Theses

• Habilitation
• Phd thesis
• Master thesis
• Bachelor thesis
• Diploma thesis

2020

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

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2017

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

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2016

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

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2014

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

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2005

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

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2024

• A Novel Multianalyte Lateral Flow Immunoassay for the Simultaneous Detection of Protein and Nucleic Acid Biomarkers
  2024, Anna Klebes
  First reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK - Chemie und Physik von Grenzflächen
  

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• Investigation of salt precipitation and water management in zero-gap CO2 electrolyzers producing CO
  2024, Joey Disch
  First reviewer: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Ingo Krossing, Institut für Anorganische und Analytische Chemie Albert-Ludwigs-Universität Freiburg
  

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2023

• Development and quantitative assessment of generic processes to enable 3D-bioprinting of arti cial tissue
  2023, Fritz Koch
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Günter Finkenzeller, Uniklinik Freiburg
  

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• Electrode engineering for anion-exchange membrane water electrolyzers
  2023, Susanne Koch
  First reviewer: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung/Elektrochemische Energiesysteme
  Second reviewer: Prof. Dr. Stefan Glunz, INATECH, Freiburg
  

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• Fantastic nanoparticles and where to find them: Advanced electron microscopy for the investigation of novel electrocatalysts
  2023, Philipp Heizmann
  First reviewer: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Anna Fischer, Institut für Anorganische und Analytische Chemie, Uni Freiburg
  

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• Optimization and characterization of proton-exchange membrane fuel cells based on novel hydrocarbon ionomers
  2023, Hien Nguyen
  First reviewer: Dr. Severin Vierrath, EES-Group, IMTEK
  Second reviewer: Prof. Dr. Thomas Hanemann, IMTEK - Werkstoffprozesstechnik
  

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2022

• Automation of library preparation for next generation sequencing by centrifugal microfluidics
  2022, Jacob Hess
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Melanie Börries, Institut für Medizinische Bioinformatik und Systemmedizin, Uni Freiburg
  

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• Bound-free phase detection immunoassay compatible with nucleic acid amplification point-of-care devices
  2022, Benita Johannson
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
  

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• Characterization of Membrane Electrode Assemblies for Vanadium Redox Flow Batteries
  2022, Brian Shanahan
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
  

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• Detection and Enumeration of Bacteria by Pump-free Digital Droplet Assays
  2022, Yu-Ting Kao
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Piotr Garstecki, ICHF, Warschau
  

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• Liquid Biopsy for colorectal cancer – from cfDNA extraction to the quantification of point mutations by multiplexed ddPCR
  2022, Franziska Schlenker
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: PD Dr. Cornelia Eckert, Charite Universitätsmedizin Berlin
  

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• Microfluidic Automation of Sample Preparation for Mass Spectrometry Based Proteomics
  2022, Niklas Klatt
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Oliver Schilling, Inst. Für Molekulare Medizin, Uni Freiburg
  

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2021

• Fabrication of hollow 3D tissue models with drop-on-demand bioprinting and controlled cellular self-assembly
  2021, Kevin Tröndle
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Soeren Lienkamp, Universität Zürich, Schweiz
  

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• Microfluidic system integration for droplet based digital nucleic acid testing
  2021, Martin Schulz
  First reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK - Chemie und Physik von Grenzflächen
  

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• Normally-closed dispensing valve and in-line pressure and flow sensor fabricated in low-cost polymer technology
  2021, Sabrina Kartmann
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Joost Lötters, University of Twente
  

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2020

• Development of Novel Electrode Concepts for Proton Exchange Membrane Water Electrolyzers
  2020, Melanie Bühler
  First reviewer: Prof. Dr. Stefan Glunz, INATECH, Uni Freiburg
  Second reviewer: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
  

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• Mediator displacement LAMP: A novel method for universal sequence-specific detection of isothermal nucleic acid amplification
  2020, Lisa Becherer
  First reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK - Chemie und Physik von Grenzflächen
  

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  Abstract

  Nucleic 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: PD Dr. Cornelia Eckert, Charité, Berlin
  

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  Abstract

  Minimal 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
  First reviewer: Prof. Dr. Stefan Glunz, INATECH, Uni Freiburg
  Second reviewer: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
  

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  Abstract

  Sustainably 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
  First reviewer: Prof. Dr. Stefan Glunz, INATECH, Uni Freiburg
  Second reviewer: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
  

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  Abstract

• Reduction of system complexity in centrifugal microfluidics by magnetophoresis at continuous rotation and thermo-pneumatic bubble mixing
  2020, Sebastian Hin
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Bastain Rapp, IMTEK - Prozesstechnologie
  

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  Abstract

  Centrifugal 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
  First reviewer: Prof. Dr. Lars Pastewka, IMTEK - Simulation
  Second reviewer: Prof. Dr. Simon Thiele, Helmholtz-Institut Erlangen-Nürnberg für Erneuerbare Energien (HI ERN)
  

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  Abstract

2019

• Characterization and optimization of anodes for microbial electrochemical technologies treating wastewater
  2019, Joana Madjarov
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
  Second reviewer: Prof. Uwe Schröder, TU Braunschweig
  

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• Frequenzgesteuertes Öffnen von Stickpacks in zentrifugalen Mikroanalysesystemen
  2019, Daniel Baumann
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jens-Peter Majschak, TU Dresden
  

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• Handling of Microparticles for Assay Automation in Centrifugal Microfluidics
  2019, Yunpeng Zhao
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Ulrike Wallrabe, IMTEK - Mikroaktorik
  

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  Abstract

• Highly parallel microbioreactors for cell line development based on well plates with functional lids
  2019, Cheng-Han Tsai
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
  Second reviewer: Prof. Dr. Joost Lötters, University of Twente, Niederlande
  

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• LabSlice XL – A centrifugal microfluidic cartridge for the automated bio-chemical processing of industrial process water
  2019, Stefan Burger
  First reviewer: Prof. Dr. Roland Zengerle, Anwendungsentwicklung
  Second reviewer: Prof. Dr. Anja Boisen, Technical University of Denmark
  

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• Lumped model simulation for the fast development of robust centrifugal-microfluidic lab-on-a-chip systems
  2019, Ingmar Schwarz
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jens Ducrée, Dublin City University, Irland
  

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• Pneumatic operations in centrifugal microfluidics - An enabling technology for assay automation
  2019, Steffen Zehnle
  First reviewer: Prof. Dr. Roland Zengerle, Anwendungsentwicklung
  Second reviewer: Prof. Dr. Ulrike Wallrabe, Mikroaktorik
  

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• Screening of electrode materials for microbial fuel cells operating with S. oneidensis and G. sulfurreducens
  2019, Elena Kipf
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. J. Gescher, KIT Karlsruhe
  

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• Single-cell dispensing for isolation and analysis of individual mammalian and microbial cells
  2019, Julian Riba
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: PD Dr. Heiko Becker, Uniklinik Freiburg
  

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  Abstract

• StarJet printheads for printing molten solder jets at 320°C and molten aluminum alloy droplets at 950°C
  2019, Björn Gerdes
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jürgen Wilde, IMTEK / Aufbau- und Verbindungstechnik
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
  

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  Abstract

2018

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

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  Abstract

2017

• Advances in Fabrication and Characterization of Functional Layers in Polymer Electrolyte Fuel Cells
  2017, Matthias Klingele
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Richard Hanke-Rauschenbach, IfES - Leibniz Universität Hannover
  

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  Abstract

  Hydrogen 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
  

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  Abstract

  The 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Stefan Glunz, Fraunhofer ISE
  

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• Microfluidic Apps for centrifugal thermocyclers at constant rotational frequency
  2017, Mark Keller
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
  

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  Abstract

  Lab-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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Richard Hanke-Rauschenbach, IfES - Leibniz Universität Hannover
  

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  Abstract

  In 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Ulrike Wallrabe, IMTEK - Mikroaktorik
  

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  Abstract

  Die 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof.in Dr.-Ing. Ellen Ivers-Tiffée , KIT Karlsruhe
  

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  Abstract

  In 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Norbert Willenbacher, KIT, Karlsruhe
  

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  Abstract

  Within 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jens Ducrée, Dublin City University, Dublin/Irland
  

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  Abstract

  Diagnostics 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jens Ducrée, Dublin City University, Dublin/Irland
  

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  Abstract

  The 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Piotr Garstecki, Polish Academy of Sciences, Warschau/Polen
  

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  Abstract

  Medical 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
  First reviewer: PD Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: PD. Dr. Fritz Schwarzmann, Uni Regensburg
  

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  Abstract

  Mediator 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Wilfried Weber, Biologie, Uni Freiburg
  

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  Abstract

  Metabolites 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK - Sensorik
  

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  Abstract

  IntelliDrug 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. M. Reth, MPI für Immunbiologie und Epigenetik
  

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  Abstract

  Signaling 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Holger Reinecke
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK - Chemie und Physik von Grenzflächen
  

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  Abstract

  Molecular 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Alexander Rohrbach, IMTEK - Bio- und Nanophotonik
  

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  Abstract

  The 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
  First reviewer: Prof. Dr. Roland Zengerle
  Second reviewer: Prof. Dr. Gerald Urban
  

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  Abstract

  Trends 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
  First reviewer: Prof. Dr. Roland Zengerle
  Second reviewer: Prof. Dr. Gerald Urban
  

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  Abstract

  Botulinum 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Johannes Gescher, KIT/IAB, Karlsruhe
  

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  Abstract

  Biofuel 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Andreas Guber, KIT, Mikrofluidik
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Two phase flow drop formation in StarJet system: Simulations for molten metal and development of superhydrophobic nozzle for aqueous liquids dosage
  2014, Artur Tropmann
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jan Korvink, IMTEK
  

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  Abstract

  The 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

  Assumptions 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Hans Peter Wendel, Universitätsklinikum Tübingen
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK
  

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  Abstract

  All 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK
  

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  Abstract

  A 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

  Implantable 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: PD Dr. habil rer. nat. Manfred Weidmann , Georg-Augustus-Universität Göttingen
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jens Ducrée, School of Physical Sciences, Dublin City University
  

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  Abstract

  Countless 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Leonahrd Reindl, IMTEK
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Margit Zacharias, IMTEK
  

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  Abstract

  Polymer 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

  A 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Ulrike Wallrabe, IMTEK
  

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  Abstract

  The 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

• Non-contact Dispensing Technologies for Printing of Single Cells
  2012, Azmi Bin Mohamed Yusof
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jan G. Korvink, IMTEK
  

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  Abstract

  Key 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Peter Woias, IMTEK
  

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  Abstract

  The 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

  Two 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

  The 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

  A 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

• Passive and self-regulating fuel supply in direct methanol fuel cells
  2010, Nils Paust
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

• Passive components for the control of bubbles and droplets in microstructures
  2010, Tobias Metz
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  

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  Abstract

  In 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jan G. Korvink, I
  

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  Abstract

  One 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Hermann Sandmeier, IFF, Uni Stuttgart
  

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  Abstract

  The 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK
  

• The Twin Epipoly Process: A Foundry Process Flow for a Micrfluidic Application Kit in Silicon/Glass
  2008, Tjalf Pirk
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jürgen Wilde
  

2007

• Droplet Generation From the Nanoliter to the Femtoliter Range
  2007, Timo Lindemann
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jan G. Korvink, IMTEK
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  

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  Abstract

  Subject 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Ullrich Certa, F. Hoffmann La-Roche, Basel
  

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  Abstract

  The 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Ulrike Wallrabe, IMTEK
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jan G. Korvink, IMTEK
  

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  Abstract

  This 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. W. Menz, IMTEK
  

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  Abstract

  In 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.

2024

• Automation of DNA Clean-Up by Centrifugal Microfluidics
  2024, xx
  

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  Abstract

• Hybrid 3D-bioprinting towards an advanced bone healing model and in vivo testing
  2024, xx
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Optical detection of multicellular spheroids in nanolitre sized free falling droplets
  2024, xx
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: PD. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  

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  Abstract

2023

• Automation of hybrid capture based target enrichment method for whole exome sequencing by centrifugal microfluidics
  2023, XX
  First reviewer: PD Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK - Chemie & Physik von Grenzflächen
  

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  Abstract

• Design, Manufacturing and Evaluation of a Microfluidic Cartridge for Molecular Sample-to-Answer Analysis
  2023, XX
  First reviewer: Apl. Prof. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Development of a non-contact capacitive sensor for in-flow microparticle detection
  2023, XX
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Jochen Kieninger, IMTEK - Elektrische Messtechnik und Eingebettete Systeme
  

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  Abstract

• Development of carbon supported Ag nanoparticles as catalyst for CO2 electrolysis
  2023, XX
  First reviewer: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung/Elektrochemische Energiesysteme
  Second reviewer: Dr. Jochen Kieninger, IMTEK - Sensoren
  

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  Abstract

• Entwicklung eines auf Calciumphosphat-Zement (CPC) basierenden hybriden 3D-Bioprinting-Verfahrens zum Drucken von Knochenersatzprodukten
  2023, XX
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Evaluation of cfMeDIP-qPCR for colorectal cancer detection in liquid biopsy samples
  2023, xx
  First reviewer: Prof. Dr. Bernd Kammerer, Organische Chemie, Universität Freiburg
  Second reviewer: PD. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Fully functional hybrid 3D-printing of multi-layered electrical circuit
  2023, XX
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Bastian Rapp, IMTEK - Prozesstechnologie
  

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  Abstract

• Integrated reference electrode for anion-exchange membrane water electrolysis
  2023, XX
  First reviewer: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Jochen Kieninger, IMTEK - Elektrische Messtechnik und Eingebettete Systeme
  

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  Abstract

• Konstruktion eines thermo-pneumatisch aktuierten Labormusters für die mobile Diagnostik
  2023, XX
  First reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Screening of protein biomarkers for hypomethylating agent resistance in acute myeloid leukemia using absolute quantification by protein interaction cou-pling
  2023, XX
  First reviewer: PD. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Vanadium redox flow battery performance of pretreated Aemion+™
  2023, XX
  First reviewer: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung/Elektrochemische Energiesysteme
  Second reviewer: Dr. Jochen Kieninger, IMTEK - Sensoren
  

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  Abstract

2022

• A multi-cellular 3D bioprinting approach to monitor cell-cell interactions and to enhance understanding of vascularization processes
  2022, Joshua Weygant
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Günter Finkenzeller, Uniklinik Freiburg
  

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  Abstract

• Development of a microfluidic channel structure to increase the interspacing between micro tumors in a fluid flow
  2022, Trong Nguyen
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: PD. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Development of porous Bipolar Membranes for the Application in CO2-Electrolysers producing CO
  2022, Stefan Ingenhoven
  First reviewer: Dr. Severin Vierrath, EES-Group, IMTEK
  Second reviewer: Dr. Jochen Kieninger, IMTEK - Elektrische Messtechnik und Eingebettete Systeme
  

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  Abstract

• Ein mikrofluidischer Chip für die automatische Sample-to-answer-Analyse am Beispiel eines MRSA Nachweises
  2022, Benedict Martens
  First reviewer: apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK - Sensoren
  

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  Abstract

• 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
  First reviewer: Prof. Dr. Winfried Römer, Centre for Biological Signalling studies - BIOSS, Uni Freiburg
  Second reviewer: apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Normalization of DNA for Library Preparation for Next Generation Sequencing using Carboxylated COC Surfaces
  2022, Moritz Huber
  First reviewer: PD. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Optimization of the cathode catalyst layer composition of a zero-gap CO2 electrolyzer
  2022, Luca Bohn
  First reviewer: Dr. Severin Vierrath, EES-Group, IMTEK
  Second reviewer: Dr. Jochen Kieninger, IMTEK - Elektrische Messtechnik und Eingebettete Systeme
  

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  Abstract

2021

• A novel microporous layer with integrated flow field structures in PEMFCs
  2021, Neethu Philip Thombra
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Stefan Glunz, Fraunhofer ISE
  

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  Abstract

• Anode Development for Alkaline Direct Alcohol Fuel Cells
  2021, Edgar Cruz Ortiz
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Hanemann, IMTEK - Werkstoffprozesstechnik
  

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  Abstract

• Automated generation of platelet poor plasma in a microfluidic cartridge
  2021, Lena Karkossa
  First reviewer: PD Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Ulrich Walker, Universitätsspital Basel / CH
  

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  Abstract

• Development and characterization of Pt/C catalysts for proton-exchange membrane fuel cells
  2021, Miriam von Holst
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Hanemann, IMTEK - Werkstoffprozesstechnik
  

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  Abstract

• Entwicklung und Charakterisierung einer Multiplex-LAMP zur Differenzierung von Makrolid-Resistenzen bei Treponema pallidum subsp. Pertenue
  2021, Helena Gmoser
  First reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK - Sensoren
  

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  Abstract

• Improvement of the Water Management of Anion Exchange Membrane Fuel Cells
  2021, Jakob Salewsky
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Machine-learning for enhanced single spheroid detection
  2021, Sarah Eisenkolb
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Measuring the Volume of Liquid Jets with Computer Vision
  2021, Guilherme Miotto
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Brox, IMTEK - Computer Science
  

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  Abstract

• Mechanisch verstärkte Kohlenwasserstoffmembran für die Polymerelektrolytbrennstoffzelle
  2021, Clara Schare
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  

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  Abstract

• On the development of fluorine free membrane electrode assemblies towards scalable production of proton exchange membrane fuel cells
  2021, Farmal Khan
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Paste Transfer Simulation in Stencil Printing
  2021, Kiarash Karimi
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Lars Pastewka, IMTEK - Simulation
  

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  Abstract

• Thermal cycling by centrifugation-controlled convection for ultra-fast polymerase chain reaction
  2021, Niklas Virks
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Wilfried Weber, Inst. Für Biologie II, Uni Freiburg
  

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  Abstract

2020

• Efficient solid phase extraction of short DNA fragments on carboxylated surfaces using centrifugal microfluidics
  2020, Sophie Jenne
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Entwicklung und Validierung eines Augmented Reality Reportingtools
  2020, Darko Ilić
  First reviewer: Dr. Philipp M. Scholl, TF, Uni Freiburg - Rechnerarchitektur
  Second reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  

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  Abstract

• 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
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Roman Keding, Fraunhofer ISE
  

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  Abstract

• Evaluation of the production and opening dynamics of double chamber stickpacks
  2020, Hedieh Ludwig
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Ulrike Wallrabe, IMTEK - Mikroaktorik
  

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  Abstract

• Herstellung von Reaktionsgefäßen für die Miniaturisierung von Einzelzell-Analyse-Workflows auf planaren Oberflächen
  2020, Merit Sieben
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Materailien der Mikrosystemtechnik
  

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  Abstract

• Hybrid 3D Printing: A coalesced 3D printing of polymer and solder
  2020, Zeba Khan
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Bastian Rapp, IMTEK - Prozesstechnologie
  

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  Abstract

• Hybrid bioprinting of thermoplastics and hydrogels for 3D tissue cultures
  2020, Ole Thaden
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Influence on Break-in Process of PEM Fuel Cell Materials and Hot Press Production Conditions
  2020, Irene Franzetti
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Michael H. Eikerling, Institute of Energy and Climate Research, Forschungszentrum Jülich
  

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  Abstract

• Study on the implementation of direct hybrid fuel cell/battery systems for different electrically assisted bicycle types
  2020, Leonardo Ranck Zingano
  First reviewer: Jun-Prof. Dr. Stefan Pauliuk, Nachhaltiges Energie- und Stoffstrommanagement, Uni Freiburg
  Second reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  

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  Abstract

2019

• Characterization of rheological properties of liquid during dispensing using deep learning
  2019, Pranshul Sardana
  First reviewer: Dr. Peter Koltay, IMTEK / Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Brox, Informatik / Pattern Recognition and Image Processing
  

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  Abstract

• Dependency of Polymer-Polymer Adhesion on Process Parameters in 3D-Printing of Polycaprolactone
  2019, Stefan Conrad
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Development of a tubular flow sensor
  2019, Mona Masouni Goudarzi
  First reviewer: Prof. Dr. Hans Zappe, IMTEK / Mikrooptik
  Second reviewer: Dr. Peter Koltay, IMTEK / Anwendungsentwicklung
  

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  Abstract

• Evaluation of StarJet temperature process window for maskless solder printing on ENIG
  2019, Michael Fechtig
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jürgen Wilde, IMTEK - Aufbau- und Verbindungstechnik
  

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  Abstract

• Evaulation of a homogenous, digital single-cell transcriptomic assay
  2019, Shadi Edalati
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  

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  Abstract

• LabSlide für die hochparalelle, LAMP-basierte Auswertung digitaler Immunoassays
  2019, Julian Rüdiger
  First reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK / Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jenny Kehrbusch, Hochschule Kaiserslautern
  

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  Abstract

• Lensiess imaging of prokaryiotic cells for single cell printing
  2019, Kerstin Thiemann
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Microfluidic Perfusion Functional Lid for Organ-on-a-chip Applications
  2019, Ahmad Itani
  First reviewer: Dr. Peter Koltay, IMTEK / Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK / Simulation
  

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  Abstract

• Reinforced membranes for polymer electrolyte membrane water eiectrolyzers
  2019, Dunja Abed el Hafez
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  

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  Abstract

2018

• An Optical Method for Quantitative Volume Determination of Liquid Jets
  2018, Kevin Pfleghar
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Bioprinting of endothelial cells towards controlled vascularization structures in artificial tissues
  2018, Kevin Tröndle
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. rer. nat. Günter Finkenzeller, Klinik für Plastische und Handchirurgie Freiburg
  

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  Abstract

• Development of a tubular flow sensor
  2018, Mona Masoumi Goudarzi
  First reviewer: Dr. Peter Koltay, IMTEK - Uni Freiburg
  

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  Abstract

• Recognition of living and dead cells for automated cell sorting
  2018, Mais Hanoon
  First reviewer: Dr. Peter Koltay
  

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  Abstract

• Towards Direct Membrane Deposition and Noble Metal Free Cathodes for PEM Water Electrolysis
  2018, Peter Holzapfel
  First reviewer: Prof. Dr. Simon Thiele, Electrocatalytical Interface Engineering, Friedrich-Alexander University Erlangen-Nürnberg
  Second reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwedungsentwicklung
  

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  Abstract

• “Developing of a signal fixation strategy tor the proximity ligation assay“
  2018, Uljana Botscherowa
  First reviewer: Dr. Matthias Meier, IMTEK - Uni Freiburg
  Second reviewer: Prof. Roland Zengerle, IMTEK - Uni Freiburg
  

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  Abstract

2017

• Automatisierte DNA-Extraktion durch den Einsatz von Membranen im LabTube für die Tuberkulosediagnostik
  2017, Leonard Kost
  First reviewer: Prof. Dr, Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Alexander Rohrbach, IMTEK
  

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  Abstract

• Effect of Coriolis Force and Flow Rate on Binding Efficiency of Biomolecules in Centrifugal Microfluidics
  2017, Ahmad Saleem Akhtar
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Alexander Rohrbach, IMTEK - Bio- und Nanophotonik
  

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  Abstract

• Guidelines für das MRD-Monitoring mittels biplex Mediatorsonden-PCR bei akuter lymphoblastischer Leukämie
  2017, Franziska Schlenker
  First reviewer: PD Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: PD Dr. Cornelia Eckert, Campus Virchow-Klinikum (CVK), Charité Berlin
  

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  Abstract

• Segmentierung von makroporösen Materialien mittels Infiltration und maschinellem Lernen
  2017, Arne Götze
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
  

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  Abstract

• 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
  First reviewer: PD Dr. Sven Kerzenmacher, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Siegfried Fink, Forstbotanik, Uni Freiburg
  

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  Abstract

2016

• Analytische Proteinaffinitätschromatographie in der offenen Mikrofluidik
  2016, Boris Hamouda
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Oliver Ambacher, Fraunhofer-Institut für Angewandte Festkörperphysik IAF
  

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  Abstract

• Artifcial 3-D cell culture on a microfluidic chip
  2016, Kerstin Schlegel
  

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  Abstract

• Development and evaluation of a methane-producing microbial electrolysis cell
  2016, Marielle Fink
  First reviewer: Prof. Wilfried Weber , BIOSS und Institut für Biologie II, Uni Freiburg
  Second reviewer: Prof. Matthias Boll, Institut für Biologie II, Uni Freiburg
  

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  Abstract

• Development of a pneumatic Coriolis mixer in centrifugal microfluidics
  2016, Pawel Ranoszek
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK-Simulation
  

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  Abstract

• Development of a synthetic switch for measuring in situ dynamics of protein turnover rates
  2016, Christoph Gäbelein
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Development of techniques for the in-line viscosity determination in a low-cost flow sensor
  2016, Fritz Koch
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Entwicklung eines Begasungssystems für den Betrieb abiotischer Glukosebrennstoffzellen in simulierter Gewebeflüssigkeit und Serum
  2016, Melanie Bühler
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
  

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  Abstract

• Erforschung stabiler Emulsionen auf mikrofluidischen Chips für eine Lab-on-a-Chip-Anwendung
  2016, Anja Schachtschneider
  

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  Abstract

• Konzeptionierung, simulationsgestützte Auslegung und Charakterisierung von Fluidikstrukturen für schnelle Zonen-PCR auf der zentrifugalen LabDisk Plattform
  2016, Peter Berli
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. P. Woias, IMTEK - Konstruktion von Mikrosystemen
  

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  Abstract

2015

• Development of a microfluidic disk for digital droplet PCR
  2015, Marcel Geltman
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: PD Dr. Albrecht Brandenburg, Fraunhofer Institute for Physical Measurement Techniques (IPM)
  

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  Abstract

• Gasblasenbasiertes Mischen für beadbasierte Affinitätsreaktionen in der zentrifugalen Mikrofluidik
  2015, Martin Schulz
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK-Sensoren
  

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  Abstract

• Herstellung eines Sensors zur ortsaufgelösten Messung des elektrischen Potentials in einer Brennstoffzellen-Katalysatorschicht
  2015, Michaela Frase
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
  

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  Abstract

• Open subtrate microbatch protein crystallization using the PipeJet™ technology
  2015, Jonathan Kottmeier
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Oliver Einsle, Lehrstuhl für Biochemie
  

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  Abstract

• Zentrifugo-thermopneumatisches Wachsventil auf der zentrifugalen mikrofluidischen LabDisk Plattform
  2015, Anna Drzyzga
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Dr. Andreas Greiner, IMTEK
  

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  Abstract

2014

• Aerosolbedingte Kontaminationsanalyse Mediatorsonden-PCR basierter Geneslices
  2014, Alexander Heyne
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Charakterisierung und Optimierung des blasengestützten Mischens in LabTube-Kartuschen
  2014, Maximilian Grösche
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, I
  

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  Abstract

• Charakterisierung und Optimierung des Nukleinsäureassays für neonatale Sepsis auf einer zentrifugal-mikrofluidischen Foliendisk
  2014, Thorsten Meßinger
  

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  Abstract

• Conceptual study of LabTube based automation of a specific protocol for bacterial DNA extraction from whole blood samples
  2014, Ardavan Shabanian
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Peter Woias, IMTEK
  

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  Abstract

• Herstellung und Charakterisierung von Platin- Legierungskatalysatoren für den Einsatz in der implantierbaren Glukose-Brennstoffzelle
  2014, Felix Wiedenmann
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK, IMTEK
  

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  Abstract

• Kühlraten-Ventilaktuator auf der zentrifugalen LabDisk Jakob Schott
  2014, Jakob Schott
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Thomas Hanemann, IMTEK
  

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  Abstract

2013

• Aufbau mehrlagiger Mikrofluidikstrukturen aus thermogeformten Foliensubstraten am Beispiel der SAXS-LabDisk
  2013, Dirk Buselmeier
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Automatisierung magnetpartikelbasierter Immunoassays auf zentrifugal-mikrofluidischem Lab-on-a-Chip System
  2013, Jing Jin
  First reviewer: Prof. Dr. Michael Köhler, Technische Universität Ilmenau
  Second reviewer: Prof. Dr. Roland Zengerle, IMTEK
  

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  Abstract

• Cartridge development for automated immunoassays in standard laboratory centrifuges
  2013, Vaskar Gnyavali
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Charakterisierung der Bead-basierten Nukleinsäure-Extraktion auf der zentrifugal-mikrofluidischen Foliendisk
  2013, Sergej Keil
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Enhanced in-situ generation of polonies in picowell arrays by solid phase PCR for sequencing
  2013, Sebastian Hin
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Entwicklung eines integrierten Verschlusses für Lab-on-a-Chip Systeme aus thermogeformten Folien
  2013, Jan Smetana
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Gas bubble based mixing for DNA extraction on centrifugal microfluidic platforms
  2013, Jens Liebeskind
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Imaging technology for volumetric characterization of non-contact micro-dosages
  2013, Muniyogeshbabu Thanikhatla Govindaiah
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Alexander Rohrbach
  

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  Abstract

• Implementierung und Optimierung eines Mikrothermoformprozesses mit dem Einsatz starrer Formwerkzeuge
  2013, Janika Bischoff
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Claas Müller
  

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  Abstract

• Large volume handling on LabDisk
  2013, Johannes Wöhrle
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

2012

• Characterization and Optimization of DNA extraction in the LabTube cartridge
  2012, Shou Zhang
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Charakterisierung und Optimierung unabhängiger Prozessparameter für einen Mikrothermoformprozess Marion
  2012, Marion Finkbeiner
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Contact replication of DNA microarrays
  2012, Andreas Grabow
  First reviewer: Prof. Dr. rer. nat. Christiane Zell, Hochschule Offenburg
  

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  Abstract

• Design and Elevation of Tempered PDMS Flow Cell for Protein Microarray Generation
  2012, Suleman Shakil
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jürgen Wilde, IMTEK
  

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  Abstract

• Development of Pneumatic Pumping Unit Operations on Centrifugal Platforms and Comparative Study to a Network Model Approach
  2012, Steffen Zehnle
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Digital solid-phase PCR in picowell arrays to generate DNA colonies for sequencing
  2012, Martin Trotter
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Electrophoretic Separation of DNA in Written Gel Lines on Planar Substrates
  2012, Ludwig Gutzweiler
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Margit Zacharias, IMTEK
  

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  Abstract

• Kontaktfreies Drucken von Zellen und Hydrogelen für Tissue Engineering und Einzelzellanalyse
  2012, Sonja Niekrawietz
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK
  

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  Abstract

• Neukonzeptionierung und Charakterisierung einer Fluidik zur PCR-basierten DNA-Analytik
  2012, Miriam Kräft
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Quantitative measurement system for noncontact micro dispensers based on QCM technology
  2012, Jin Zhang
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK
  

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  Abstract

• Weiterentwicklung und Automatisierung eines kontaktfreien Dosierers für den sub-μL Bereich
  2012, Imad Malki
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jürgen Wöllenstein, IMTEK
  

2011

• Development of a Fluorescence detection system using a semi-conductive photomultiplier for centrifugal microfluidic platform
  2011, Muthukaruppan Gnanadesigan
  First reviewer: Prof. Dr. Bernhard Vondenbusch, Hochschule Furtwangen
  Second reviewer: Prof. Dr. Roland Zengerle, IMTEK
  

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  Abstract

• Development of an Active Micromixer Using an External Mechanical Actuator Array
  2011, Yawar Abbas
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Development, fabrication and characterization of a low-cost electromagnetic dispensing valve for the sub-μl range
  2011, Sabrina Kartmann
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Dr. Andreas Greiner, IMTEK
  

• High Throughput Cell Lysis Based on Integrated Bubble-Jet Actuators
  2011, Vitaliy Kondrashov
  First reviewer: Prof. Dr. Ulrich Mescheder, Hochschule Furtwangen University
  Second reviewer: Prof. Dr. Dietrich Kühlke, Hochschule Furtwangen University
  

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  Abstract

• High Throughput Cell-Sorter based on Integrated Bubble-Jet Actuators
  2011, Natalia Andreevna Bakhtina
  First reviewer: Prof. Dr. Ulrich Mescheder, Hochschule Furtwangen University
  Second reviewer: Prof. Dr. Dietrich Kühlke, Hochschule Furtwangen University
  

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  Abstract

• Imaging of Immobilization Processes of Molecules with iRIfS
  2011, David Lämmle
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Günter Gauglitz, Eberhard-Karls- Universität Tübingen
  

• Mechanical Lysis on a Centrifugal Microfluidic Disk
  2011, Stefan Schuhladen
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Network Simulation of a Solenoid Dispensing Valve
  2011, Shivam Dwivedi
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jürgen Wilde, IMTEK
  

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  Abstract

• Nukleinsäure-Extraktion auf einer zentrifugal-mikrofluidischen Foliendisk mit Hilfe magnetischer Partikeln
  2011, Bülent Kanat
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

2010

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

2006

• Development of a Device for Integrated Cell Culturing and Stimulation
  2006, Markus Wintermantel
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  

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  Abstract

• Development of a valve-less micropump for fuel cell applications
  2006, Yasir Osman Mustafa Elnager
  

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  Abstract

2024

• Development of an automated optical fill level detection in microfluidic channels under rotation using image recognition
  2024, xx
  First reviewer: Apl. Prof. Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Evaluating the Impact of Different Factors on Organoid Functionality
  2024, xx
  

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  Abstract

• Implementierung und Charakterisierung eines optischen Füllstandsensors für das automatisierte Packen von "Dual-Mode"-Chromatographiesäulen
  2024, xx
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Untersuchung des Salzausfalls in einem zerogap CO2 Elektrolyseur bei gepulstem Betrieb
  2024, xx
  First reviewer: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung
  

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  Abstract

2023

• Conductivity evaluation of anion-exchange membranes for water electrolyzers
  2023, XX
  First reviewer: Dr. Severin Vierrath, EES-Group, IMTEK
  

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  Abstract

• Rissfreie Anodenkatalysatorschicht auf Kohlenwasserstoffmembran durch Reduzierung der Membranquellung
  2023, xx
  First reviewer: Dr. Severin Vierrath, IMTEK - Anwendungsentwicklung
  

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  Abstract

2022

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

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  Abstract

2021

• Charakterisierung der UV-induzierten Freisetzung von Hydrogel-Bead gekoppelten Primern für eine digitale PCR mittels eines neuronalen Netzes zur Tropfenklassifizierung
  2021, Daniela Rassler
  First reviewer: PD Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Charakterisierung eines dampfdruckunterstützten thermopneumatischen Pumpmechanismus
  2021, Maren Kirste
  First reviewer: PD Dr. Nils Paust, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Charakterisierung von Alginathydrogelen zur Herstellung einer Referenz-Biotinte für die Standardisierung von DoD-Bioprinting-Verfahren
  2021, Lea Zausch
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Entwicklung neuer Wasserstoff-Brennstoffzellen mit fluorfreien Ionomeren und reduzierter Platin-Katalysatorbeladung
  2021, Andreas Fischbach
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Etablierung und Optimierung einer 4-plex Mediatorsonden-PCR zur simultanen Detektion von DNA und RNA
  2021, Larissa Graner
  First reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: PD Dr. rer. nat. Stefanie Eyerich, Helmholtz Zentrum München
  

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  Abstract

• Kostengünstige funktionalisierte Elektroden für den elektrochemischen Nachweis elektroaktiv gelabelter DNA Sonden
  2021, Dalina Dler Boya
  First reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK - Chemie und Physik von Grenzflächen
  

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  Abstract

2020

• Druckkontrolliertes Direktvolumen - Verdrängersystem zur kontaktfreien Dosierung für Nano und Mikrolitertropfen
  2020, Florian Lux
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Properties of solder metal bumps printed by StarJet technology on ENIG surfaces for backside contacting
  2020, Caoting Li
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Lars Pastewka, IMTEK - Simulation
  

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  Abstract

• Untersuchung der Lagerstabilität von fluorogenen Reportermolekülen
  2020, Sebastian Dominik Huber
  First reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Lisa Becherer, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Verbesserung eines Ultraschall-Sprühprozesses zur Herstellung von PEM Brennstoffzellen mit optimierter Leistung
  2020, Souaad Saoud
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Patrick Ruther, IMTEK - Materailien der Mikrosystemtechnik
  

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  Abstract

2019

• Bakterien-Detektion mittels Dunkelfeldmikroskopie für den Einzelzelldruck
  2019, Raphael Dammann
  First reviewer: Dr. Peter Koltay, Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, Simulation
  

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  Abstract

• Nanostrukturierte Keramiken für elektrochemische Energieanwendungen
  2019, Luca Bohn
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
  Second reviewer: Dr. Patrick Ruther, IMTEK / Materialien für Mikrosystemtechnik
  

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  Abstract

• Trocknungspuffer für partikelgekoppelte C-reaktive Proteine im Point-of-Care
  2019, Lena Karkossa
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK / Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK / Simulation
  

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  Abstract

2018

• CHARAKTERISIERUNG VON INDUSTRIELLEN AL-PASTEN ZUR EVALUATION DER Dispenstechnologie für die Rückseitenmetallisierung von bifacialen PERC Solarzellen
  2018, Marcel Klemens
  First reviewer: Prof. Dr. Roland Zengerel , IMTEK - Uni Freiburg
  Second reviewer: Dr. Andreas Greiner, IMTEK - Uni Freiburg
  

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  Abstract

• Charakterisierung von Hydrogelen für die Herstellung von 3D-Strukturen mittels Extrusion
  2018, Bushra Banu Ansari
  First reviewer: Dr. Peter Koltay, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK - Simulation
  

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  Abstract

• Charakterisierung von Stützmaterialien aus bioresorbierbarem Kunststoff für das 3D-Bioprinting
  2018, Nicolas Bignani
  First reviewer: Prof.Dr. Roland Zengerel , IMTEK - Uni Freiburg
  

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  Abstract

• Hochdynamische Multianalyt-Assays –Gleichzeitige Detektion von Proteinen und Nukleinsäuren
  2018, Angela Schade
  First reviewer: Prof. Dr. Dieter Stoll , Hochschule Albstadt-Sigmaringen
  Second reviewer: Apl. Prof. Dr. Felix von Stetten, IMTEK, Uni Freiburg
  

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  Abstract

• Validierung der Fixierung von diagnostischen Reagenzien in Stickpacks
  2018, Michael Eisele
  

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  Abstract

2017

• Microbial Electrolysis Cells for the Treatment of Industrial Wastewater: Evaluating the Effect of Different Separators on Reactor Performance
  2017, Pia Kolb
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr, Sabine Sané, University College Freiburg
  

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  Abstract

• Verifikation eines Simulationsmodells zur Folienausdehnung auf mikrofluidischen Testträgern
  2017, Valentin Dimitrov
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  

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  Abstract

2016

• Charakterisierung zentrifugal mikrofluidischer Testträger zur DNA-Extraktion von Bakterien aus Wasserproben
  2016, Michael Zehnle
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK-Simulation
  

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  Abstract

• Einzelzelldruck für die Analyse zirkulierender Tumorzellen
  2016, Kevin Pfleghar
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK-Simulation
  

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  Abstract

• Elektrochemischer Hybridisierungsnachweis mit Mediatorsonden auf Inkjetgedruckten Strukturen für die Anwendung als E-DNA Sensor
  2016, Monika Farina
  First reviewer: PD Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK - Sensorik
  

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  Abstract

• Increasing current production in microbial electrolysis cells by adding granular activated carbon to the anode
  2016, Veronika Vazhnik
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  

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  Abstract

• Influence of different separators on the performance of microbial electrolysis cells
  2016, Dunia Abed el Hafez
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Dr. Andreas Greiner, IMTEK-Simulation
  

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  Abstract

2015

• Aufbau eines Prüfstandes zur Untersuchung der Präzision bei der kontaktfreien Tropfenpositionierung im Nano- und Pikoliterbereich
  2015, Matthias Körner
  First reviewer: Prof. Dr.-Ing. Volker Hirsch
  Second reviewer: Prof. Dr.-Ing. habil. Catherina Burghart
  

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  Abstract

• Digital droplet loop-mediated isothermal amplification (ddLAMP) on standard laboratory devices
  2015, Clara Siber
  First reviewer: PD Dr. Felix von Stetten
  Second reviewer: Prof. Dr. Gerald Urban
  

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  Abstract

• Membran-basierter Probeneinlass für pathogenes Vollblut auf einer zentrifugalen mikrofluidischen LabDisk Plattform
  2015, Manuel Loskyll
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Oliver Paul, Materialien der Mikrosystemtechnik, IMTEK-Sensoren
  

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  Abstract

• Nachweis von respiratorischer Pathogen DNA auf einer zentrifugal-mikrofluidischen LabDisk Plattform
  2015, Franziska Schlenker
  First reviewer: PD Dr. Felix von Stetten, IMTEK - Anwendungsentwicklung
  Second reviewer: PD Dr. A. Brandenburg, Fraunhofer Institute for Physical Measurement Techniques (IPM)
  

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  Abstract

• Voruntersuchungen zur Behandlung von synthetischem Brauereiabwasser mit der filtrierenden mikrobiellen Brennstoffzelle
  2015, Jonas Rapp
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK - Anwendungsentwicklung
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK - Biomedizinische Mikrotechnik
  

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  Abstract

2014

• Charakterisierung des Ausdehnungsverhaltens einer flexiblen Messzelle unter Druckbeaufschlagung
  2014, Malena Kellermann
  

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  Abstract

• Charakterisierung unterschiedlicher Elektrodengeometrien eines kapazitiven Einweg-Drucksensors
  2014, Julian Rüdiger
  

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  Abstract

• Development of Detection Methods for Mediator Probe Cleavage during Recombinase Polymerase Amplification
  2014, Laura Leonhardt
  

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  Abstract

• Grundlegende Untersuchungen zur Machbarkeit der filtrierenden mikrobiellen Brennstoffzelle
  2014, Arne Götze
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK
  

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  Abstract

2013

• Electrospinning of anode materials for microbial fuel cell applications
  2013, Alexander Sido
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK
  

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  Abstract

• Geometrische Multiplex Mediator-Sonden PCR in GeneSlices
  2013, Sandra Cindric
  First reviewer: Prof.Dr. Ulrike Salat, Hochschule Furtwangen University
  Second reviewer: Prof. Dr. Roland Zengerle, IMTEK
  

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  Abstract

• Mikrofluidische Integration und Charakterisierung einer Phagenselektion
  2013, Tatiana Serr
  First reviewer: Prof.Dr. Ulrike Salat, Hochschule Furtwangen University
  Second reviewer: Prof. Dr. Roland Zengerle, IMTEK
  

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  Abstract

2012

• Entwicklung von luftatmenden Kathoden für die mikrobielle Brennstoffzelle
  2012, David Thissen
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK
  

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  Abstract

• Etablierung eines Referenzprotokolls zur Pestiziddetektion mittels eines enzymatischen Biosensors
  2012, Martin Schulz
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Untersuchung des Einflusses von Aminosäuren auf die Glukose-Oxidation an hochporösen Platin-Katalysatoren
  2012, Lena Bleck
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK
  

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  Abstract

2011

• Anodenmaterialien für die hocheffiziente Mikrobielle Brennstoffzelle
  2011, Julia Koch
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK
  

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  Abstract

• Beschichtungen für Lab-on-a-Chip-Systeme
  2011, Dirk Rappenecker
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Charakterisierung eines beadbasierten Immunassays auf Foliendisk
  2011, Dominik Feldmeier
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Dr. Andreas Greiner, IMTEK
  

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  Abstract

• Die Anwendung von Verbundfolien zur Reagenzienvorlagerung in LoaC-Systemen
  2011, Yannick Barb
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Dr. Andreas Greiner, IMTEK
  

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  Abstract

• Experimentelle Charakterisierung und Parameteroptimierung eines Dosiersystems für den sub μl-Bereich
  2011, Johannes Aleker
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Herstellung von hochporösen Platin-Elektroden mittels gepulster galvanischer Abscheidung als Anode für die implantierbare Glukose- Brennstoffzelle
  2011, Anna Drzyzga
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK
  

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  Abstract

• Langzeitstabilität Aquacer behandelter Oberflächen & Optimierung einer mikrofluidischen Blutplasma-Separation
  2011, Johannes Fries
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Mikrofluidische Gradientenerzeugung
  2011, Robert Bergmann
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  

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  Abstract

• Rekombinase Polymerase Amplifikation im kontinuierlichen, mikrofluidichen Fluss
  2011, Alexander Heyne
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Universeller DNA-Nachweis mittels Mediator-Sonden-Technologie
  2011, Lucas Dreesen
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Jürgen Rühe, IMTEK
  

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  Abstract

• Verschließbare Einfüllöffnungen für Lab-on-a-Foil-Systeme
  2011, Sebastian Reiter
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

2010

• Charakterisierung und Verbesserung der PipeJet™ eines Dispensers
  2010, Ktaich Rabih
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Entwicklung eines Braille - Display betriebenen, mikrofluidischen PDMS - Pump- und Ventilsystems
  2010, Henning Meier
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Immobilisierung von Primern auf Poly(-dimethylsiloxan) (PDMS) für Primer-Verlängerungsreaktionen
  2010, Sebastian Hin
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Dr. Oswald Prucker, IMTEK
  

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• On-Chip Lyse von E. coli im kontinuierlichen Fluss
  2010, Johannes Wöhrle
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Reagenzienvorlagerung in delaminierbaren Folienbeuteln
  2010, Jan Smetana
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Ulrike Wallrabe, IMTEK
  

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  Abstract

• Thermisches Deckeln von Foliendisks
  2010, Dirk Buselmeier
  First reviewer: Prof. Dr. Claas Müller, IMTEK
  Second reviewer: Prof. Dr. Roland Zengerle, IMTEK
  

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2009

• Detection of Bacteria with Magnetic Beads on a Centrifugal Microfluidic Cartridge
  2009, Dureid Michael Qazzazie
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Entwicklung eines Teststands und einer Testfluidik für die Manipulation magnetischer Partikel auf einer zentrifugalmikrofluidischen Plattform
  2009, Alexander Emplerle
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Claas Müller, IMTEK
  

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  Abstract

• Fluoreszenzdetektionsstrategien für folienbasierte Lab-on-a-Chip-Systeme
  2009, Patrick Reith
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Leonhard Reindl, IMTEK
  

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  Abstract

• High resoluted chemical Cell Transfection in confluent Cell Cultures
  2009, Moritz Thielen
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Implementierung eines kompetitiven und Sandwich Immunoassays in ein zentrifugal-mikrofluidisches System
  2009, Imad Malki
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Kontrolliertes Einbringen hoch benetzender Flüssigkeiten in Lab-on-a-Foil-Systeme
  2009, Roland Feuerstein
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Polymere Schutzschicht für implantierbare Glukose Brennstoffzellen
  2009, Stanislav Sherman
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Thomas Stieglitz, IMTEK
  

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  Abstract

• Prozessentwicklung für mikrofluidische Chips auf Basis von vorstrukturierten Thermoplasten und Trockenresisten
  2009, Ludwig Gutzweiler
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Gerald Urban, IMTEK
  

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  Abstract

• Selektive Oberflächenmodifizierung und verbesserte Prozessdynamik für die TopSpot Technologie
  2009, Michael Bleile
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Specific Chemical Stimulation of Cells for Patch Clamp Assays
  2009, Olga Sobolev
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract

• Untersuchung eines neuen mikrofluidischen Ventils für hochbenetzende Flüssigkeiten
  2009, Richard Jahner
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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2008

• Processing Optimization of the μ-Active NASBA Chip
  2008, Marius Clad
  First reviewer: Prof. Dr. Roland Zengerle, IMTEK
  Second reviewer: Prof. Dr. Holger Reinecke, IMTEK
  

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  Abstract