stimulation of single cells gains more importance in the field of microsystem technology due to the rapid progression in pharmacology and fundamental biological research. This work focuses on the development of a dosage unit for the chemical stimulation of in-vitro cells which is the prime component of a novel cell stimulation tool (picoinjector). It enables the precise dispensing of sample volumes in the range of V < 10pl with a high spatial and temporal resolution and thus allows for the stimulation of single cells. The contact-free on-demand sample delivery is based on the bubble-jet principle derived from the ink-jet technology. The manufactured and characterized dosage units feature a nozzle diameter of dD = 10μm and dD = 20μm and dispense a droplet volume of VT = 5.9pl and VT = 9.0pl with a very good reproducibility of CV < 9% in the respective volume range. Further, an actuation frequency of up to νmax = 11kHz is achieved thus allowing for the synchronization with the switching times of e.g. neuronal cells which are in the range of ν = 1-10 kHz. Furthermore, the dosage unit features a very long lifetime of up to 2 million dispensing cycles. The characterization is conducted with ink as well as water-based buffer solutions used for cell stimulation (e.g., 10 mM adenosine triphosphate in phosphate buffered saline). The dosage unit can dispense two chemical samples in serial as well as parallel operation. Different designs (priming as well process optimized) are evaluated in a thorough concept study and are validated with computational fluid dynamics (CFD) leading to these exceptional results. Here, the experiments are in very good agreement with the simulation data (VT = 8,7pl @ dD = 20μm; νmax = 11,2kHz). Further, a minimal nozzle diameter of dD = 7 μm for the dimensioning of the fluidic structures is determined with the use of an analytical model. Based on these designs, a suitable fabrication process is developed. The metal of the heater structures for the thermal actuation is deposited and subsequently structured via silicon micromachining. A robust operation is enabled by heaters made of titanium with a layer thickness of dTi = 100 nm, featuring highly reproducible properties (RHeater = 11 ± 0,5Ω). As a transparent material is required for the fluidic structures, a new fabrication process is developed based on SU-8 photoresist. The best results for sealing of the fluidic structures are achieved with a SU-8/SU-8 bond which is evaluated also in regard to the optimal process parameters by pull tests. With the use of these results as well as an optical quality control by scanning electron microscopy (SEM), an optimal bonding force of F = 0,5kN and bonding temperature of T = 125°C-150°C are determined. With these parameters, a very high bond strength of BS = 50MPa is achieved.
Entwicklung eines fluidischen Mikrochips zur Zellseparation
2007, Thorsten Borchardt
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Alexander Rohrbach, IMTEK
KurzfassungA field of enormous importance in microsystem technology is dedicated to the development of portable laboratories, so called lab-on-a-chip systems. These systems provide cheap, portable and miniaturized solutions, which are of great relevance in the field of chemistry, biology, analysis and medical technology. In this work a low-cost chip for continuous separation of blood cells, i.e. erythrocytes and leukocytes, is presented. The cell separation relies on a complete passive, hydrodynamic principle, where the first part focuses cells and aligns them to the wall. Due to the focusing unit the center of masses follow different stream lines according to the shape and size of the particles. The subsequent branching of the channel network is used to separate two different types of cells. The size of the cells that can be separated is determined by the volume flow rate into each outlet and is thus directly related to the fluidic resistances in the channel network. In the design presented in this work, the chip allows separating cells between 2-5 μm from larger cells depending on the position of the punch holes (fluidic connection) and is thus especially suited for the range of human blood cells. Different designs with characteristic channel dimensions of 30 μm and 50 μm have been characterized and evaluated to find the optimum working range. For the best design investigated in this work a separation efficiency of ~87 % was achieved.
Flexible Hydrophobe Diffusionsbarriere für Drop-on-Demand Dispenser
2007, Monika S. Straßer
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Holger Reinecke, IMTEK
KurzfassungThe chemical stimulation of individual cells in physiological (liquid) environments with a high temporal and spatial resolution is of increasing impact in the field of fundamental biological research as well as pharmaceutical applications. This thesis is to present a new flexible and easy to handle approach for the transfer of pl-droplets into a liquid environment next to any individual cell by avoiding any leakage by diffusion. A disposable, self-aligning hydrophobic cap from polydimethylsiloxane (PDMS) that can be easily attached to any drop-on-demand dispenser was developed. Inserting the modular system into liquids traps the air inside the cap and forms two stable menisci at the nozzle and the outlet. This way, the phase-gap is created and works as a reversible burst valve that prevents any leakage by diffusion. Only droplets ejected with high kinetic energy are able to overcome the surface tension and pass the phase-gap. In this thesis two different setups e.g. for dispensing 500 pl with high spatial resolution (50 μm, ~ 200 ms) and alternatively with high temporal resolution (~ 100 μm, << 1 ms) are presented. First, the drug could be directly injected into the liquid environment making the defined initial drug concentration instantaneously available in a time frame t0 << 1 ms (limited frame rate of high speed camera). In contrast, the drug release could be spatially focused correlating the design of the phase-gap to enhance the spatial resolution. This way, the drug inside the droplet with an initial diameter of d = 100 μm is spatially focused to the smaller outlet (w = 50 μm). The volume stability of the phase-gap was characterized with the piezo-stack actuated NanoJet dispenser featuring a large volume range (V = 0.07 nl - 1.2 nl) with a very high linearity (R2 = 0.99) and an excellent reproducibility of CV < 3%. When the dispenser is operated, the injection rate is limited in order to avoid flooding of the phase-gap. We achieved a maximum volume injection rate of up to 137 nl/s at a resolution of 250 pl at 475 Hz which is the limit of our driving electronics (typical injection rates in applications are well below 0.1 nl/s). Furthermore, the maximum dispensable volume could be increased up to 70 % by implementing a venting channel. By using the fixed volume pico-injector dispenser (V = 11 pl, CV < 9 %) the release of two fluids at the same time through the phase-gap was successfully shown. To ensure a flexible, low-cost fabrication process, a silicon master featuring the geometry of the dispenser and the phase-gap was fabricated via dry etching, fixed in a drilled aluminum mold, casted with PDMS (Sylgard 184) and cured by applying a force of F = 95 N. Outlet widths down to w = 20 μm and array structures with outlet width w = 50 μm and a pitch of 100 μm were realized. We have proven that the “phase-gap“ exhibits an excellent long-term stability of more than 30 hours against capillary priming.
Integrated DNA Extraction on a Centrifugal Microfluidic Platform
2007, Sarah Pausch
Erstgutachter: PD Dr. rer. nat. Jens Ducrée, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungAn essential objective in nucleic acid based diagnostics is an integrable and reliable DNA extraction method from a sample of whole blood. Such a method enables the implementation and automation of the full process chain, starting from the withdrawal of a patient’s blood to an analytical result. This work systematically develops a fundamental understanding of DNA extraction on a centrifugal microfluidic platform. Therefore, a market research on silica particles as solid phase for the integrated DNA extraction is conducted. In proof-of-principle experiments three different silica particle types are tested in combination with extraction buffers of two commercially available extraction kits. These off-disk experiments lead to a binding capacity of 0.02 μg/mg for the combined particle- buffer system and of 0.2 μg/mg for the particles and buffers originating from the same kit supplier. Different microfluidic channel layouts for the aggregation of the silica particles in miniaturized columns are designed and fabricated. A well defined amount of solid phase is an important prerequisite for reproducible DNA extraction results which leads to the development and testing of two particle injection protocols. The amount of particles inside the column can be controlled by injecting them within a suspension but big pin-holes and less dense particle packaging are observed. A denser packed column can be achieved by injecting the particles in the dry state. This method of choice is evaluated regarding the centrifugal flow rates through different sized particle columns over the frequency of rotation. Although there are fluctuations towards higher flow rates, they are comparable in the important range for the proposed DNA extraction of < 2 μl/sec. The downscaling of the extraction assay towards an integrated nucleic acid extraction system is accomplished in a step-wise approach. Firstly, only the binding step is realized within the extraction chamber featuring the silica particle column while all subsequent process steps are still processed off-disk. The amount of DNA that binds onto the silica particles is eluted externally and is comparable to the amount eluted in off-disk experiments proving the binding under quasi-static flow conditions. These experiments show no dependency of the amount of bound DNA from the flow rate within the chosen range of < 2 μl/sec. Finally, the complete extraction protocol is run fully integrated within the centrifugal microfluidic structure. The characteristic extraction curves are measured for two systems, featuring a 16% yield and 0.71 μg maximum binding capacity for the commercially available system and a 7.4% yield and 0.12 μg maximum binding capacity for the combined particle-buffer system, respectively. Additionally, the possibility to spatially separate the extracted DNA within the elution buffer from the waste liquids via a novel microfluidic droplet switch is demonstrated during the on-disk extractions. Potential error mechanisms in the assay protocol, e.g. inhomogeneous flow conditions, non-optimal washing procedures and DNA affinity to the polymer channels are identified and will fundamentally support future work.
Rotationally Enhanced Mixing, Dissolving and Valving for Automated Immunoassay Diagnostics
2007, Philipp Lang
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
KurzfassungThe crucial factor for the success of lab-on-a-chip technologies is the ability to miniaturize, parallelize, and automate complex assay protocols on a disposable cartridge. This master thesis presents the technical achievements towards an integrated immunoassay platform technology for multi-analyte point-of-care diagnostics. This platform is based on a centrifugal microfluidic cartridge for competitive immunoassays within an backend laboratory device of the ZentriLab joint research project. Accordingly to immunoassay protocols, the fluidic structure on the cartridge is subdivided into individual fluidic unit operations which are divided into multiple modules. These unit operations are based on reproducibly known microfluidic systems and are improved for the use of biological reagents. The fluidic unit operation mixing is extended by dissolving of prestored reagents. The dissolution time is reduced by a alternating spinning procedure which is called shake-mode. The best performance of dissolution for an overall volume of 50 μl is reached by: optimized aspect ratio of 0.26 (depth/width) for the chamber, dissolving temperature of 30°C, and alternating spinning acceleration of 32 Hz/s. This efficient method allows a 16-fold acceleration of the dissolution for a lyophilizated bead [P1]. Resulting from this experiments the unit operations of incubation and washing are also accelerated by a shake-mode induced stirring with 20 Hz/s acceleration. New concepts of geometrical barriers are developed to avoid bubble formation in a fluidic chamber which are generated due to the properties of biological fluids. These barriers are designed with protruding edges of the side wall combined with a recess in the top of the chamber. Therefore, it is possible now to realize a readout of the chemilumenescence signal at rest or during marginal rotation. Biological requirements of the assay impose a challenge for the unit operation switching. Hydrophilic surfaces and fluids with solved tensides and salts make it difficult to realize a robust valving which also withstands shake-mode. In this work it is shown that only combinations of siphon valving with capillary valving or hydrophobic valving enable a robust and reproducible switching. Two combinations of siphons are developed, a single use capillary siphon with an hydrophobic patch and a reusable volume triggered siphon with a capillary valve. These knowledges of unit operations for biological applications are used to assemble a complete microfluidic system which uses the benefits of a centrifugal system to produce a disposable cost-efficient cartridge for competitive immunoassays.

2006

Developing a Sensor to detect free flying droplets
2006, Andreas Ernst
Erstgutachter: Prof. Dr. Bernhard Vondenbusch
KurzfassungDosing liquids in very small amouts plays an important role in many different kinds of industries. For example in the chemical, biological and pharmaceutical industry the importance of the application of highly precise dosage systems increases enourmously. However in the classical field of mechanical engineering small dosands are of interest. Due to the constantly research more and more newly designed dispensing systems are placed on the market. According to the small size of the dosand, which lies often in the nanoliter range, controlling the dispensed media is very difficult and thus associated with high costs. The currently used sensor systems are very comprehensive and increase the costs of a dispensing system to a multiple. Furthermore the most of the available systems process their measurement in contact with the dispensed droplet, which leads to a contamination of the liquid. Also a direct in time measurement is often impossible. The problems of detecting small amounts of liquid are handled in this work. The sensoric concept handles especially the dispensed media and is focused on the dosing process. Particularly a low cost method should be developed wich provides an in time measurement to detect, in the best case, the volume of the dispensed liquid. A non-contact working mode is required, which means that the droplets don´t get in contact with the sensor unit in order to avoid contamination. Due to the diversity of designs and systems a concept should be acquired which can be made compatible to any non-contact dispensing system. To avoid contamination and evaporation the installation size shall be as small as possible keeping the distance between the dispensing nozzle and the substrate small. Two principle ideas were given by the IMTEK, which were invented before this work. These are: • using the beahviour of a light beam • exploiting the characteristics of an electric field Beside the development of the theoretical concepts, first experiments should be done. Based on the found conclusions, suggestions of improvements and further developments shall be given for the treated sensor units.
Development And Characterization Of A PDMS Microvalve
2006, Ramasubramanian K.Jaganatharaja
KurzfassungThis thesis is to present a novel hydraulic actuation mechanism for modular microvalves based on hydraulically amplified deflection of a piezostack actuator using a solid silicone elastomer (PDMS) as a hydraulic fluid. Reducing the flow resistance with a conical nozzle shaped hydraulic chamber, the challenge of replacing conventional hydraulic liquids with a solid silicone elastomer has been successfully overcome and proved to work. The whole setup is easy to fabricate by molding of low-cost polymers like epoxy-resin and PDMS. The maximum deflection of 9 µm of a piezostack actuator was amplified to 31.6 µm using the presented hydraulic actuator setup. Outstanding features of this new device are: high actuation frequencies up to 500 Hz with a shortest response time of 1 ms, an excellent volume efficiency of roughly 85 %, combined with an extraordinary reproducibility of CV = 2%. For larger response times of 50 - 60 ms the volume efficiency was found to increase up to 92%. The hydraulic actuator is able to hold the amplified deflected state for even 1000 s and has been deflected for more than 6.5 million cycles without any degradation or drift in the amplification (CV = 1%). A second actuator with similar design was constructed and was found to reproduce the same result. The amplification of the deflection is proportional to the cross-sectional area ratio between the piezostack actuator and the conical nozzle exit of the hydraulic chamber. In a systematic study, different hydraulic actuator configurations having different area ratios of 4, 8 and 16 were constructed and characterized. The hydraulic actuator with an area-ratio of 4, which was discussed above, achieved a maximum amplified deflection of 31.6 µm with an amplification factor of 3.6 and a volume efficiency of roughly 86 %. The hydraulic actuator with an area ratio of 8 had a maximum amplified deflection of 59.1 µm with an amplification factor of 6.5 times and a volume efficiency of roughly 82 % with a CV less than 2 % in terms of deflection. The hydraulic actuator with an area ratio of 16 obtained a maximum deflection of 75.5 µm with an amplification factor of 8 and volume efficiency of about 52%. A linear degradation of the volume efficiency with increasing area ratio was observed. A conceptual design for a modular microvalve based on the novel actuation mechanism by hydraulically amplified piezo deflection using solid silicone elastomer (PDMS) is proposed. The design comprises of independent modules of different functionalities which offers the advantages of using disposable fluidic components and easy replacement of modules upon misfunctions. The amplified deflection from the modified micro hydraulic chamber was characterized using white light interferometry. Few design modifications to avoid the stray protrusion of the silicone elastomer have been suggested.
Development of a Test System to Prove Neuronal Activity of Stimulated Cells
2006, Ronny Pfeifer
Erstgutachter: Prof. Dr. Karin Mittmann
Zweitgutachter: Prof. Dr. Roland Zengerle, IMTEK
KurzfassungThe selective stimulation, as well as the functional inhibition of certain areas of the brain establishes broad fields for diagnosis, therapy and for the scientific understanding of the cerebral system and associated diseases. The aim of the European “Neuroprobes” project is the development of a platform, based on “multielectrode arrays” (MEAs), for selective stimulation of certain brain areas, with the simultaneous recording of the produced electrical and chemical signals. On the basis of this diploma, an adequate test system, for the microfluidic platform of this system, which is developed at IMTEK, is to be established. This system should allow a characterization as well as an examination of the functional capability of the needles of the MEA. The first part of this work introduces the PC12 cell line as a neuronal model system. PC12 cells in particular, are commonly appointed in the neuroscience research. In terms of reliable neuronal stimulation, the effect of various neurotransmitters (ATP, Bradykinin, Carbachol and Glutamate) on PC12 cells is tested. It is shown that, through stimulation by ATP, the highest signaling intensity as well as a response of almost 100% of the cells can be reached. For further characterisation of the test system, on the one hand, the concentration dependent reaction of the PC12 cells to ATP in the range of 0,1 - 500 μM is shown, on the other hand the possible inhibition through an ATP receptor antagonist in the range of 10 - 1000 μM. For evidence of the neuronal activity the high sensitive “Ca2+- Imaging” method is used, whereas for evaluation the free accessible software “ImageJ”, which is based on Java, is used as an alternative for expensive commercial software. In the second part of this work, using the cost-effective as well as highly flexible “Microcontact Printing”, protein patterns are generated through microstructuring, which allow the culturing of PC12 cells in a structured way. In doing so, on the one hand a defined arrangement of the cells is possible, on the other hand a approach is created, that allows to study the spacial- as well as the temporal neuronal signal dispersion in a defined system. After characterization of the printing process with the help of fluorescence labeled antibodies, extracellular matrix (ECM) protein is used for the defined adjustment of the cells. First experiments show the potential of the above mentioned systems for future applications.
Development of an In-Vitro Biocompatibility Test for Materials with Respect to its Applicability for the Implantable Direct Glucose Fuel Cell Elisabeth
2006, Elisabeth Rosen
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Dr. Michael Grahn, Queen Mary University, London
KurzfassungAn essential question that needs to be answered for the increasing number of biomedical MEMS implants prior to contact with the human body is weather they are biocompatible. In this thesis an adequate testing procedure for both, the Direct Glucose Fuel Cell (DGFC) in operation and its materials, was implemented. Due to the complex assembly of the DGFC and its interaction with the biological environment in form of redox reactions, special considerations in selection and design of an adequate test are required. First, the state-of-the-art possibilities for testing biocompatibility in-vitro were reviewed. Afterwards the innate toxicity of the materials was determined. For this, the metabolism of 3T3 mouse fibroblasts in direct (DC) and indirect contact (IC) with material samples was assayed with alamarBlue™. In these test protocols, materials based on activated carbon absorbed this dye completely. Therefore, the need to design a novel and appropriate testing protocol, capable of investigating the cytotoxicity of activated carbon and complex devices including such highly porous materials, occurred. A newly developed Indirect Contact Test with Regularly Exchanged Culture Media (ICE) successfully solves the problem of dye absorption. Furthermore this test method allows combination of two different evaluation methods, the alamarBlue™ and the Lactated Dehydrogenase (LDH) assay. Application of the DC, IC, and ICE cytotoxicity tests revealed, that formaldehyde crosslinked hydrogels are less toxic than thermally crosslinked hydrogels. It was also shown, that platinum and the electrodes, as essential components of the DGFC, showed only slightly cytotoxic potential. It is suggested to apply the Direct Contact Test (DC) for testing of materials with tissue contact, and the newly developed test (ICE) for testing of the DGFC in operation. For materials not in contact with tissue, an elution assay may easier to perform than the Indirect Contact Test (IC).
Dynamics of Electrowetting – Compact Modeling
2006, Lucas Frenz
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Dr. Andreas Greiner
KurzfassungThis thesis presents a systematic analytical investigation on electrowetting on dielectrics (EWOD). The fundamental processes of electrowetting (moving, splitting and merging of fluid droplets) are described analytically. In particular, it is shown that a second order differential equation, balancing viscous, inertial and capillary forces, is adequate to describe the equation of motion. This leads to a spring-massdamper- analogy, which in turn is used to derive dynamic models for electrowetting. These analytical models are suitable in order to analyze the characteristic process times of electrowetting devices, resulting for example in a linear dependency on the viscosity, a strong (quadratic) dependency on the pad width and a reciprocal dependency on the surface tension. Parameter studies with CFD-simulations confirm these findings. The analytical models for the single processes have been incorporated into a general simulation framework formulated in the hardware description language VHDLAMS. In contrast to other simulation techniques it is possible to simulate a long sequence of processes with this compact model, as it is necessary for complex analysis and synthesis protocols. Therefore, these models allow one to describe the electrowetting devices adequately, as it is essential for an efficient designing process. Finally, an experimental setup is presented that can be used to study the behavior of electrowetting devices. Additionally, guidelines are proposed of how to achieve reliably working devices.
Herstellung von Biopolymer-Mikrosphären auf einer zentrifugalen mikrofluidischen Plattform
2006, Lars O. Nägele
Erstgutachter: Prof. Dr. Hermann Sandmeier, Uni Stuttgart
Zweitgutachter: PD Dr. Jens Ducrée, IMTEK
Kurzfassung
Herstellung von Biopolymer-Mikrosphären auf einer zentrifugalen mikrofluidischen Plattform
2006, Lars O. Nägele
Erstgutachter: Prof. Dr. Hermann Sandmeier, Uni Stuttgart
Zweitgutachter: PD Dr. Jens Ducrée, IMTEK
Simulation of Drug Delivery through Nanoporous Membranes
2006, Thomas Wielath
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Dr. Andreas Greiner, IMTEK
KurzfassungThis thesis presents a fast simulation model which was created and used to understand the transport mechanisms of small amounts of solved drugs (fl - pl range) through a nanoporous membrane, to receive temporally and spacially resolved information about the concentration of a drug in a liquid on the membrane and to determine the dependency of the geometric membrane parameters on the concentration profile in the liquid layer. The presented model is benchmarked with an analytical model and a commercially available simulation tool (CFDRC). In comparison to the CFD model the simulation time is reduced by one third by separate calculation of the convectional and diffusional fluid transport. The resulting routine is a multipurpose tool with an user friendly interface which is used to reconstruct experiments to receive exact data about the conditions of the fluids (e.g. maximum concentration, diffusion radius), it is used to find unknown parameters of an experiment by fitting the simulation results to experimental data (e.g. diffusion constant) and it can be used as layout tool to find a membrane for the application which provides a concentration profile that meets the demands. Limiting values for the influence of the membrane are found as well as for the maximum concentration and the spacial and temporal resolution. A membrane will not influence the delivery of a drug over a specific but unexpected low porosity which is depending on other parameters, too. The maximum reachable concentration is governed by the transport of the drug through the membrane. Although, diffusive and convectional transport through the membrane decreases linear with increasing membrane thickness, the maximum achievable concentration shows exponential decreasing behavior what means convergence to a defined value. As well the size of an area within a concentration higher than a threshold value has emerged to obtain a nonintuitive developing. The largest area can be affected by slow release of a drug through a membrane so that the concentration will propagate on a relative low level. This new information are used to plan experiments and design custom-made membranes which meet the demands to adjust concentration profiles. To adapt the membrane parameters, a process for the fabrication of custom made nanoporous membranes is shown. Furthermore, a concept for the experimental evalutation of the simulation results is presented.
TopSpot E-Vision: Optical Quality Control for TopSpot E
2006, Nicolai Wangler
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Hermann Sandmeier, Uni Stuttgart
KurzfassungIn the last years the microarray technology has revolutionised the fields of biotechnology and drug discovery. Due to its high parallelity of different analyses can be accomplished in one single experiment to generate tremendous mounts of data. Microarray technology has paved the way in decoding the human genome and alternative therapeutic methods can be developed with pharmacogenomic approaches as well using this technology. With the growing diversification of microarray applications low cost technologies for the production of microarrays become more and more important. One commercially available microarrayer is the TopSpot E from BioFluidiX [15], a semi-automated entry level arrayer for small lot manufacturing in research institutes and laboratories. The TopSpot technology uses a pressure pulse generated by a piezo driven actuator to dispense an array of free flying droplets in a highly parallel manner. The quality of the printed array strongly depends on the liquid as well as the driving parameters of the piezo so that a parameter optimization is necessary for unknown sample liquids. The aim of this work is to broaden the field of applications for the TopSpot E microarrayer by integrating an optical quality control system into the current device to monitor and optimise the microarray fabrication process. Due to the continuous observation of the printing process a high quality standard can be assured and appropriate print parameters can be determined for unknown sample solutions easily. The presented thesis describes a cost-effective implementation of an optical quality control system into the TopSpot E device. The implementation of which had to be realised without compromising on the design and functionality of the TopSpot E microarrayer in its present form. To achieve this a 1.3 mega-pixel camera was used to capture each printed array and a high performance CCD-camera was applied for imaging the flying droplets during the printing process. In addition to the assessment of methods applicable for imaging of droplets in flight and on microarray substrates, also a design for the dedicated optical quality control system was developed for TopSpot E. Finally first results obtained with the prototype disposing of fully integrated cameras are presented. These successfully tested optical qualtiy control features will open up new fields of applications for the improved TopSpot E microarrayer - termed TopSpot E-Vision.

2005

Development of a Manufacturing Method for Direct Glucose Fuel Cells Incorporating Different Polyelectrolytes
2005, Natsuki Miyakawa
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jürgen Rühe, IMTEK
KurzfassungThe feasibility of several different polymer crosslinking methods was investigated to produce poly(vinyl alcohol) (PVA)-based hydrogels (i.e. very hydrophilic polymer networks) incorporating different polyelectrolytes (PELs) for the applications in implantable direct glucose fuel cells (DGFCs): physical crosslinking methods such as freeze-thaw and annealing, and chemical crosslinking methods including esterification, crosslinking by heating, UV-, proton beam-, X-ray-, -ray-, and electron beam irradiation, etherification by dibromoethane vapor, acetalization by aldehydes like glutaraldehyde and formaldehyde. Crosslinking of different polymer blends made from PVA and different PELs using formaldehyde in a salt solution resulted in hydrogels of acetalized PVA with entrapped PELs, so-called semi-interpenetrating polymer networks (sIPNs). This method allowed for the incorporation of different PELs in the hydrogels. Moreover, the method was found to be favorable because of its simple setup and process, low crosslinking temperature, possibility to crosslink wet samples, potential biocompatibility, good mechanical properties of the resulting polymer networks. A simple and reproducible method to produce homogeneous polymer blend films with desired thickness was developed. Using this method, PVA-based polymer blend films containing strongly anionic PEL poly(sodium 4-styrenesulfate) (PSSNa), weakly anionic PEL poly(acrylic acid) (PAA), weakly cationic PEL poly(ethyleneimine) (PEI), and strongly cationic PEL poly(diallyldimethyl ammonium chloride) (PDDMACl) were fabricated. They were crosslinked to form sIPN-based hydrogels, and their properties such as swelling ratio, gel fraction, tensile strength, and ion exchange capacity (IEC) were investigated. Based on the established polymer crosslinking method, a method to assemble DGFCs was developed. Using this method three DGFCs were fabricated, and their performance was characterized by measuring the cell voltage under galvanostatic load.
Development of a peristaltic bubble jet pump
2005, Thomas Schillak
Dichtemessung mit dem Vibrationsgrenzschalter Liquiphant M
2005, Stanislav Herwik
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Leonhard Reindl, IMTEK
KurzfassungLiquiphant M is a universal limit switch for liquids that is used for level detection in tanks and pipes. The resonance frequency of Liquiphant M is sensitive to the density of surrounding liquid. This property makes it suitable to act as density transmitter. The objective of this work is to investigate and compensate the disturbances that occur during the density measurement with the Liquiphant. The occurring disturbances maybe classified in two categories. On the one hand there are irreversible disturbances, which cannot be compensated. The ageing of the sensor is such a disturbance, it cause a permanent shift of the resonance frequency and reduce thus the accuracy of the density measurement. The resonance frequency can be stabilized by artificial ageing. An other unexpected irreversible disturbance are sporadic hops of the resonance frequency during temperature change. This occurs just with bimorph-drivers, therefore for the density measurement just stack-drivers can be used. The other category are reversible disturbances, that can be compensated in different manner. For Density measurement the temperature, pressure and viscosity are significant disturbances. Temperature and pressure may be compensated by calibration of sensor. The temperature hysteresis can be eliminated just by keeping the temperature nearly constant. The compensation of viscosity efforts changes on the electronic circuit. The frequency measurement in the circuit is also disturbed by the ambient temperature, so this temperature range is to be constricted. After compensation of all reversible disturbances, the density of liquids in the range 0,5g/cm³ to 1,2g/cm³ can be measured with an accuracy about 0,3%.
Hemoglobin Determination and Processing of Microarray Experiments on a Centrifugal Microfluidic Platform
2005, Markus Dube
KurzfassungThis diploma thesis focuses on two novel assay formats for the Bio-Disk as centrifugal microfluidic platform. The first assay format measures human hemoglobin and is based on optical absorption as detection principle. To this end, a toxic-free and environmental friendly assay kit with sodium lauryl sulfate as reagent is used. Light is guided by total internal reflection at a V-groove into a reaction chamber and attenuated depending on the initial concentration of hemoglobin. At a second V-groove, the light is then deflected towards a detector. Advantages of this method are the integration in the existing readout concept of the Bio-Disk, as well as a good linearity of R2 = 0.993 and a reproducibility CV of 2.9%. Additional benefits are a small sample volume (2 μL) and a short time-to-result of 100 s. The second assay format combines the benefits of the microarray technology with the strengths of the centrifugal microfluidic platform. In contrast to common methods, the microarray is not printed onto slides, but into a reaction cavity of a polymer disk which follows the format of a standard compact disk. This way, error prone handling steps are reduced, and continuous control of the filling of the reaction cavity is achieved. Additionally, the sample volume is cut to 50 μL, and a time-to-result of only 45 min is accomplished. The competitiveness of this approach was demonstrated with a three step BSA fluorescence immunoassay. A good coefficient of variation of less than 20% and a good linearity of R2 = 0.993 is achieved. For the readout of the assay, a cost-efficient and simple optical concept is developed, thus allowing a further miniaturization of a corresponding reader. The two novel assay formats prove the flexibility of the Bio-Disk, since an easy integration of the hemoglobin detection in the previously developed concept is possible. The microarrays-on-disk with an automated assay processing and readout opens up new application fields of point-of-care and personalized diagnostics.
High Performance Processing of Microarrays on Centrifugal Platforms
2005, Sascha Lutz
Integrated Sample Metering for Immunoassays on a Lab-on-a-Disk
2005, Joachim Harter
Erstgutachter: PD Dr. rer. nat. Jens Ducrée
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
KurzfassungAn important objective in medical diagnostics is the integration of the full process chain from the preparation of a patient’s whole blood to an analytical result. Rotating disks are a promising approach for liquid handling due to the intrinsic pumping, valving and particle separation by the interplay of the centrifugal force field and capillary action. Aliquoting of processed samples to defined volumes for subsequent detection is a crucial step in highly automated and parallelized assay protocols. This work presents microfluidic networks for on-disk metering and aliquoting of plasma samples using the concept of hydrophobic valving. To facilitate automatization of parallel processes, tolerances resulting from all steps of the technology chain are systematically investigated and minimized. Soft embossing from a silicone rubber (PDMS) mold generated from a photolithographically structured multilayer structure (SU-8 photoresist) is used to precisely replicate channels onto polymer disks (COC). The disk is coated with hydrophilic and hydrophobic polymers. Felt-pen writing techniques are developed for precise defintion of hydrophobic patches. A rigid multilayer lid ensures optimal sealing of the disks without any sagging of the cover. The prototyping technologies prove to deliver high accuracy regarding channel dimensions and positioning of the hydrophobic patches which are key to a reliable volume definition. Before parallelization, a single metering unit is optimized for small tolerances in metered volumes and valving frequencies. Tolerances in metered volumes of typically 650nl are decisively minimized to coefficients of variation (CV) of less than 1% by tailoring the menisci of the liquid plug to control the surface phenomena. Hydrophobic valves with burst frequencies ranging from 10Hz to 50Hz have been fabricated and characterized. The CVs in burst frequencies are less than 5% and 1% in volume definition. Sample aliquoting is achieved by parallel assembly of several single valves which are linked by a distribution channel. In order to minimize the dead volume of liquid in channel segments between the metering units, a zig-zag structure incorporating both liquid distribution and metering is introduced. The whole process is governed by a designated frequency protocol. Upon stopping the disk, the aliquoting zig-zag structure is primed by capillary action while avoiding gas pockets. The zig-zag structure exhibits hydrophobically blocked radial outlet valves and inner air vents which are shared with adjacent subunits. The volume of each subunit geometrically defines the volume of each aliquot. While spinning at 20 Hz, the hydrophobic valves at the outlets still hold while the liquid in the upstream capillary is pulled back into the plasma reservoir. During this phase, the plug splits at a defined position under the vent and retracts into the plasma chamber under the influence of the centrifugal force. Upon elevating the frequency to 35 Hz, the hydrophobic valves break and the plug is split into individual volume fractions. The precision as well as the reproducibility of the metered aliquots is decisively impacted by the course of the liquid break-off on the inner vents of the zig-zag structure. Volume repartitioning is suppressed by tailoring small menisci and by the sharpness of the inbound edges. The fabricated structures feature aliquot nominal volumes of 250 nl and 125 nl at CVs better than 6%.
Integrated Sample Metering for Immunoassays on a Lab-on-a-Disk
2005, Harter Joachim
Erstgutachter: Prof. Dr. Roland Zengerle
KurzfassungAn important objective in medical diagnostics is the integration of the full process chain from the preparation of a patient’s whole blood to an analytical result. Rotating disks are a promising approach for liquid handling due to the intrinsic pumping, valving and particle separation by the interplay of the centrifugal force field and capillary action. Aliquoting of processed samples to defined volumes for subsequent detection is a crucial step in highly automated and parallelized assay protocols. This work presents microfluidic networks for on-disk metering and aliquoting of plasma samples using the concept of hydrophobic valving. To facilitate automatization of parallel processes, tolerances resulting from all steps of the technology chain are systematically investigated and minimized. Soft embossing from a silicone rubber (PDMS) mold generated from a photolithographically structured multilayer structure (SU-8 photoresist) is used to precisely replicate channels onto polymer disks (COC). The disk is coated with hydrophilic and hydrophobic polymers. Felt-pen writing techniques are developed for precise defintion of hydrophobic patches. A rigid multilayer lid ensures optimal sealing of the disks without any sagging of the cover. The prototyping technologies prove to deliver high accuracy regarding channel dimensions and positioning of the hydrophobic patches which are key to a reliable volume definition. Before parallelization, a single metering unit is optimized for small tolerances in metered volumes and valving frequencies. Tolerances in metered volumes of typically 650nl are decisively minimized to coefficients of variation (CV) of less than 1% by tailoring the menisci of the liquid plug to control the surface phenomena. Hydrophobic valves with burst frequencies ranging from 10Hz to 50Hz have been fabricated and characterized. The CVs in burst frequencies are less than 5% and 1% in volume definition. Sample aliquoting is achieved by parallel assembly of several single valves which are linked by a distribution channel. In order to minimize the dead volume of liquid in channel segments between the metering units, a zig-zag structure incorporating both liquid distribution and metering is introduced. The whole process is governed by a designated frequency protocol. Upon stopping the disk, the aliquoting zig-zag structure is primed by capillary action while avoiding gas pockets. The zig-zag structure exhibits hydrophobically blocked radial outlet valves and inner air vents which are shared with adjacent subunits. The volume of each subunit geometrically defines the volume of each aliquot. While spinning at 20 Hz, the hydrophobic valves at the outlets still hold while the liquid in the upstream capillary is pulled back into the plasma reservoir. During this phase, the plug splits at a defined position under the vent and retracts into the plasma chamber under the influence of the centrifugal force. Upon elevating the frequency to 35 Hz, the hydrophobic valves break and the plug is split into individual volume fractions. The precision as well as the reproducibility of the metered aliquots is decisively impacted by the course of the liquid break-off on the inner vents of the zig-zag structure. Volume repartitioning is suppressed by tailoring small menisci and by the sharpness of the inbound edges. The fabricated structures feature aliquot nominal volumes of 250 nl and 125 nl at CVs better than 6%.
Notfall-Disk Integrierte Detektion von notfallrelevanten Parametern auf einem zentrifugalen mikrofluidischen System
2005, Thomas A. Brefka
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
KurzfassungThe use of centrifugal, microfluidic platforms, as the Bio-Disk platform, brings along various advantages. Process integration leads to simple workflow for the operator. Parallelization of different or equal analysis structures offers a high efficiency on small space. This diploma thesis deals with the development of analysis structures and reactions for the integrated detection of emergency parameters on the microfluidic Bio-Disk platform. The design, development, and characterization of an analysis structure for the hematocrit determination is presented. Experimental characterization for the implementation of an analysis system for the determination of hemoglobin and alcohol, respectively, will also be presented. With an overview over emergency relevant parameters, the parameters Hematocrit, Hemoglobin, Alcohol and Troponin are selected for further investigation, according to the selection criteria high relevance, optical detection principle and amenability for integration on the Bio-Disk platform. An U-shaped channel was chosen for the implementation of the hematocrit measurement on the Bio-Disk platform. The working range of this method lies between 18% and 72%, exceeding the physiological range (30% – 68%). Our results feature a good reproducibility, with a coefficient of variation of CV = 5% and a linearity of R2 = 0,9985. The integrated metering structure reached a maximum coefficient of variation of CV = 13%. Thus the feasibility of hematocrit determination on a centrifugal, microfluidic platform is shown. By applying a data layer, this platform renders the usage in a CD-drive possible. Various parameters can be combined on one disk. The determination of hemoglobin and alcohol is carried out by homogeneous assays. Thereby, the blood cells are lysed chemically (SLS) and hemoglobin is determined by a reaction followed by an optical absorption measurement. The determination of the concentration of alcohol is carried out by an enzyme assay, with alcohol oxidase in combination with the enzyme peroxidase and a dye. Both detection methods are readily integrable. The Bio-Disk platform is a modular platform with simple interfacing. It consists of a cheap passive disk for disposal after use and a reusable drive unit. Sedimentation of blood, simple mixing and optical detection are integrable. Der Einsatz von zentrifugalen, mikrofluidischen Plattformen, wie die Bio-Disk Plattform, bringt für eine Analyse viele Vorteile mit sich. Durch Prozessintegration werden Abläufe für den Anwender vereinfacht und durch Parallelisierung verschiedener oder gleicher Analysestrukturen wird eine enorme Leistungsfähigkeit auf kleinstem Raum ermöglicht. Im Rahmen dieser Diplomarbeit werden Analysestrukturen und Nachweisreaktionen zur integrierten Detektion von notfallrelevanten Parametern auf der Bio-Disk Plattform entworfen und die Herstellung und Charakterisierung einer Analysestruktur zur Bestimmung des Hämatokritwertes dargelegt. Des Weiteren wird die experimentelle Charakterisierung eines Analysesystems zur Konzentrationsbestimmung von Hämoglobin bzw. Ethanol präsentiert. Anhand einer Übersicht von notfallrelevanten Parametern der klinischen Chemie werden die Parameter Hämatokritwert, die Hämoglobin-, Ethanol- und die Troponin T beziehungsweise Troponin I Konzentrationen nach den Kriterien hohe Relevanz, optisches Detektionsprinzip und Implementierbarkeit auf der Disk für weitere Untersuchungen ausgewählt. Um die Bestimmung des Hämatokritwertes Hkt auf die Bio-Disk Plattform zu transferieren, wird ein U-förmig gestalteter Kanal als grundlegende Analysestruktur gewählt. Der Arbeitsbereich der Hämatokritwertbestimmung liegt zwischen 18% und 72% und deckt damit den physiologischen Wertebereich (30% – 68%) ab. Dabei wird eine gute Reproduzierbarkeit, mit einem Variationskoeffizienten von VK = 5% und einer Linearität von R2 = 0,9985 erreicht. Die integrierte Meteringstruktur erreicht einen Variationskoeffizienten von maximal VK = 13%. Die Machbarkeit einer Bestimmung des Hämatokritwertes auf Basis einer zentrifugalen, mikrofluischen Plattform konnte damit gezeigt werden. Die Plattform bietet darüber hinaus die Möglichkeit einer Verwendung in einem CD-Laufwert durch Aufbringen einer Datenschicht. Dabei können verschiedene Parameter auf eine Disk kombiniert bestimmt werden. Das erarbeitete Konzept zur Konzentrationsbestimmung von Hämoglobin und Ethanol erfolgt auf Basis eines homogenen Assays. Dabei werden die Blutzellen chemisch aufgeschlossen (SLS) und das Hämoglobin umgesetzt. Das Produkt wird über eine Absorptionsmessung detektiert. Ethanol wird mit Hilfe eines Enzymassays detektiert. Zur Verwendung kommen Alkoholoxidase, sowie Peroxidase und ein Farbstoff. Beide Nachweismethoden sind einfach in vorhandene Strukturen im Bio-Disk System integrierbar und erweitern damit das Spektrum dieser Plattform. Die Bio-Disk Plattform ist eine modulare Plattform mit einfach gehaltenen Schnittstellen. Sie besteht aus einer kostengünstigen, passiven Disk und einer wiederverwendbaren Antriebseinheit. Die Disk kann nach Abschluss der Messung entsorgt werden. Die Sedimentation von Blut, einfaches Mischen und ein optisches Detektionssystem sind integrierbar.
Notfall-Disk - Integrierte Detektion von notfallrelevanten Parametern auf einem zentrifugalen mikrofluidischen System
2005, Brefka Thomas
Erstgutachter: Prof. Dr. Roland Zengerle
Zweitgutachter: Prof. Dr. Thomas Stieglitz
KurzfassungThe use of centrifugal, microfluidic platforms, as the Bio-Disk platform, brings along various advantages. Process integration leads to simple workflow for the operator. Parallelization of different or equal analysis structures offers a high efficiency on small space. This diploma thesis deals with the development of analysis structures and reactions for the integrated detection of emergency parameters on the microfluidic Bio-Disk platform. The design, development, and characterization of an analysis structure for the hematocrit determination is presented. Experimental characterization for the implementation of an analysis system for the determination of hemoglobin and alcohol, respectively, will also be presented. With an overview over emergency relevant parameters, the parameters Hematocrit, Hemoglobin, Alcohol and Troponin are selected for further investigation, according to the selection criteria high relevance, optical detection principle and amenability for integration on the Bio-Disk platform. An U-shaped channel was chosen for the implementation of the hematocrit measurement on the Bio-Disk platform. The working range of this method lies between 18% and 72%, exceeding the physiological range (30% – 68%). Our results feature a good reproducibility, with a coefficient of variation of CV = 5% and a linearity of R2 = 0,9985. The integrated metering structure reached a maximum coefficient of variation of CV = 13%. Thus the feasibility of hematocrit determination on a centrifugal, microfluidic platform is shown. By applying a data layer, this platform renders the usage in a CD-drive possible. Various parameters can be combined on one disk. The determination of hemoglobin and alcohol is carried out by homogeneous assays. Thereby, the blood cells are lysed chemically (SLS) and hemoglobin is determined by a reaction followed by an optical absorption measurement. The determination of the concentration of alcohol is carried out by an enzyme assay, with alcohol oxidase in combination with the enzyme peroxidase and a dye. Both detection methods are readily integrable. The Bio-Disk platform is a modular platform with simple interfacing. It consists of a cheap passive disk for disposal after use and a reusable drive unit. Sedimentation of blood, simple mixing and optical detection are integrable.
Optimierung der Reaktionsbedingungen in Durchflusszellen für Bead-basierte Immunoassays auf mikrofluidischen Zentrifugalplattformen
2005, Axel Kaltenbacher
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Claas Müller, IMTEK
KurzfassungCentrifugal microfluidic platforms have the potential for a complete integration of all relevant process steps for diagnostic tests of whole blood. Apart from the automation of the workflow, particular application potential emerges from parallelization of tests by arrangement of multiple analytic devices. One of the major objectives of the Bio-Disk project is the determination of the immune status of human whole blood by processing a sandwich immuno-assay on one single disk. Compared to batch mode reaction on titerplates, binding between antibodies and antigens on the disk happens in flow cells. The capture proteins, in this case the antigens, are immobilized on micro-spheres which are held back in a flow cell. A significant parameter of the binding reaction is the flow rate of the prepared antibody solution in the flow cells. Thus, the flow rates have to be adjusted by frequency and control of the hydro-dynamic resistance to offer the antibodies enough time to link with the immobilized antigens. For the adjustment of the hydro-dynamic resistance in the flow cells, it is crucial to control the channel dimensions with aid of an appropriate manufacturing process. The structures are replicated into polymer discs (cyclo-olefin-copolymers, COC) by an existing hot embossing process using micro-structured silicone rubber inserts. In this thesis, the subsequent temperature diffusion bonding procedure to seal the channels has further been developed by processing a two-component-foil with COC-polymers with different glass-transition temperatures. This mechanically stable foil allows a more robust process control of the bonding and almost entirely reduces sagging of the cover caused by thermal deformation into channels with 100 μm height and width of 1200 μm. Additionally, compared to formerly used foils, the two-component foil has an increased bond strength between cover and substrate by factor 8. Furthermore, a coating process for COC substrates has been developed in order to position hydrophilic surface conditions. These relevant improvements allow the precise control of hydro-dynamic resistance of fluids in micro-channels. The design of the flow cells has been optimized in order to avoid trapped air in the channels; the flow rates within one chamber and within several chambers of one single disk have been investigated. The flow rates can be reproducibly controlled by rotation frequency in parallel chambers without any bead column. Without the influence of the beads, the deviations within one chamber can be reduced from more than 100 % to less than 5 %. The deviations between different chambers on one single disk can be reduced to less than 20%. For the first time, this method allows the realization of 16 parallel immuno-assays on one disk. The impact of a bead column on the flow rate is investigated. Beads packed in a monolayer suffer from poor regularity and lead to high deviations in flow rates. Multilayer beads arrangements give a more homogeneous assembly leading to more constant flow rates in the cells. In the optimized flow cells, 3-step immune fluorescence tests of hepatitis-A, labeled with fluospheres®, have been accomplished. Thereby, probe volumes of 1 μl serum are detectable. By optimizing the concentration of detection antibodies and using multilayer bead columns, a signal-to-noise-ratio of 7:1 has been achieved for a standard serum of the "Paul-Ehrlich-Institut". A signal ratio of 4:1 has been achieved of 1μl serum to 1/50th μl serum with a fifty fold thinning of 1μl of the serum. Finally, further improvement measures have been established which are essential for the immuno-assay regarding the possibility to test also blood samples with a lower difference of the immune titers.
Optimierung der Reaktionsbedingungen in Durchflusszellen für Bead-basierte Immunoassays auf mikrofluidischen Zentrifugalplattformen
2005, Kaltenbacher Axel
Erstgutachter: Prof. Dr. Roland Zengerle
Optimierung von Systemkomponenten der integrierten Prozesskontrolle für die TopSpot-Technologie
2005, Antje Kurowski
KurzfassungIn the last years the developement of molecular biological technology for sequencing genomes has increased very fast. Key-technologies like increasing efficiency, miniaturizing, parallelization and automatisation will be applied more and more within the microarray-technology. During this diploma-thesis the non contact printing technology TopSpot used as TopSpot 4R4 IntPro (with integrated pressure sensor) developed by HGS-IMIT and IMTEK was used to produce microarrays. To guarantee a stabilized and reproducible printing process in a wide range of print media, the correlation between the system components have to be analysed. The best combination of the applied ring components, were determined. The x – countered o-ring which couple the moving of system piston and piezo-actor vibration, in combination with a 27,4 shored PDMS sealing-ring, with a ringwall diameter of 800μm and a 150μm sealling-lip height, gave the best results. The pressure signal in the diagram follows now the piezo-actor amplitude. The vibration of the piston were absolutely oppressed during the complete printing process and a complete controll of the pressure profile is now possible.
Verfahrensentwicklung zur Herstellung einer implantierbaren Direkt-Glucose-Brennstoffzelle
2005, Artur Lorenz
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Peter Woias, IMTEK
KurzfassungImplantable energy converter systems which transform the body’s energy into electrical energy offer a great potential for modern minimal-invasive medicine. Direct-glucose-fuel-cells allow the electrochemical conversion of glucose stored in body fluids, thus providing the electrical energy for multiple applications. The functional principle was first described 1911 and has been proven multiple times [1]. The fuel cell comprises a total of three electrodes which are separated by ion conducting and glucose permeating membranes. The electrodes are arranged in a way that the solved “fuels” glucose and oxygen are first separated from each other. This is achieved by completely reducing the oxygen at selective cathodes. Thus, only the glucose can reach the anode and releases its energy by oxidation. In this work, fabrication processes for a direct glucose fuel cell were established and validated. These fabrication processes were documented in standard operating procedures (SOPs). The electrodes consist of a catalyst which is applied on a powdery carrier and linked to a silver screen with a polymer. To achieve this, the catalyst carrier is suspended in a watery polymer solution and is degassed. After centrifugation of the suspension, the “polymer-catalyst- paste” is ready for the fabrication of the electrodes. Spreading of the paste on a foil and the integration of the silver screen completes the production of the electrodes. For the casting of the membrane, a mold is filled with a polymer solution and water is evaporated subsequently. The assembly of the components is achieved by etching the polymers again. For this, the electrode-membrane-unit is placed between wetted filter paper. The diffusion of water thus induces the gluing of the components by solving the polymers. After the thermal crosslinking of the polymers, the electrode-membrane-unit is packaged and the cable connections are contacted and isolated. At the subsequent voltage and current measurements, the prototype provided a total power of 1.26 µW with a voltage of 43 mW while using a 3.9 kΩ load resistance. This result differs from the literature value by a factor of 40. In the final progress of this work, the weak points were identified and the first measures were taken.

2004

Blood Separation and Bead-based Immunodiagnostics for Centrifugal Microfluidics
2004, Stefan Häberle
Erstgutachter: Dr. Jens Ducrée
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungAn essential objective in medical diagnostics on centrifugal microfluidic platforms is an integrable, robust separation procedure for whole blood. This first step is crucial for a full process chain from the preparation of a patient´s whole blood to an analytical result. This work systematically develops a fundamental understanding of blood separation in centrifugal microfluidics. Extensive experimental investigations on centrifugal batch separation of blood in microchambers and continuous blood flow in microchannels are carried out. Based on these results, a novel centrifugal process for the continuous extraction of plasma from the sediment by a 3-stage microfluidic structure is presented. The course of the separation procedure terminates with metered plasma that is available for subsequent on-disk processing. The functionality has been proven and widely characterized by numerous experiments and simulations. The developed technique supplies 2μl plasma from 5μl whole blood at moderate frequencies of 40Hz within 20s, only. The residual cell concentration in the purified plasma amounts to less than 1%, independent of the frequency of rotation. This novel structure allows a 100% reliable extraction of plasma from raw blood. It expands the microfluidic toolbox for centrifugal platforms by a new, very important element. The second part of the thesis focusses on the transfer of standard immunoassays from conventional titerplates to the novel bead-based format of the centrifugal platform. Structures for aggregation of beads in monolayer columns on the disk are designed and fabricated. As a prerequisite to the assays, innovative solutions for trouble-free injection of pre-coated beads into the channel network on the disk are developed. Several capture assays with mouse IgG immobilized on the surface of polystyrene beads of 50μm in diameter are carried out on the disk. FITC labeled anti-IgG at concentrations between 70nM and 7μM are bound to the immobilized IgG. The experimental curve of fluorescent intensity from bound IgG/anti-IgG complexes over concentration of supplied anti-IgG follows the typical sigmoidal curve of standard assays. The limit of detection is 133nM and enables a significant reduction of sample volumes from 50μl in titerplates to 1μl in disk format. The main parameters affecting the improvable reproducibility of the fluorescent signal are subject to detailed investigations. All potential error mechanisms in the assay protocol comprising unstable flow conditions, pipetting errors, non-optimal washing procedures, fluctuations of the fluorescence detector and the data processing are quantified according to their impact on the error. Based on these investigations, a package of measures for a significantly improved assay performance is proposed.
Blood Separation and Bead-based Immunodiagnostics for Centrifugal Microfluidics
2004, Haeberle Stefan
Erstgutachter: Prof. Dr. Roland Zengerle
Characterisation of a Nanolitre Dispenser Based on PipeJet(TM) Technology
2004, Gracki Sebastian
Erstgutachter: Prof. Dr. Roland Zengerle
Characterisation of a nanolitre dispenser based on PipeJet™ Technology
2004, Sebastian Kamil Gracki
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Hermann Sandmeier, Uni Stuttgart
KurzfassungThe PipeJet™ system was invented in 2003 at the IMTEK. The dispenser allows for the contact-free dosage of small amounts of liquids in the range from a few nanolitres up to some microlitres. The key element of the system is an elastic tube with a well-defined inner diameter that is squeezed with high dynamics. The liquid is driven out through the end of the tube. The dosed volume is independent of the viscosity or the surface tension over a certain range. Due to the simple geometry PipeJet™ is a low cost and very robust device. Since it is a new system its optimal design parameters has not been analysed yet. This is the subject of this diploma thesis. The behaviour of the PipeJet™ dispenser is analysed with special attention to the repeat accuracy and the dependency of the dispensed droplet volume on the dynamic parameters of the pistons stroke. Applying small modifications to the system a highly linear dosage line is obtained. Concurrent, changing the setup, the PipeJet™ can be adjusted to a fixed volume dispenser. Volumes of some microlitres can be obtained by consecutive dispensing of smaller nanolitre drops. The limiting factor thereby is the refilling of the dispensed liquid volume from the reservoir. An adequate model to predict the maximum shot frequency and flow rate is confirmed. The independency of the system’s dispensing behaviour concerning higher viscous fluids and those with different surface tension is examined. Finally a proof of principle for pipetting with the system is given. Based on the gained insight design rules for a fixed and a variable volume dispenser are presented.
Compact Model of a Laminar Micromixer
2004, Claudio G. Cupelli
KurzfassungThis work presents how the complex task of micromixing can be mapped onto a (lumped) network model. A systematic analysis of the operation of microfluidic structures will lead to design rules how a concept based on modular components, needed for a network implementation, can be achieved. Based on the modularity of these components a split-and-recombine mixer will be designed, that systematically combines, splits and twist fluid streams in order to enhance mixing. It will be shown that the modularity of the concept can be extended to fluids of strongly varying viscosities. The principle idea for the development of the compact model for a multi-laminar mixer is based on a zero order model, which allows to estimate the progress of diffusion with a semianalytical approach. Essential quantities that are adequate to quantify the progress of mixing within a network of these components will be outlined and investigated through a series of CFD-simulations for multi-laminar flow with fluids of different viscosities. Essential aspects of the network implementation will be discussed and highlighted at the example of a complete step of a split-and-recombine mixer. The experimental part of this work comprises the design of microfluidic structures and the validation of essential quantities that are described by the network model. Diese Arbeit zeigt auf, wie die komplexe Fragestellung des Mikromischens auf ein Netzwerkmodell abgebildet werden kann. Eine systematische Analyse der Funktionsweise mikrofluidischer Komponenten führt zu Designregeln, die zeigen, wie ein Konzept basierend auf modularen Komponenten, die für die Netzwerkimplementierung notwendig ist, erreicht werden kann. Basierend auf der Modularität dieser Komponenten wird ein Splitund- Recombine Mischer aufgebaut, der systematisch Fluidströme vereint, auftrennt und dreht und somit die Voraussetzung für ein effizientes und homogenes Durchmischen schafft. Es wird gezeigt, dass Konzept auf Fluide stark unterschiedlicher Viskositäten ausgedehnt werden kann. Der Grundgedanke bei der Entwicklung eines Kompaktmodells für einen Multilaminarmischer ist dabei, ein Modell 0-ter Ordnung zu finden, das es erlaubt den Mischfortschritt in einem semi-analytischer Ansatz zu beschreiben. Wesentliche Gesichtspunkte der Netzwerkimplementierung werden diskutiert und exemplarisch für einen kompletten Schritt einer Split-und-Recombine Stufe aufgezeigt. Der experimentelle Teil dieser Arbeit umfasst den Aufbau von mikrofluidischen Strukturen und die Validierung der Größen, die im Netzwerkmodell verwendet werden.
Compact Model of a Laminar Micromixer
2004, Cupelli Claudio
Erstgutachter: Prof. Dr. Roland Zengerle
Design and Characterization of Novel Rotational Micro Mixers
2004, Schlosser Hans-Peter
Erstgutachter: Prof. Dr. Roland Zengerle
Design and characterization of novel rotational micro mixers
2004, Hans-Peter Schlosser
KurzfassungThe presented diploma thesis was performed from December 2003 to June 2004 at the Institute of Microsystem Technology (IMTEK), University of Freiburg, Germany. It was supervised by Prof. Dr. Roland Zengerle, head of chair for MEMS-Applications and Dr. Jens Ducrée, assistant. In this work a novel concept of a centrifugal platform for high throughput reactive micro mixing is developed, implemented, and investigated by numerous experiments and extended CFD simulations. The modular concept of the platform is based on a mixing unit with microchannels rotating on a conventional laboratory centrifuge. The mixing unit is connected to a reservoir which features several concentric cavities. Educts of a chemical reaction are continuously injected into these cavities by a dispenser. The centrifugal force induced on liquids in rotating channels, propulses the educts outwards through planar microchannels. The educts are mixed in the channels and react to products which are expelled from the rotating unit into a resting receiving vessel. Compared to mere diffusion, mixing is improved by various effects appearing only on centrifugal platforms. First, the velocity-dependent Coriolis pseudo force results in a transverse advection of adjacent educts within the channel. This stirring current leads to a drastic enlargement of the interface. Second, the expelled streams are sheared, thinned out and multi-laminated on the non-spinning wall of the receiving vessel. Third, an intertwining of phases directly at the junction of the channels is experimentally observed. These effects are subject to intensive investigations and optimized towards an ultra-fast, high throughput mixing scheme. The most relevant parameters of mixers are quality of the mixture and throughput. The mixing performance is quantified by the selectivity of an adapted test reaction. Mixing quality up to a micromixedness ratio of 3,000 for spinning rates beyond 120 Hz are obtained by centrifugal mixing. The dependency of mixing quality from throughput and pressure drop was experimentally investigated and ranks among other high-performance micromixers. Following these results, the mixing performance is further improved by finding an optimal channel geometry. A characteristic mixing time is derived from extensive simulations on the chemical kinetics of the measured mixing parameters. It is shown that the characteristic time for proper mixing exceeds the mean residence time of educts in the spinning channel. Further improvement thus demands an essential enlargement of the channel length and corresponding residence time, respectively. A considerable throughput measured to be 1 ml/s per channel is evidence to the great potential of this centrifugal mixing concept. It can massively be enhanced by generic parallelization of the radial channels. The modular platform allows high-throughput mixing for a manifold of applications since the mixing unit can be adopted to individual needs. First experiments indicate novel flow control procedures based on the appearing Coriolis force. It is observed that the flow rates in channels merging at the junction on the spinning disk can be controlled by the spinning rate. This leads to a simple, active tuning of mixing ratios between educts by means of the spinning rate. Evident relations between rotational frequency and the flow rates through two merging channels are experimentally derived. CFD simulations support these experimental findings. The results show the high potential of the micromixing platform, featuring major throughput and high grade quality. Actuation is sourced out by the system partitioning and the modular setup particularly enables multipurpose applications.
Entwicklung eines miniaturisierten Glukose-Assays auf einer zentrifugalen mikrofluidischen Diagnostikplattform
2004, Jürgen Steigert
Erstgutachter: PD Dr. rer. nat. Jens Ducrée
Zweitgutachter: Prof. Dr. Thomas Stieglitz, IMTEK
KurzfassungIn dieser Diplomarbeit wird ein neues Konzept zur Verlängerung optischer Strahlwege durch Messkammern in mikrofluidischen Analysegeräten (Lab-on-a-Chip) vorgestellt und evaluiert. Grundlage hierfür sind monolithisch in die Rückseite des Polymer-Chips eingearbeitete V-Gräben, die einen Anstellwinkel von 45° aufweisen. Trifft ein Lichtstrahl, der senkrecht über die Vorderseite des Chips eingekoppelt wird, auf die Seitenfläche eines V-Grabens, so ist hier der Einstrahlwinkel von 45° größer als der Grenzwinkel der Totalreflektion der gebräuchlichen Polymere und der Lichtstrahl wird deshalb vollständig um 90° in die Chip-Ebene umgelenkt. In unserem Konzept trifft nun der geführte Lichtstrahl einer roten Laserdiode auf eine Messkammer, in der ein Absorptions-Assay durchgeführt wird. Entsprechend dem Gesetz von Lambert-Beer ist die Abschwächung des Strahls ein Maß für die Konzentration eines Reaktionsprodukts, woraus sich wiederum die ursprüngliche Konzentration des Analyten bestimmen lässt. Der abgeschwächte Lichtstrahl wird nachfolgend von einem identischen V-Graben erneut um 90° umgelenkt und tritt aus dem Chip in Richtung Detektor aus. Dieses Konzept lässt sich für die meisten für mikrofluidische Strukturen genutzten Polymere aufgrund der gegebenen Brechungsindizes nutzen und erlaubt die Festlegung der optischen Weglänge durch eine auf dem Chip befindliche Messkammer unabhängig von der Höhe des Chips. Konkret wurde eine optische Weglänge von 10 mm in einem Chip der Höhe 1,5 mm implementiert. Im Gegensatz zu direktem Durchstrahlen der Messkammer (optische Weglänge 1 mm) konnte die Sensitivität des Analyse-Chips ebenfalls um einen Faktor 10 gesteigert werden. Um die Eigenschaften des Konzeptes zu demonstrieren, wurde ein Glukose-Assay mit Pferdeserum als Analyt auf einer Lab-on-a-Disk Plattform entwickelt. Zusätzlich wurden die biochemischen Prozesse und das Mischverhalten der Probe und der Reagenzien in der Disk-basierten Messkammer charakterisiert. Dabei ergaben sich hervorragende Sensoreigenschaften, wie ein geringes unteres Detektionslimit (cmin = 124 μM) zusammen mit einer ausgezeichneten Linearität (R2 = 0,998) über einen Arbeitsbereich von nahezu 3 Grössenordnungen. Weiterhin zeigt sich eine sehr gute Reproduzierbarkeit (CV = 2 %) und eine hervorragende Auflösung (Δc = 320 μM). In einem nächsten Schritt wurde ein Assay zur Bestimmung der Glukosekonzentration von unbehandeltem menschlichem Vollblut etabliert. Dabei erfordert das optische Auslesen die Verdrängung der Erythrozyten aus dem Strahlweg, was über einen zusätzlichen Zentrifugationsschritt zur Sedimentation der Erythrozyten in der Messkammer erreicht wird. Auch hier ergaben sich ausgezeichnete Sensoreigenschaften, wie eine Reproduzierbarkeit von CV = 4 %, ein unteres Detektionslimit von cmin = 199 μM, eine hervorragende Auflösung (Δc = 500 μM) und einem ausgezeichneten linearen Verhalten (R2 = 0,997) über einen Arbeitsbereich, der den klinisch geforderten Bereich vollständig abdeckt. Ein weiterer Vorteil des entwickelten Glukose-Assays, speziell im Hinblick auf kommerziell erhältliche Geräte zur Glukosebestimmung, ist die Unabhängigkeit der Ergebnisse gegenüber Schwankungen des Hämatokritwertes. Das evaluierte System bietet die Möglichkeit, das Auslesen der Assay-Ergebnisse statisch durchzuführen (Disk befindet sich in fixer Position) oder unter Rotation der Disk, was zusätzlich eine zeitaufgelöste Analyse der Reaktionsprozesse ermöglicht. Zur internen Kalibration wurde das System durch eine zusätzliche Referenzkammer erweitert, so dass eine Rausch-Kompensation möglich ist. Des Weiteren weist das System einen Streulichtanteil von < 1 % auf. Weitergehend wird ein Integrationskonzept zur Blutgruppenbestimmung auf der Disk vorgestellt. This diploma thesis is to evaluate and represent a new concept of extending optical pathways through measurement chambers in microfluidic analytic devices (labs-on-a-chip). Substantial for this are V-grooves that are embedded at the reverse side of a polymer substrate, having an opening angle of 45°. The optical beam of a standard red laser diode is directed at perpendicular incidence on the flat upper side of the chip. As the beam has entered the polymer chip it is reflected at the symmetric side face of the triangular V-groove. To ensure reflection the angle of incidence α has to exceed the critical angle αc for total internal reflection (TIR) which is governed by the refractive indices of the applied polymer substrate and the surrounding air. In our concept the guided beam passes a measurement chamber in which an absorption-assay is carried out. According to the law of Beer Lambert the beam attenuation is a measure for the concentration of a reaction product, that in turn is directly correlated to the original concentration of the analyte to be determined. In succession the attenuated beam is redirected by 90° and leaves the chip towards the detector. Because of the given refractive indices this concept can be applied to most of the polymers used for microfluidic structures and allows the fixture of the optical path length of the measurement chamber, fitted on a chip regardless its height. An optical path length of 10 mm is implemented in a chip having 1,5 mm of height. In contrast to a direct beam incidence through the measurement chamber (optical path length of 1 mm) the sensitivity of the analytical chip is increased by a factor of 10. In order to demonstrate the features of the concept a glucose-assay with horse serum as sample is developed on a lab-on-a-disk platform. Furthermore, the biochemical processes and the mixing behaviour of the sample and the reagents are characterized in the disk-based measurement chamber. Outstanding sensor features are achieved such as a significantly low limit of detection (cmin = 124 μM) as well as an excellent linearity (R2 = 0,998) within a working range extending over nearly three orders of magnitude. In addition the sensor features a very high reproducibility (CV = 2 %) and an exceptional resolution (Δc = 320 μM). In a further step an assay aiming at the determination of the glucose concentration of untreated human whole blood is established. The optical read-out requires the removal of the red blood cells (RBC) from the optical pathway which is achieved by an additional centrifugation step to sediment the RBCs within the measurement chamber. Again excellent sensor features are achieved, such as a high reproducibility of CV = 4 %, a low limit of detection of cmin = 199 μM, a very good resolution (Δc = 500 μM) with an outstanding linearity (R2 = 0,997) within a working range, fully covering clinical requirements. Another advantage of the developed glucose-assay, with regard to commercially available devices of glucose determination, is the independence of the results towards fluctuations of the haematocrit value. The evaluated system offers the opportunity to read-out the assay results statically (disk is in a fixed position) or under rotation, which in addition enables a real-time analysis of reaction kinetics. For an internal calibration a reference chamber is integrated so that a noise compensation is possible. Moreover, the system holds a stray light percentage of < 1%. Besides, an integration concept for blood group determination on the disk is presented.
Entwicklung eines miniaturisierten Glukose-Assays auf einer zentrifugalen mikrofluidischen Diagnostikplattform
2004, Steigert Jürgen
Erstgutachter: Prof. Dr. Roland Zengerle
Fast Production of Cell Microarrays Using a Highly Parallel Pressure Driven Nanoliter Dispenser
2004, Wintermantel Markus
Erstgutachter: Prof. Dr. Roland Zengerle
Functional Langmuir-Blodgett Films for Biosensor Applications
2004, Dorrer Christian
Erstgutachter: Prof. Dr. Roland Zengerle
Integrierte Prozesskontrolle für TopSpot 4
2004, Waldemar Honstein
KurzfassungThe presented diploma thesis was carried out from January to August 2004 at the institute for microsystem technology (IMTEK), university of Freiburg, Germany. Tutors were Prof. Dr. Roland Zengerle, head of chair for application development and Dipl-.Ing. Remigius Niekrawietz. An integrated process control for the TopSpot 4 microarrayer with a new method for sealing of air chamber is presented. A massive parallel analysis of thousands of different substances is one need of biotechnology. Microarrays are a great number of known substances, deposited in a regular pattern on a small area, enabling high throughput analysis. There are several approaches for the manufacturing of such microarrays. One of them is the TopSpot technology, developed with IMTEK and HSG-IMIT. It is a non-contact microarray printing technology based on pneumatic actuation. A pressure pulse is generated by a piston, which acts on all ejection channels simultaneously, causing the ejection of the substances. Up to 384 droplets can be dispensed in parallel from the silicon micromachined TopSpot printhead. The newly developed TopSpot module uses a pressure sensor for integrated process control. During the piezo stack actuation the sensor detects the generated pressure pulse in the air chamber. The measured signal can be used to control the dispensing process for errors like air chamber overflow and emptying of nozzles. The new TopSpot module also uses a new and more effective sealing of air chamber by using a PDMS-ring. This technique allows reducing the initial air volume to a minimum. By the use of new sealing technique the dispensible media bandwidth was expanded. In particular media with higher viscosity are now dispensible up to 10.8 mPas. The working frequency was also increased from 2 to 50 Hz. The system works at an actuation amplitude up to 80 μm. A minimum volume of 1 nl can be achieved for the parallel droplet ejection out of 24 nozzles. Optimal parameters for droplet ejection were found like rising edge of 380 μm/ms and using a optimised sealing ring hardness of 29 shore. With these parameters the dispensing frequency could be extended to 30 Hz and for special applications at even 50 Hz. With this design it was possible to reduce the piston flight, which was previously a major problem. Another improvement of the dispenser was the fabrication and optimisation of PDMSrings, which can seal the generated air pressure directly on silicon layer. Properties of some PDMS-types were investigated for best air chamber sealing and low strain of the printhead. For the validation of the TopSpot 4 modules diluted glycerine was used in most of the experiments. The validation comprises an investigation of cross contamination, printing precision and the reproducibility of the process. The standard deviation of the dosage volume, which was measured by an automated image processing system on the fly, shows the reproducibility of droplet volumes within one single channel to be better then 1 %. The outstanding performance of this printhead was also verified by a fluorometric method. We found an overall CV of the experiment to be better than 3 %.
Integrierte Prozesskontrolle für TopSpot 4
2004, Honstein Waldemar
Erstgutachter: Prof. Dr. Roland Zengerle
Magnetic-Bead Enhanced Mixing for Bioassays in Centrifugal Microfluidics
2004, Andreas Geipel
Erstgutachter: Dr. Jens Ducrée
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungMixing is a key process step in all bioanalytical assays. This work outlines a novel batch-mode mixing concept which is implemented on our centrifugal microfluidic Bio-Disk platform. This concept is based on two fundamental schemes to induce advection: the magneto-hydrodynamically interaction of magnetic beads with an external magnetic field and the inertia of the liquid. For the first scheme, magnetic beads are filled in a disk-based mixing chamber. The disk is exposed to a magnetic multipole field created by a set of permanent magnets resting in the lab frame. Setting the disk into spinning motion, the beads follow the orbit of the chamber to experience a time-oscillating magnetic field. By choosing an optimum alignment of the magnets and frequency of rotation, the suspended beads are periodically deflected within the chamber to induce advection by means of the Stokes drag in the liquid phase. Periodic alternate spinning clearly turned out to amplify the bead-based mixing as the trajectory of the beads covers almost the entire mixing chamber. In the latter mixing scheme, advective currents are induced by means of the inertia of the liquid upon periodic reversal of the sense of rotation. Combining both schemes, mixing of a 25-µl volume within less than 1second has been achieved! With respect to a mixing time of 7minutes for mere diffusion, this indicates an enhancement by three orders of magnitude! The feasibility of the mixing concept has been proven in a disk-based glucose assay. Applying magnetic beads in the described scheme, the overall time-to-result has been reduced by a factor of three. The presented concept displays several advantages compared to other active micromixing schemes: First, it involves a simple and modular setup which avoids 3D-micromachining, moving microparts, on-chip actuators and current controlled microcoils to invoke deflection of magnetic beads in a chamber at rest. Additionally, rather low spinning frequencies in the range of 5-10Hz, only, are required which is below typical burst frequencies of hydrophobic valves. Finally, the volume to be mixed is scalable between the nl- and the ml-range.
Magnetic-Bead Enhanced Mixing for Bioassays in Centrifugal Microfluidics
2004, Geipel Andreas
Erstgutachter: Prof. Dr. Roland Zengerle
Multichannel Dispenser based on PipeJet™ Technology
2004, Stephanie Wandres
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Hermann Sandmeier, Uni Stuttgart
KurzfassungThe highly precise and reliable control of small amounts of liquids in the nanoliter range (1nl = 10-6 cm³) has become increasingly important in the chemical, biological and pharmaceutical industry. In addition to the applications in life-science, the handling of small liquid volumes is also of interest in classical mechanical engineering or in the development of new inkjet print-heads. During this work, a prototype of an 8-channel dispenser based on the PipeJet™ technology is designed, fabricated and tested. The dispensing device relies on a direct displacement principle and delivers droplet volumes down to 500 pl as a free flying liquid jet. The key element of the multichannel dispenser are commercially available polymer tubes filled with the dosing medium by capillary forces. Each channel is individually controlled by a piezoelectric actuator. The tube with a well-defined inner diameter is squeezed with high dynamics and the liquid is driven out through its end. In addition to the design of the 8-channel dispensing system, the characterization of one channel is performed. The dosed volume of different media is analyzed on important influencing parameters such as the piezo stroke or the dosage frequency. Furthermore, the printing of droplets on different surfaces is investigated for evaluating the droplet’s quality. Based on these results, some modifications of the design are suggested for improvement. Finally, an outlook on possible concepts concerning the solution of a 32-channel dispenser are presented.
Multichannel Dispenser Based on PipeJet(TM) Technology
2004, Wandres Stephanie
Erstgutachter: Prof. Dr. Roland Zengerle
Produktion von Zell-Microarrays mit einem hochparallelen druckgetriebenen Nanoliter Dispenser
2004, Markus Wintermantel
KurzfassungIn last few years miniaturization and parallelization of biological assays has become a more and more important development in biomedical technology. Despite all technological progress in production of DNA and Protein microarrays, the production of cell microarrays remained exceedingly difficult. Due to the high biological demands of living cells. But with cell microarrays characterizing the binding of membrane protein-specific antibodies to cell surface markers is possible [8].In this work the noncontact printing technology TopSpot was used to produce mammalian cell microarrays in a highly parallel manner. Elementary demands of cells on printing technology were solved. The evaporation of cells suspension during printing and immobilization on the slides was solved by adding 30% (v/v) glycerol to the printing solution. The analyses of the cell vitality showed that less than 10% of the cells were lysed during the priting process. Immobilisation of mammalian cells on different substrates was achieved by a drop-in-drop technique using the EDC-NHS system. In contrast to most other immobilization methods it was possible to immobilize adherent and non-adherent cells. The results indicate that mammalian cells can be printed highly parallel on different substrates along with keeping their vitality and function.
Read-out Concepts for Luminescence-Based Bioassays on Centrifugal Microfluidic Platforms
2004, Lutz Riegger
Erstgutachter: Dr. Jens Ducrée
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungThis work outlines two different optical read-out strategies adapted for a parallel fluorescence immuno-assay (FIA) and an enzyme-assay (EA) performed on the centrifugal microfluidic Bio-Disk platform. For the parallel FIA, an ensemble of beads acts as a solid-phase carrier. In contrast to conventional bead-based assays, a color-coding is used to distinguish different types of capture-antibodies which are coated on the surface of the beads. An additional fluorescent tag indicates binding events on such a bead. To evaluate this parallel assay, both, the color tag for identification and the spatial distribution of the fluorescence intensity at the surface of the bead have to be determined. As a consequence, the spatial resolution of the optical setup must be below the diameter of the beads and an additional step is required to identify all beads located in a designated detection chamber. A first successful technical implementation is based upon high-power LEDs to excite fluorescence and a color CCD-camera together with a set of a suitable excitation- and emission-filter to suppress non-fluorescent signal and background fluorescence. An alternative implementation featuring a fiber-probe spectrophotometer proves to exhibit an insufficiently high limit of detection of the read-out device for the fluorescence measurements. Different concepts for color tagging of the beads such as incorporated dyes and incorporated quantum- dots are analyzed and evaluated. Dyed beads show the most promising results since they are readily distinguishable and feature an excellent stability. Beads with incorporated quantum-dots feature excellent luminescence characteristics. However, these beads exhibit a rather limited life-time and reproducibility, which could not be improved, yet. Additionally, a survey is conducted to enhance the brightness of conventional fluorochromes applied as labels of the detection-antibodies by alternative labels such as quantum-dots or Fluospheres. It turns out that the highest fluorescence intensity can be achieved with Fluospheres, although efficient long-term coupling to the detection- antibodies could not be implemented within the time-frame of this work. To enable automatization, read-out strategies are realized for both the tag-determination and the fluorescence read-out. For the enzyme-assay (EA), a long optical path through the sample solution is required for sensitive detection. Thus, a novel method for coupling the incident light into the disk-plane is implemented. A first successful technical setup is based upon a red laser diode as a light source, and a fiber-probe spectrophotometer to detect changes in the optical density in the sample solution. Furthermore, the enzyme-assay setup is calibrated with a conventional cuvette spectrophotometer. Finally, a glucose- assay in a buffer solution is realized.
Read-out Concepts for Luminescence-Based Bioassays on Centrifugal Microfluidic Platforms
2004, Riegger Lutz
Erstgutachter: Prof. Dr. Roland Zengerle

2003

Economics of Plastics Microfabrication for Microfluidics in the Life Sciences
2003, Johannes Grabowski
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. W. Menz, IMTEK
KurzfassungThis thesis is part of the FlowMap project. FlowMap is an explorative technology roadmap investigating markets for microfluidic technologies in the life sciences, such as biotechnology, drug discovery, medical therapeutics and diagnostics as well as environmental monitoring. By interviewing more than 150 major players in the field, we found out that a rapidly increasing number of microfluidic applications utilises polymers as a substrate for microfluidic applications. On the one hand, the use of plastics opens up new options for material characteristics to adjust physico-chemical surface interactions which, for instance, affect adsorption, surface coatings and capillary forces. There are also proven devices made of medical grade plastics which have passed the strict medical regulation. On the other hand, the moderate material cost as well as the amenability to mass fabrication make plastic technologies the most promising candidate for the production of low-cost disposable devices. Key questions addressed in this work are: • What are the characteristics of the plastics as a material? • Which plastics technologies are utilised in microfluidics-based life science applications and how can they be characterised? • What is the cost structure of plastics microfabrication? An overview of common plastics and important characteristics is provided. Structuring technologies are categorised according to standardised characterisation sheet which allows a systematic choice and comparison of the technology. These sheets are set up for hot embossing and injection moulding of polymers as well as technologies for master fabrication such as laser ablation, micromilling, UV-LIGA, EDM and DRIE of silicon. By conducting interviews with more than 35 vendors and users of these technologies, information about relevant costs is gathered. For replication technologies, a parametric cost model is developed which allows an estimation of manufacturing cost as a function of the production volume and other impact factors. These models provide good agreement with validated costs for a hot embossed CE structure and the injection moulded TopSpot chip. This supplement to FlowMap serves as a valuable basis for making economical and technological decisions at a very early conceptual stage of his project for a potential user of polymers.
Economics of Plastics Microfabrication for Microfluidics in the Life Sciences
2003, Grabowski Johannes
Erstgutachter: Prof. Dr. Roland Zengerle
Entwicklung eines Prüfmesstandes für hydrophobe Oberflächen, mikrofluidischer Druckköpfe
2003, Haselberger Harald
Erstgutachter: Prof. Dr. Hermann Sandmaier
Zweitgutachter: Prof. Dr. Roland Zengerle
Entwicklung und Evaluierung eines Messplatzes zur Charakterisierung mikrofluidischer Strukturen auf einer rotierenden Scheibe
2003, Christian Beer
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Hermann Sandmeier, HSG-IMIT, Villingen-Schwenningen
KurzfassungThis diploma thesis includes the development of an experimental setup which makes it possible to characterise microfluidic processes on a rotating disc in the CD-format. High resolution images of structures on the micrometer scale can be recorded from the CDs that rotate with frequencies of up to 10.000 rpm. This has been achieved by devising and realising an adequate concept of the experimental setup. A particular CD motive power drives the structured CDs with a high frequency stability. The number of revolutions of the player is governed by a PC and the current rotation frequency is given out as a TTL-compatible trigger signal. The CCD-camera „PCO SensiCam FastShutter SVGA“ whose exposure time goes down to 100 ns can be used for the image pickup. Furthermore the experimental setup consists of a stroboscope (DRELLO 255-01) for sufficient light intensity and a linear drive to position the microscope and the attached camera precisely along the radial direction of the CD: For the detection of the rotation frequency as well as for the trigger signal for the stroboscope and the camera a special real-time-card (ADwin-light-16) is used which has got its own processor and can therefore perform its tasks in real time without depending on the PC. This card had to be programmed in a programming language that is close to Basic. A software panel in TestPoint has been developed for the steering of the whole system. The linear drive and a controllable temporal delay of the camera exposure as against the motive power makes it possible to observe the complete surface of the CD during the rotation. The present thesis shows the underlying concept of the experimental setup as well as the evaluation of the single components according to certain parameters. Great importance has be attached to the behaviour of the single components that is decisive for the experimental setup as a whole system. It is only after a certain time that the stroboscope used in the experimental setup reaches the maximal light intensity. This delay of the flash had to be determined first at it is necessary to steer the camera this amount of time earlier composed to the stroboscope. Without the compensation that has been carried out this constant delay would lead to the fact that the structure that is to observe would move out of the image when the frequency changes. The occurring azimuthal picture shift has been analysed and assessed as well.
Entwicklung und Evaluierung eines Messplatzes zur Charakterisierung mikrofluidischer Strukturen auf einer rotierenden Scheibe
2003, Beer Christian
Erstgutachter: Prof. Dr. Roland Zengerle
Evaluation of Highly Dynamic Actuation Concepts for DWP and PCR-Slide Dispensers
2003, Moosmann Christian
Erstgutachter: Prof. Dr. Roland Zengerle
Evaluation of highly dynamic actuation concepts for DWP and PCR-Slide dispensers
2003, Christian Moosmann
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jan G. Korvink, IMTEK
KurzfassungMicrofluidic dosage systems can be coarsely separated in direct displacement dispensers and pressure driven dispensers. Direct displacement dispensers assign a force to the liquid, usually over a membrane or an elastomer. Pressure driven dispensers build up a pressure in a pressure chamber above a dosage chip, which then causes the liquid to be ejected. Since the media used for the pressure generation are gases (mostly air or nitrogen), which are highly compressible, exact control of the pressure run is rather demanding. However, to achieve a reproduceably high dosage quality this is essential. While this is obvious for variable volume dispensers, fixed volume dispensers like the DWP (dispensing well plate) should not be affected by varying pressure runs too much. While the dispensed volume indeed varies only little, the quality of the jet, especially of unwanted spraying after the jet tear-off, depends heavily on the pressure history. This work aims at the improvement of the pressure control for dosing the DWP dispensers and the PCR-Slide dispensers. Therefore the pressure run that can be achieved with several concepts are analytically computed to gain comprehension for the important parameters. With the gained knowledge a dispensing station is constructed to realize the concepts of interest. Experimental analysis is used to verify the influence of the pressure run on the dosage quality.
Herstellung von Protein-Microarrays nach dem TopSpot 4-Prinzip
2003, Ruben Kuehlewein
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
KurzfassungA system realizing liquid handling for a bio-sensor is developed in this work. This way, it is possible to detect pathogens according to their genetic code. The invented fluidic processor is able to perform the manual steps automatically. The automatization reveals problems not experienced in the manual process. Processes like mixing of fluids and washing the sensor have been investigated. The samples to be analyzed are saliva and urine. The pretreatment of saliva is complicated and done by another group. The pretreatment if urine is easier. Just the concentration of the bacteria needs to be raised. Thus, the focus of this work is: • realizing of a miniaturized automated concept of existing manual processes • investigating mixing and washing processes • realizing of a cell concentrator
Herstellung von Protein-Mikroarrays nach dem TopSpot 4-Prinzip
2003, Kuehlewein Ruben
Erstgutachter: Prof. Dr. Roland Zengerle
Hydrodynamics in Rotating Systems - Coriolis-Induced Switching and Patterning of Laminar Flows in Rotating Microchannels
2003, Thomas Galtzel
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Kurzfassung
Hydrodynamics in Rotating Systems - Coriolis-Induced Switching and Patterning of Laminar Flows in Rotating Microchannels
2003, Thomas Glatzel
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
Hydrodynamics in Rotating Systems - Coriolis-Induced Switching and Patterning of Laminar Flows in Rotating Microchannels
2003, Glatzel Thomas
Erstgutachter: Prof. Dr. Roland Zengerle
Hydrodynamischer Aufbau von Monolagen aus Bead-Suspensionen in mikrofluidischen Kammern
2003, Michael P. Dobmeier
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Engelbert Westkämper, Uni Stuttgart
KurzfassungThe miniaturisation of conventional medical and protein laboratory procedures gets stronger impact on fundamental research. The central demands made on this miniaturised analysis systems (bio chips) are price reduction compared to conventional methods and time saving by faster analysis at higher quality standards. Two major categories of such analysis systems are distinguished. These are chip based and bead based systems. Beads are spheres with a diameter in the range of μm. They are made of plastic or glass. Beads are favorable in terms of costs and flexible production of individual assays. Fluidic chips, allowing a more conveniently handling of bead based assay systems, need to be developed to enable simple and fast analysis. The goal of this work was to achieve a model-system to provide calculations and to predict the microfluidic behaviour and the aggregation of beads at geometrical barriers in microfluidic chips with commercial CFD (computational fluid dynamics) software. There is no commercial CFD tool available mastering direct simulation of spatial expanded particles. Hence, there is no numerically calculation of bead aggregation (as of July 7th 2003). Three different models were developed and verified to visualize bead aggregation numerically. The model based on coupled two-phase flow with flow through porous media is most feasible using FIDAP (Fluent Inc.) as the most appropriate simulation tool. Experimentally and simulative investigations were carried out to demonstrate that aggregated beads show the same hydrodynamic properties as porous media. The hydrodynamic resistance of aggregated beads was determined experimentally by flow rate measurements and compared to simulations of the flow through porous media. The physical foundations, which this model is based on, are described by the Navier-Stokes-Equation, Stoke's law of friction (two-phase flow simulation) and Darcy's law of flow through porous media. Darcy's law provides a linear relationship between the filter speed of a liquid through a porous media and the pressure gradient at laminar conditions. The two phase flow simulation was used to investigate the flow properties of the beads in the chamber and to calculate their particle trajectories. The regions where the beads aggregate were determined by spatial analysis of trajectories. To adjust the hydrodynamic properties, the regions were switched to porous media. New particle trajectories were calculated based on this new domain. This method was iteratively applied with the use of a developed JAVA program and FIDAP in order to visualize the aggregation of the beads. The simulation results were sucessfully verified by experimental measurements. In conclusion, the specified simulation-model was able predict aggregation patterns close to reality.
Hydrodynamischer Aufbau von Monolagen aus Bead-Suspensionen in mikrofluidischen Kammern
2003, Dobmeier Michael
Erstgutachter: Prof. Dr. Roland Zengerle
Hydrophobic Flow Control on a Rotating Microfluidic Disk
2003, Meike Moschallski
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Gerald Urban, IMTEK
KurzfassungThis thesis investigates technological concepts for the realization of hydrophobic valves on a rotating microfluidic disk. Several types of hydrophobic valves in microstructured channels are designed, fabricated and characterized. The burst frequency of the valve represents the angular velocity, at which the centrifugal force induced on a liquid plug in a rotating channel exceeds the counteracting capillary force at a hydrophobic barrier. The burst frequency of each valve depends on the height and width of the channel as well as the contact angle of the hydrophobic patch, the radial length of the water column acting on the barrier and also the radial position on the disk. Since the impact of each parameter strongly depends on the capabilities of the employed technology, two different technological options are characterized. At first, channels are structured in dry resist technology, locally coated with hydrophobic Teflon solutions and sealed by a dry resist film. In the second approach, microchannels are CNC-milled in PMMA disks. These structures are locally coated and then sealed by a self-adhesive PDMS cover. The functionality of several types of valve structures is tested and characterized with water according to their burst frequencies. Hydrophobic barriers in simple radial channels stop the liquid up to a certain frequency. However standard deviations of even 10 Hz are too high to allow a precise flow control. Extensive investigations show that instable plug lengths caused by vaporization effects at the unsealed inlet decisively affect the frequency characteristics. The strong standard deviation in the plug length can be avoided by decoupling the liquid plug from the inlet reservoir. Based on this, innovative microfluidic metering structures are presented that define a fix plug length of a specific volume and rule out any effects from the inlet. Thus the standard deviation of burst frequencies is reduced from 10 Hz to 0.8 Hz. The implemented burst frequencies range from 7 Hz to 22 Hz in different valves. Sealing of the channels is critical since a deflection of the cover leads to a shift in capillary pressure and thus also to deviations between designed and measured burst frequencies. Therefore, the deflection of the dry resist cover is discussed in detail. Bending of the cover is avoided by a mechanically more stable PDMS cover on microstructures in PMMA. Using this technology, valves are realized featuring exactly the designed burst frequencies. Finally, valves with different burst frequencies are connected in series and in parallel building simple process chains for flow control in a rotating lab-on-a-disk.
Hydrophobic Flow Control on a Rotating Microfluidic Disk
2003, Moschallski Meike
Erstgutachter: Prof. Dr. Roland Zengerle
Konzeption, Umsetzung und Charakterisierung eines TopSpot 5-Gerätes zur hochparallelen Dosierung im Picoliter-Bereich
2003, Ingo Goutier
KurzfassungDie TopSpot-Technologie ist ein Verfahren zur hochparallelen Herstellung von Bio- Chips nach dem Spotting-Verfahren. Das Verfahren bringt ähnlich wie ein Tintenstrahldrucker kleinste Tropfen berührungslos, parallel und in Hochgeschwindigkeit auf den Chip auf. Mehrere hundert oder tausend Spots bilden ein sogenanntes Microarray, mit dessen Hilfe sich biologische und medizinische Proben mit hohem Durchsatz entschlüsseln lassen. Herzstück der Technologie ist ein mikrosystemtechnisch hergestellter Druckkopf, der eine Formatwandlung zwischen eingangsseitigen Reservoiren und dicht angeordneten Düsen vollzieht, durch die pneumatisch Flüssigkeitstropfen ejektiert werden. Das TopSpot-5- Verfahren stellt die Erweiterung des Systems dar und funktioniert nach einem Direktverdrängungsmechanismus, bei dem ein Elastomer in die Düsen verdrängt und dadurch Flüssigkeit ejektiert wird. Es wurde an der Albert-Ludwig-Universität Freiburg, Institut für Mkrosystemtechnik, am Lehrstuhl für Anwendungsentwicklung in Zusammenarbeit mit der Hahn-Schickard Gesellschaft, Villingen-Schwenningen entwickelt. Durch niedrigere Stellwege des Aktors und eine wesentlich höhere Dynamik des Systems lässt sich das Dosiervolumen unterhalb eines Nanoliters variabel einstellen. Die vorliegende Diplomarbeit beschäftigt sich mit der Ausarbeitung und Umsetzung eines Konzeptes für einen funktionalen Prototypen nach dem TopSpot-5-Prinzip, der einen anwendungsbezogenen Betrieb erlaubt. Das neukonzipierte Modul ist kompatibel mit dem TopSpot-4-Modul und erlaubt daher eine einfache Integration in die verschiedenen Top- Spot-Arrayer. Der Prototyp stabilisiert den Druckvorgang bis zu einer Taktrate von zehn Hertz ohne Qualitätsverlust. Zusammen mit den neukonzipierten Druckköpfen konnte die Homogenität der geprinteten Tropfenreihen stark verbessert werden. Schwachpunkte des Systems sind die Kreuzkontamination der Medien beim Einlegen des Elastomers in den Druckkopf und die schlechte Reproduzierbarkeit. Durch die gewonnenen Ergebnisse aus der Charakterisierung lassen sich genaue Forderungen hinsichtlich Anpassung des Druckkopfdesigns und der Ansteuerung formulieren, um die Effizenz, die Zuverlässigkeit und die Dosiergenauigkeit zu erhöhen. Vor allem konnten den Systemgrenzen Ursachen zugewiesen und Methoden zur Erweiterung des Dosierspektrums evaluiert werden. Durch die Nutzung unterschiedlicher Elastomere und Druckköpfe kann mit dem TopSpot-5-Modul eine variable Dosierung von 0,08 bis 1 nl vorgenommen werden. Die im Vorgängerprojekt festgestellten Zusammenhänge konnten verifiziert werden. Wichtigster Punkt ist die proportionale Abhängigkeit zwischen dem ejektierten Tropfenvolumen und der Piezoauslenkung. Durch Kenntnis des dynamischen Verhaltens des Piezoaktors konnten Kennlinien ermittelt werden, die eine direkte Einstellung des zu dosierenden Volumens ohne iteratives Nachregeln ermöglichen.
Konzeption, Umsetzung und Charakterisierung eines TopSpot-5-Gerätes zur hochparallelen Dosierung im Picoliter-Bereich
2003, Goutier Ingo
Erstgutachter: Prof. Dr. Roland Zengerle
Messplatz zum Aufbau von Monolagen aus Strömungen von Bead-Suspensionen in mikrofluidischen Kammern
2003, Patric Schippers
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Engelbert Westkämper, Uni Stuttgart
KurzfassungAutomated processing and analysis of biological fluids (e.g. human blood) is a major goal of medical diagnostics. Lab-on-a-chip concepts are subject of extensive R&D and first products have been launched on the market. They allow point-of-care applications which are preferable in terms of costs, ease of handling and time-to-results. Biomolecular layers on surfaces or beads are a common means for detection. Bead based detection concepts show on the one hand reduced diffusion times and avoids, via an offchip preparation of the beads, the exposure of biosensitive layers to the typically harsh conditions during microfabrication and sealing. On the other hand, bead-based assays are amenable to multiplexing, i.e., the read-out of multiple tests in the same biochemical assay in parallel. This work is part of the novel "Bio-Disk" initiative where the concentration of various antibodies in whole blood is determined by a biochemical ELISA immunoassay in parallel. The „Bio-Disk“ is a centrifugal "lab-on-a-disk" platform which have in recent years been considered as an elegant means for processing fluids without pumps and other active on-chip components. Also this work is a part of the investigation of a planar fluidic chip of the Fraunhofer IPA. This work presents the investigation of hydrodynamic filling of a flat detection chamber. Highly periodic and mechanically stable monolayers of beads were created. The main aggregation patterns are hexagonal and single cubic with corresponding packing densities of 90,69% and 78,54 % respectively. The filling ratio depends on the inlet pressure and on the chamber design. An average filling ratio of more than 94% has been achieved reproducably. By elevating the inlet pressure, we achieved short filling times in range of a few seconds. In the theoretical part of this work, the beads have been handled as a discrete problem. The dimensions of the chamber was conditioned by the diameter of the beads. Since the form of the chamber had an impact on the aggregation pattern, several microfluidic devices for bead-based analyses were designed and fabricated in PMMA, based on theorethical geometrical considerations. Each microfluidic device consists of one inlet channel, one flat aggregation chamber, measuring 190 μm in depth, for the beads with 176 μm in diameter and several outlet channels. Suspensions of beads are loaded into the flat chamber by a pressure driven flow. With the depth smaller than a bead diameter, the outlets act as barriers to the beads and force them to accumulate in the chamber. Therefore, the decisive impact parameters are the geometry, the particle concentration of suspension and the inlet pressure. It has been discovered, that a chamber with one outlet and a parallelogram form with an angle of 60° enhanced the hexagonal aggregation pattern. In order to avoid disturbances by centrifugal forces on the bio-disk a simplified model sytem was established. Therefore the development, the construction and the evaluation of an experimental set-up was needed, which had enabled the controlled initiation and monitoring of the beads in the aggregation chamber. For each measurement cycle, the reservoirs are initially filled and then the gas-pressure is set to the nominal value. After reaching the nominal level, the two valves are opened and the filling process is observed with a 3CCD-Camcorder (Canon XL 1) at 24 frames/sec and a spatial resolution of 720 x 480 pixel. Special attention is paid to local voids and boundary lines between areas of high periodicity. It was found out that during filling process, local voids are partially cured. The boundary lines, however, remained stable. To determine the number of aggregated beads, an image of the completely filled chamber was taken by a digital camera (Nikon Coolpix 995). The image file is then processed and the number of beads is calculated automatically (NeuroCheck, rel. 5.0, NeuroCheck GmbH, Germany).
Messplatz zum Aufbau von Monolagen aus Strömungen von Bead-Suspensionen in mikrofluidischen Kammern
2003, Schippers Patric
Erstgutachter: Prof. Dr. Roland Zengerle
Microfluidic Sample Preparation System for Cellular DNA detection
2003, Michael Vosseler
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Chih-Ming Ho, School of Engineering and Applied Science Engineering IV, Los Angeles, USA
KurzfassungA system realizing liquid handling for a bio-sensor is developed in this work. This way, it is possible to detect pathogens according to their genetic code. The invented fluidic processor is able to perform the manual steps automatically. The automatization reveals problems not experienced in the manual process. Processes like mixing of fluids and washing the sensor have been investigated. The samples to be analyzed are saliva and urine. The pretreatment of saliva is complicated and done by another group. The pretreatment if urine is easier. Just the concentration of the bacteria needs to be raised. Thus, the focus of this work is: • realizing of a miniaturized automated concept of existing manual processes • investigating mixing and washing processes • realizing of a cell concentrator
Microfluidic Sample Preparation System for Cellular DNA detection
2003, Vosseler Michael
Erstgutachter: Prof. Dr. Roland Zengerle
Nanoliterdispenserarray Dispensing Well Plate: Entwicklung eines neuen Fertigungsverfahrens und Designoptimierung
2003, Bohl Benjamin
Erstgutachter: Prof. Dr. Roland Zengerle
Optimierung der Microarrayqualität & Erweiterung des Printmedienbereichs mit TopSpot4
2003, Remigius Niekrawietz
Optimierung der Microarrayqualität & Erweiterung des Printmedienbereichs mit TopSpot 4
2003, Niekrawietz Remigius
Erstgutachter: Prof. Dr. Roland Zengerle
Zweitgutachter: Prof. Dr. Hermann Sandmaier
Optimization of the TopSpot/4 Microarray Printing Principle
2003, Jürgen Steindl
KurzfassungTopSpot technology is a method for highly parallel and simultaneous delivery of a multitude of reagents in the nanoliter range for producing microarrays. With microarrays, also called biochips, biological probes can be analyzed or decoded. They consist of a substrate (e.g. glass slide) as ground plate, and a large number of known substances, placed in regular pattern on a small area, enabling high throughput analysis. The TopSpot technology is in the group of non-contact microarray printing devices. The actuation principle is based on piezo excitation and pneumatic compression of an air chamber through a piston. The core piece is a micromachined printhead, which can deliver up to 384 droplets at once. It fulfils the format conversion from a standard reservoir pattern to a 0.5 mm pitch pattern, which enables packing up to thousands of little droplets onto a small area. This diploma thesis presents new kinds of piston designs for this device. Three main changes have been approached and characterized. It ranged from minor changes on the existing device, to major piston changes. The key was to reduce the dead volume in the existing system. In the existing system, a lot of different parts are involved in the actuation process, which have to have very tight tolerances to make the device working. This leads to a very cost intensive production. The presented pistons break this ’tolerance- chain’ and could make the device much cheaper. Also efforts were made to eliminate some of the parameters on the existing device. At present time, there are two different main parameters, which change the stroke of the piston, and which have influences on each other. The controllability of the piston, which does the compression of the air chamber was not achieved, but with one of the new piston designs, it is possible to realize that very easy, but due to lack of time this was not finished. A ventilation drilling, which is in the actual piston could be proven to be unnecessary, which would simplify the production. It was found out, that the droplet size is hardly adjustable with the actual parameters.
Optimization of the TopSpot/4 Microarray Printing Principle
2003, Steindl Jürgen
Erstgutachter: Prof. Dr. Roland Zengerle
Parameterstudie mittels CFD - Simulation über den Dosiervorgang beim Nanomapdispenser
2003, Kalix Jan
Erstgutachter: Prof. Dr. Roland Zengerle
Parameterstudie mittels CFD – Simulation über den Dosiervorgang beim Nanomapdispenser
2003, Jan Kalix
KurzfassungDie Analysesysteme in der modernen Biotechnologie sind einer immer weitergehenden Miniaturisierung unterworfen. Zusammen mit einer steigenden Automatisierung ist so eine parallele Prozessierung tausender Proben möglich, die auch die Ansprüche der Pharmaforschung an einen hohen Probendurchsatz befriedigt. Um die dabei auftretenden Flüssigkeitsmengen im Nanoliterbereich zu prozessieren werden höchste Ansprüche an die Integration und Genauigkeit der Dosiersysteme gestellt Für diese Anforderungen wird im Nanomap-Projekt eine multifunktionale, modulare Analyseplattform entwickelt, auf der biochemische Prozesse zur Analyse von DNAProben ablaufen. Integriert ist eine Dispensiereinheit, die es erlaubt, wenige Nanoliter der prozessierten DNA-Probe auf eine externe Dekodiereinheit zu dosieren. Der Dosiervorgang erfolgt dabei nach dem DWP-Verfahren (Dispensing Well Plate), bei dem eine auf dem Nanomapchip integrierte Düse kapillar befüllt wird und nachfolgend mittels eines pneumatischen Druckpulses entleert wird. Diese Diplomarbeit untersucht den Dosiervorgang im Hinblick auf Medienunabhängigkeit und Dosiergenauigkeit. Zum besseren Verständnis der Vorgänge innerhalb des Dispensers wird eine theoretisches Modell entwickelt. Zur Bestätigung und Erweiterung dieses Modells wird eine auf numerischen Strömungssimulationen beruhende Parameterstudie durchgeführt.
Sensoric Concepts for the NanoJet Dispensing Device
2003, Wolfgang Sreule
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
KurzfassungQuality control generally plays an essential role in automated processes and especially if MST components are involved. In miniaturized devices the functionality cannot be monitored by an operator but has to be controlled by internal sensing elements. In this work concepts are studied how to implement quality control into a specific micro dispensing device termed NanoJet dispenser. This device delivers liquid volumes in the range of several nanoliters as free flying jets and is used in Life-Science and industrial applications. In contrast to inkjet dispensers the NanoJet device relies on a direct displacement principle involving a silicon membrane. This makes it possible to use a broad range of sensors like pressure, force and flow sensors to detect the dispenser’s status. But also other concepts like optical detection of the liquid jet are studied, which can be applied to dispensers and inkjet printers in general. The work provides a comprehensive assessment of possible sensor options, including feasibility study, prototype design, manufacturing and cost issues. Prototypes of selected concepts are characterized experimentally. Based on these results recommendations for reliable and cost-effective quality control concepts are given.
Sensoric Concepts for the NanoJet Dispensing Device
2003, Streule Wolfgang
Erstgutachter: Prof. Dr. Roland Zengerle
The Channel-In-Channel (CHIC) principle – A study on bubble management in microfluidic systems
2003, Christian Litterst
Erstgutachter: Prof. Dr. Roland Zengerle, IMTEK
Zweitgutachter: Prof. Dr. Jan G. Korvink, IMTEK
KurzfassungIt is well known in microsystem technology that surface effects can have a large impact on system behavior. Especially in microfluidic systems problems can be caused by gas bubbles; those bubbles are often trapped inside channels while having either a small mobility or even clog the channel completely. To manage embedded gas bubbles passively is the objective of this work. The Channelin- Channel-concept, also called CHIC studied here, is one possible concept to handle this problems owing to its outstanding feature to be tolerant for gas bubbles clogging the channel. Reason for this tolerance is the T-shaped geometry of two nested rectangular channels and the wetting angle, forcing the bubble to one part of the channel. Thus only one part of the channel is clogged while the other remains unaffected. Tenor of this diploma thesis is the evaluation of the functional principle of this concept with the aid of measurements as well as by simulation. Within those experiments different channel geometries, fluids and also environmental working conditions will be considered to determine the influence of system parameters on the performance.
The Channel-In-Channel (CHIC) principle – A study on bubble management in microfluidic systems
2003, Litterst Christian
Erstgutachter: Prof. Dr. Roland Zengerle

2002

Bubble Handling in DNA Electrophoresis with Nanotraps
2002, Thilo Brenner
Bubble Handling in DNA Electrophoresis with Nanotraps
2002, Brenner Thilo
Erstgutachter: Prof. Dr. Y. Zohar
Zweitgutachter: Prof. Dr. Roland Zengerle
KurzfassungProblems with gas bubbles in microfluidic structures are a major issue. The strategy to handle these problems is often decisive for the success of microfluidic systems. Micro Capillary Electrophoresis (MCE) Devices need solutions with the integration of electrodes which avoid channel blocking by gas bubbles generated by hydrolysis. Nanostructures used as an alternative to conventional polymer sieving structures have to be cleaned from bubbles captured in the fluidic channels. This work presents innovative concepts to avoid channel blocking by gas bubbles. The channels and chambers at the electrode are modified to guide bubbles out of the fluidic system. The principles are verified on fabricated test structures. Two solutions are presented: electrodes sitting on conical channels and electrodes in chambers with sharp corners. The idea of alternative sieving structures for DNA separation by columns and nanotraps is introduced to the reader. Taking the coil radius of the DNA fragments as the main design rule, the structures scale in nanodimensions. Successful fabrication of nanotraps is demonstrated. Shallow channels of only 100 nm are accurately formed by sacrificial etching of a locally grown oxide layer. Bubbles occupy very stable positions in these extraordinary small dimensions and thus special cleaning procedures are developed. Gas bubbles can be washed out from the traps by electroosmotic flow. DNA injection can electrophoretically form a defined plug. When the capillaries are filled with buffer solution only, the electroosmotic flow has to be suppressed. Increasing the ionic strength of the buffer from standard 0.5x TBE to 3x TBE reduces the flow rates significantly. As a result of this work, several fully working devices are fabricated which can be used for subsequent DNA experiments. The geometrical effects of 14 bp DNA fragments in capillaries with agarose gel are investigated. Mobilities and diffusion behavior under different gel concentrations are measured. Furthermore on-chip impedance measurements for DNA detection show promising results regarding an integration of DNA sensors.
Computational Fluid Dynamic (CFD) Simulation of NanoJet Dispenser
2002, Jan Hansen-Schmidt
Computational Fluid Dynamic (CFD)Simulation of NanoJet Dispenser
2002, Hansen-Schmidt Jan
Erstgutachter: Prof. Dr. Roland Zengerle
Design and Characterization of a 384 Channel Micromachined Printhead Using TopSpot Technology
2002, Wolf Wibel
KurzfassungFor bio-analyses done in life-science, like DNA analytics, a working horse to achieve a large number of analyses in parallel is the use of so-called “microarrays”. TopSpot printing technology has been developed for cheap mass-production of microarrays. It covers a family of printheads with various numbers of nozzles and a fitting print module for the actuation of the printheads to generate the microarrays. Actuation hardware using these print modules are available for small quantity production of microarrays for small laboratories up to industrial mass-production systems. An advantage of TopSpot in comparison to its competitors is the parallel non-contact spotting of all substances: The media filled into the printhead’s reservoirs are reformatted via micro-channels etched into a silicon layer to the size of the microarray. To print all spots in common one single air pulse is applied on the nozzle array of the printhead and the droplets are ejected. In this work a new printhead compatible to the TopSpot print module was developed which is capable to print 384 different substances in parallel (up to now the most complex TopSpot printhead had 96 nozzles.). For this goal first a general research of dependencies of printhead parameters as channel and nozzle dimensions and production technology was done which resulted in so-called “design rules”. These design rules can easily be used to determine parameters of future TopSpot printheads. A practical application of the design rules was the realisation of the so far largest TopSpot printhead with 384 nozzles. Using the rules a fitting layout for the new printhead was developed: To have the same arrangement as in a 384 well microtitre plate, a 24 x 16 nozzle matrix was chosen . These nozzles are connected via micro-channels to two 24 x 8 reservoir arrays. As the pitch of the nozzles had to be enlarged to 1 mm to enable the placement of all channels needed between the outer nozzles, a complete 1536 spot microarray with a pitch of 0.5 mm can be printed in only four passes, enabling the reproduction of all substances of a 1536 well microtitre plate. The diameter of the reservoirs is 1.8 mm, their pitch 2.25 mm. These are the same parameters as for the wells of a 1536 microtitre plate. Therefore easy filling of the reservoirs using pipeting robots is supported. The complex layout was drawn, masks were produced and the established clean-room process for the silicon micro-structuring of the microfluidic structures of TopSpot printheads took place. To use the new printhead in the existing print modules a fitting adapter was developed. As there were delays in the production, only two printheads could be completed within the diploma thesis. A short characterization of the printheads took place. All micro-channels showed self-filling capability, 384 spot microarrays using the new printhead and a common TopSpot print module were successfully printed, proofing the functioning of TopSpot technology even for this new large 384 nozzle printhead. Further tests showed high reproducibility of the spots as - with one filling of the printhead’s reservoirs - hundreds of equivalent microarrays were printed. Tests were done to check the prevention of cross-contamination between the 50 μm micro-channels of the printhead. No cross-contamination between the channels could be observed. The printhead’s performance and its ability for mass-production was proven as more than thousand printings could be done with one filling of the reservoirs. 1536 spot microarrays with a pitch of 0.5 mm were printed in four printing passes. An outlook for the further development of TopSpot technology is given focusing on the introduction of additional silicon layers to enable denser and larger nozzle arrays in a multi-layer construction to make the production of large microarrays even faster.
Design and Characterization of a 384 Channel Micromachined Printhead Using TopSpot Technology
2002, Wibel Wolf
Erstgutachter: Prof. Dr. Roland Zengerle
Konzeption eines bistabilen Mikroventils
2002, Christoph Brüggenolte
KurzfassungFür ein implantierbares Medikamentendosiersystem, bei dem es naturgemäß auf geringen Energieverbrauch ankommt, wurde nach Konzepten für ein bistabiles Mikroventil gesucht. Dabei wurde ausgiebig recherchiert, welche bistabilen Mechanismen in der Mikrosystemtechnik bereits angewandt wurden. Das Ventil wurde in Subsysteme zerlegt, und in einer morphologischen Matrix wurden für die einzelnen Subsysteme Lösungen erörtert. Die Subsystemlösungen wurden zu Ventilkonzepten kombiniert und theoretisch bewertet. Zwei Konzepte wurden ausgewählt und näher betrachtet: Das Konzept Selbsthaltung, das darauf beruht, dass die vom Einlassdruck angegriffene Fläche bei geeigneter Verschaltung abhängig vom Schaltzustand des Ventils auf verschieden große Flächen angreift, konnte ausgeschlossen werden. Die prinzipielle Machbarkeit eines Ventils, dessen bistabiles Verhalten auf Schnappscheiben basiert, konnte anhand eines Demonstrators gezeigt werden.
Konzeption eines bistabilen Mikroventils
2002, Brueggenolte Christoph
Erstgutachter: Prof. Dr. Roland Zengerle
KurzfassungFür ein implantierbares Medikamentendosiersystem, bei dem es naturgemäß auf geringen Energieverbrauch ankommt, wurde nach Konzepten für ein bistabiles Mikroventil gesucht. Dabei wurde ausgiebig recherchiert, welche bistabilen Mechanismen in der Mikrosystemtechnik bereits angewandt wurden. Das Ventil wurde in Subsysteme zerlegt, und in einer morphologischen Matrix wurden für die einzelnen Subsysteme Lösungen erörtert. Die Subsystemlösungen wurden zu Ventilkonzepten kombiniert und theoretisch bewertet. Zwei Konzepte wurden ausgewählt und näher betrachtet: Das Konzept Selbsthaltung, das darauf beruht, dass die vom Einlassdruck angegriffene Fläche bei geeigneter Verschaltung abhängig vom Schaltzustand des Ventils auf verschieden große Flächen angreift, konnte ausgeschlossen werden. Die prinzipielle Machbarkeit eines Ventils, dessen bistabiles Verhalten auf Schnappscheiben basiert, konnte anhand eines Demonstrators gezeigt werden.
Numerical Simulation of Flow Control and Mixing in a Centrifugally Driven Microfluidic System
2002, Volker Materna
Numerical Simulation of Flow Control and Mixing in a Centrifugally Driven Microfluidic System
2002, Materna Volker
Erstgutachter: Prof. Dr. Roland Zengerle
Plasma emission microstructures for gas chromatography
2002, Bernd Schöberle
Plasma emission microstructures for gas chromatography
2002, Schoeberle Bernd
Erstgutachter: Prof. Dr. Roland Zengerle
Simulation und Design eines mikrofluidischen Systems zur Dosierung von Volumina in Nanoliterbereich
2002, Salah Taoufik
Simulation und Design eines mikrofluidischen Systems zur Dosierung von Volumina in Nanoliterbereich
2002, Taoufik Salah
Erstgutachter: Prof. Dr. Roland Zengerle
Zweitgutachter: Prof. Dr.-Ing. J. Betten

2001

DWP Dispensing Well Plate - Untersuchungen an einem Mikrosystem zur hochparallelen Nanoliterdosierung
2001, Bastian Birkenmeier
Large Drop Actuators - Characterization and Improvement of Xaar Printhead
2001, Kaack Rolf
Erstgutachter: Prof. Dr. Roland Zengerle
Tunable and Highly Parallel Picoliterdispensing Based on Direct Liquid Displacement
2001, Chris Steinert
KurzfassungThe presented diploma thesis was carried out from may to november 2001 at the institute for microsystem technology (IMTEK), university of Freiburg, Germany. Tutors were Prof. Dr. Roland Zengerle, head of chair for application development and Dr. Bas de Heij. A new method for a tunable, highly parallel and simultaneous delivery of a multitude of reagents in the picoliter range is presented. A massive parallel analysis of thousands of different substances is one need of biotechnology. Microarrays are a great number of known substances, deposited in a regular order on a small area, enabling high throughput analysis. There are several approaches for manufacturing microarrays. One of them is the already used TopSpot method, developed by HSG-IMIT in association with BioChip Technologies GmbH. It represents a non-contact microarray printing technology basing on pneumatic actuation, which generates the pressure pulse for droplet ejection. Up to 96 droplets can be ejected in parallel with the silicon micromachined TopSpot printhead. The next generation of the TopSpot driving mechanism should use a more effective direct liquid displacement method instead of pneumatic actuation. Therefore, an incompressible, but deformable, elastomer replaces the air chamber. During a piezo stack actuation a certain amount of the elastomer is pushed into the nozzles and generates droplet ejection. Compared with the pneumatic actuation method, which needs a 80 μm piezo stroke, even a 1 μm stroke is sufficient to force a droplet ejection. The amount of elastomer displaced into the nozzles can be controlled, in a certain range, by the piezo stroke. Latter can be tuned by a defined piezo voltage amplitude and improves the new driving mechanism to a tunable liquid dispensing method. Droplet volumes of 0.05 nl to 0.83 nl are possible, if one nozzle is used. A range of 0.1 to 0.6 nl can be achieved for the parallel droplet ejection out of 24 nozzles. Main problems are placing of the elastomer stamp into the TopSpot printhead without causing cross-contamination and a homogenous, reproducible droplet ejection out of all nozzles. Properties of the piezo stack actuator, the elastomer, the TopSpot printhead and the dispensed liquid were investigated referring to their influences on the new TopSpot driving mechanism. Diluted black ink was used to print microarrays with tunable spot diameters of 80 μm to 180 μm without causing cross-contamination. The printed arrays showed a standard deviation of the spot diameter below 4 %.
Tunable and Highly Parallel Picoliterdispensing Based on Direct Liquid Displacement
2001, Steinert Chris P
Erstgutachter: Prof. Dr. Roland Zengerle
KurzfassungThe presented diploma thesis was carried out from may to november 2001 at the institute for microsystem technology (IMTEK), university of Freiburg, Germany. Tutors were Prof. Dr. Roland Zengerle, head of chair for application development and Dr. Bas de Heij. A new method for a tunable, highly parallel and simultaneous delivery of a multitude of reagents in the picoliter range is presented. A massive parallel analysis of thousands of different substances is one need of biotechnology. Microarrays are a great number of known substances, deposited in a regular order on a small area, enabling high throughput analysis. There are several approaches for manufacturing microarrays. One of them is the already used TopSpot method, developed by HSG-IMIT in association with BioChip Technologies GmbH. It represents a non-contact microarray printing technology basing on pneumatic actuation, which generates the pressure pulse for droplet ejection. Up to 96 droplets can be ejected in parallel with the silicon micromachined TopSpot printhead. The next generation of the TopSpot driving mechanism should use a more effective direct liquid displacement method instead of pneumatic actuation. Therefore, an incompressible, but deformable, elastomer replaces the air chamber. During a piezo stack actuation a certain amount of the elastomer is pushed into the nozzles and generates droplet ejection. Compared with the pneumatic actuation method, which needs a 80 μm piezo stroke, even a 1 μm stroke is sufficient to force a droplet ejection. The amount of elastomer displaced into the nozzles can be controlled, in a certain range, by the piezo stroke. Latter can be tuned by a defined piezo voltage amplitude and improves the new driving mechanism to a tunable liquid dispensing method. Droplet volumes of 0.05 nl to 0.83 nl are possible, if one nozzle is used. A range of 0.1 to 0.6 nl can be achieved for the parallel droplet ejection out of 24 nozzles. Main problems are placing of the elastomer stamp into the TopSpot printhead without causing cross-contamination and a homogenous, reproducible droplet ejection out of all nozzles. Properties of the piezo stack actuator, the elastomer, the TopSpot printhead and the dispensed liquid were investigated referring to their influences on the new TopSpot driving mechanism. Diluted black ink was used to print microarrays with tunable spot diameters of 80 μm to 180 μm without causing cross-contamination. The printed arrays showed a standard deviation of the spot diameter below 4 %.
Untersuchungen an einem Mikrosystem zur hochparallelen Nanoliterdosierung
2001, Birkenmeier Bastian
Erstgutachter: Prof. Dr. Roland Zengerle

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