Publikationsliste apl. Prof. Dr. Felix von Stetten

Originalarbeiten in wissenschaftlichen Fachzeitschriften
Reviews/Übersichtsartikel in wissenschaftlichen Fachzeitschriften
Buchbeiträge
Kurzbeiträge
Vorträge
Konferenzbeiträge

Originalarbeiten in wissenschaftlichen Fachzeitschriften

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

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2024

A. Klebes, H. Ceren Ates, Rene D. Verboket, G. A. Urban, F. von Stetten, C. Dincer, S. M. Früh
Emerging multianalyte biosensors for the simultaneous detection of protein and nucleic acid biomarkers
2024 Biosensors and Bioelectronics, Band: 244, Seite: 115800
M. Trotter, A. Schreiber, D. Kleinknecht, Z. Bagherian, F. von Stetten, N. Borst
Pathogen-Specific Electrochemical Real-Time LAMP Detection Using Universal Solid-Phase Probes on Carbon Electrodes
2024 ACS Sensors

2023

A. Klebes, H. C. Ates, R. D. Verboket, G. Urban, F. von Stetten, C. Dincer, S. M. Früh
Emerging multianalyte biosensors for the simultaneous detection of protein and nucleic acid biomarkers
2023 Biosensors and Bioelectronics, Seite: 115800
Y.-K. Lai, Y.-T. Kao, J. Hess, S. Calabrese, F. von Stetten, N. Paust
Interfacing centrifugal microfluidics with linear-oriented 8-tube strips and multichannel pipettes for increased throughput of digital assays
2023 Lab on a Chip, Band: 23, Seiten: 2623 - 2632
S. Calabrese, A. M. Markl, M. Neugebauer, S. J. Krauth, N. Borst, F. von Stetten, M. Lehnert
Reporter emission multiplexing in digital PCRs (REM-dPCRs): direct quantification of multiple target sequences per detection channel by population specific reporters
2023 Analyst, Band: 148, Seiten: 5243 - 5254

2022

E. Kipf, F. Schlenker, N. Borst, M. Fillies, R. Kirschner-Schwabe, R. Zengerle, C. Eckert, F. von Stetten, M. Lehnert
Advanced minimal residual disease monitoring for acute lymphoblastic leukemia with multiplex mediator probe PCR
2022 J. Mol. Diag, Band: 24, Nummer: 1, Seiten: 57 - 68
M. Neugebauer, C. E. Grundmann, M. Lehnert, F. von Stetten, S. M. Früh, R. Süss
Analyzing siRNA Concentration, Complexation and Stability in Cationic Dendriplexes by Stem-Loop Reverse Transcription-qPCR
2022 Pharmaceutics, Band: 2022, Nummer: 14, Seite: 1348
Y.-T. Kao, S. Calabrese, N. Borst, M. Lehnert, Y.-K. Lai, F. Schlenker, P. Juelg, R. Zengerle, P. Garstecki, F. von Stetten
Microfluidic One-Pot Digital Droplet FISH Using LNA/DNA Molecular Beacons for Bacteria Detection and Absolute Quantification
2022 Biosensors, Band: 12, Nummer: 4, Seite: 237
A. Klebes, A.-S. Kittel, R. D. Verboket, F. von Stetten, S. M. Früh
Multianalyte lateral flow immunoassay for simultaneous detection of protein-based inflammation biomarkers and pathogen DNA
2022 Sensors and Actuators B: Chemical, Band: 355, Seite: 131283
S. Hennig, Z. Shu, L. Gutzweiler, P. Koltay, F. von Stetten, R. Zengerle, S. M. Früh
Paper-based open microfluidic platform for protein electrophoresis and immunoprobing
2022 Electrophoresis, Band: 43, Nummer: 4, Seiten: 621 - 631

2021

F. Schlenker, E. Kipf, N. Borst, N. Paust, R. Zengerle, F. von Stetten, P. Juelg, T. Hutzenlaub
Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations
2021 Processes, Band: 2021, Nummer: 9, Ergänzungsband: 97
KurzfassungWe present the centrifugal microfluidic implementation of a four-plex digital droplet polymerase chain reaction (ddPCR). The platform features 12 identical ddPCR units on a LabDisk cartridge, each capable of generating droplets with a diameter of 82.7 +/- 9 µm. By investigating different oil–surfactant concentrations, we identified a robust process for droplet generation and stabilization. We observed high droplet stability during thermocycling and endpoint fluorescence imaging, as is required for ddPCRs. Furthermore, we introduce an automated process for fourcolor fluorescence imaging using a commercial cell analysis microscope, including a customized software pipeline for ddPCR image evaluation. The applicability of ddPCRs is demonstrated by the quantification of three cancer-associated KRAS point mutations (G12D, G12V and G12A) in a diagnostically relevant wild type DNA background. The four-plex assay showed high sensitivity (3.5–35 mutant DNA copies in 15,000 wild type DNA copies) and linear performance (R2 = 0.99) across all targets in the LabDisk.
M. Schulz, J. Ruediger, E. Landmann, M. Bakheit, S. Frischmann, D. Rassler, A. Homann, F. von Stetten, R. Zengerle, N. Paust
High Dynamic Range Digital Assay Enabled by Dual-Volume Centrifugal Step Emulsification
2021 Analytical Chemistry, Band: 93, Nummer: 5, Seiten: 2854 - 2860
KurzfassungWe implement dual-volume centrifugal step emulsification on a single chip to extend the dynamic range of digital assays. Compared to published single-volume approaches, the range between the lower detection limit (LDL) and the upper limit of quantification (ULQ) increases by two orders of magnitude. In comparison to existing multivolume approaches, the dual-volume centrifugal step emulsification requires neither complex manufacturing nor specialized equipment. Sample metering into two subvolumes, droplet generation, and alignment of the droplets in two separate monolayers are performed automatically by microfluidic design. Digital quantification is demonstrated by exemplary droplet digital loop-mediated isothermal amplification (ddLAMP). Within 5 min, the reaction mix is split into subvolumes of 10.5 and 2.5 μL, and 2,5k and 176k droplets are generated with diameters of 31.6 ± 1.4 and 213.9 ± 7.5 μm, respectively. After 30 min of incubation, quantification over 5 log steps is demonstrated with a linearity of R2 ≥ 0.992.
D. Baumgartner, B. Johannsen, M. Specht, J. Lüddecke, M. Rombach, S. Hin, N. Paust, F. von Stetten, R. Zengerle, C. Herz, J. R. Peham, P. N. Paqué, T. Attin, J. S. Jenzer, P. Körner, P. R. Schmidlin, T. Thurnheer, F. J. Wegehaupt, W. E. Kaman, A. Stubbs, J. P. Hays, V. Rusu, A. Michie, T. Binsl, D. Stejskal, M. Karpíšek, K. Bao, N. Bostanci, G. N. Belibasakis, K. Mitsakakis
OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice
2021 Biosensors, Band: 11, Nummer: 11, Seite: 423
L. Becherer, J. F. Hess, S. Frischmann, M. Bakheit, H. Nitschko, S. Stinco, F. Zitz, H. Hofer, G. Porro, F. Hausladen, K. Stock, D. Drossart, H. Wurm, H. Kuhn, D. Huber, T. Hutzenlaub, N. Paust, M. Keller, O. Strohmeier, S. Wadle, N. Borst, R. Zengerle, F. von Stetten
Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP
2021 Micromachines, Band: 12, Nummer: 2, Seite: 159
KurzfassungThis paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ Tlymphocytes (MT-2 cells) yielded 8 +/- 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 +/- 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 +/- 3 copies per MT-2 cell after a run time of two hours and 10 min.
A. Brunauer, R. D. Verboket, D. M. Kainz, F. von Stetten, S. M. Früh
Rapid Detection of Pathogens in Wound Exudate via Nucleic Acid Lateral Flow Immunoassay
2021 Biosensors, Band: 11, Nummer: 3, Seite: 74
KurzfassungThe rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 105 CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy.
F. Schlenker, E. Kipf, M. Deuter, I. Höffkes, M. Lehnert, R. Zengerle, F von Stetten, F. Scherer, J. Wehrle, N. von Bubnoff, P. Juelg, T. Hutzenlaub, N. Borst
Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA
2021 cancers, Band: 13, Nummer: 22, Seite: 5742
F. Schlenker, E. Kipf, N. Borst, T. Hutzenlaub, R. Zengerle, F. von Stetten, P. Juelg
Virtual Fluorescence Color Channels by Selective Photobleaching in Digital PCR Applied to the Quantification of KRAS Point Mutations
2021 Analytical Chemistry, Band: 93, Nummer: 30, Seiten: 10538 - 10545
KurzfassungMultiplexing of analyses is essential to reduce sample and reagent consumption in applications with large target panels. In applications such as cancer diagnostics, the required degree of multiplexing often exceeds the number of available fluorescence channels in polymerase chain reaction (PCR) devices. The combination of photobleaching-sensitive and photobleaching-resistant fluorophores of the same color can boost the degree of multiplexing by a factor of 2 per channel. The only additional hardware required to create virtual fluorescence color channels is a low-cost light-emitting diode (LED) setup for selective photobleaching. Here, we present an assay concept for fluorescence color multiplexing in up to 10 channels (five standard channels plus five virtual channels) using the mediator probe PCR with universal reporter (UR) fluorogenic oligonucleotides. We evaluate the photobleaching characteristic of 21 URs, which cover the whole spectral range from blue to crimson. This comprehensive UR data set is employed to demonstrate the use of three virtual channels in addition to the three standard channels of a commercial dPCR device (blue, green, and red) targeting cancer-associated point mutations (KRAS G12D and G12V). Moreover, a LOD (limit of detection) analysis of this assay confirms the high sensitivity of the multiplexing method (KRAS G12D: 16 DNA copies/reaction in the standard red channel and KRAS G12V: nine DNA copies/reaction in the virtual red channel). Based on the presented data set, optimal fluorogenic reporter combinations can be easily selected for the application-specific creation of virtual channels, enabling a high degree of multiplexing at low optical and technical effort.

2020

Y.-T. Kao, T. S. Kaminski, W. Postek, J. Guzowski, K. Makuch, A. Ruszczak, F. von Stetten, R. Zengerle, P. Garstecki
Gravity-driven microfluidic assay for digital enumeration of bacteria and for antibiotic susceptibility testing
2020 Lab Chip, Band: 20, Seiten: 54 - 63
KurzfassungThe alarming dynamics of antibiotic-resistant infections calls for the development of rapid and point-of-care (POC) antibiotic susceptibility testing (AST) methods. Here, we demonstrated the first completely stand-alone microfluidic system that allowed the execution of digital enumeration of bacteria and digital antibiograms without any specialized microfluidic instrumentation. A four-chamber gravity-driven step emulsification device generated ∼2000 monodisperse 2 nanoliter droplets with a coefficient of variation of 8.9% of volumes for 95% of droplets within less than 10 minutes. The manual workload required for droplet generation was limited to the sample preparation, the deposition into the sample inlet of the chip and subsequent orientation of the chip vertically without an additional pumping system. The use of shallow chambers imposing a 2D droplet arrangement provided superior stability of the droplets against coalescence and minimized the leakage of the reporter viability dye between adjacent droplets during long-term culture. By using resazurin as an indicator of the growth of bacteria, we were also able to reduce the assay time to ∼5 hours compared to 20 hours using the standard culture-based test.
M. Trotter, D. Juric, Z. Bagherian, N. Borst, K. Gläser, T. Meissner, F. von Stetten, A. Zimmermann
Inkjet-Printing of Nanoparticle Gold and Silver Ink on Cyclic Olefin Copolymer for DNA-Sensing Applications
2020 Sensors, Band: 20, Nummer: 5, Seite: 1333
KurzfassungInkjet technology as a maskless, direct-writing technology offers the potential for structured deposition of functional materials for the realization of electrodes for, e.g., sensing applications. In this work, electrodes were realized by inkjet-printing of commercial nanoparticle gold ink on planar substrates and, for the first time, onto the 2.5D surfaces of a 0.5 mm-deep microfluidic chamber produced in cyclic olefin copolymer (COC). The challenges of a poor wetting behavior and a low process temperature of the COC used were solved by a pretreatment with oxygen plasma and the combination of thermal (130 °C for 1 h) and photonic (955 mJ/cm²) steps for sintering. By performing the photonic curing, the resistance could be reduced by about 50% to 22.7 μΩ cm. The printed gold structures were mechanically stable (optimal cross-cut value) and porous (roughness factors between 8.6 and 24.4 for 3 and 9 inkjet-printed layers, respectively). Thiolated DNA probes were immobilized throughout the porous structure without the necessity of a surface activation step. Hybridization of labeled DNA probes resulted in specific signals comparable to signals on commercial screen-printed electrodes and could be reproduced after regeneration. The process described may facilitate the integration of electrodes in 2.5D lab-on-a-chip systems.
L. Becherer, N. Borst, M. Bakheit, S. Frischmann, R. Zengerle, F. von Stetten
Loop-mediated isothermal amplification (LAMP) – review and classification of methods for sequence-specific detection
2020 Anal Methods-uk, Band: 12, Seiten: 717 - 746
KurzfassungIn the course of the last 20 years, isothermal nucleic acid amplification tests have emerged as an important diagnostic tool, not only for clinical applications, but also for food quality control and environmental monitoring. Loop-mediated isothermal amplification (LAMP) is well known for its robust and highly sensitive and specific amplification of target DNA, which is achieved by utilizing up to six primers. Moreover, LAMP excels through its isothermal and energy efficient amplification requirements, rendering it a prime candidate for low-cost diagnostics and analysis at the point of need. Recently, methods for sequence-specific detection have gained more importance because, unlike sequence-independent detection methods, they are highly specific towards the target DNA. In the last 13 years, a variety of sequence-specific methods have emerged, based on a very diverse range of sensing techniques, including optical, magnetic, piezoelectric, electrochemical and magnetoresistive sensing. To give structure to the diverse multitude of sequence-specific methods, we created a systematic classification and provide a critical comparative evaluation according to a catalogue of criteria (analytical performance, multiplexing, quantification and instrumental requirements). Fluorescence-based detection, making up half of the methods, can be processed on open platforms and satisfies all the criteria listed before. Instrumental requirements are discussed in terms of complexity, portability and fluidic cartridges. In addition, the technological readiness level and the kind of platform (open versus method-tailored) are evaluated, the latter playing an important role in the miniaturization and automation of operational process steps. We also observe an increase in the use of smartphone-integrated sensors to improve LAMP-based point-of-need testing. In summary, recent developments in methods for the sequence-specific detection of LAMP demonstrate high potential for many future applications.
L. Becherer, S. Knauf, M. Marks, S. Lueert, S. Frischmann, N. Borst, F. von Stetten, S. Bieb, Y. Adu-Sarkodie, K. Asiedu, O. Mitjà, M. Bakheit
Multiplex Mediator Displacement Loop-Mediated Isothermal Amplification for Detection of Treponema pallidum and Haemophilus ducreyi
2020 Emerg Infect Dis, Band: 26, Seiten: 282 - 288
M. Schulz, S. Calabrese, F. Hausladen, H. Wurm, D. Drossart, K. Stock, A. M. Sobieraj, F. Eichenseher, M. J. Loessner, M. Schmelcher, A. Gerhardts, U. Goetz, M. Handel, A. Serr, G. Haecker, J. Li, M. Specht, P. Koch, M. Meyer, P. Tepper, R. Rother, M. Jehle, S. Wadle, R. Zengerle, F. von Stetten, N. Paust, N. Borst
Point-of-care testing system for digital single cell detection of MRSA directly from nasal swabs
2020 Lab Chip, Band: 20, Seiten: 2549 - 2561
KurzfassungWe present an automated point-of-care testing (POCT) system for rapid detection of species- and resistance markers in methicillin-resistant Staphylococcus aureus (MRSA) at the level of single cells, directly from nasal swab samples. Our novel system allows clear differentiation between MRSA, methicillin-sensitive S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS), which is not the case for currently used real-time quantitative PCR based systems. On top, the novel approach outcompetes the culture-based methods in terms of its short time-to-result (1 h vs. up to 60 h) and reduces manual labor. The walk-away test is fully automated on the centrifugal microfluidic LabDisk platform. The LabDisk cartridge comprises the unit operations swab-uptake, reagent pre-storage, distribution of the sample into 20 000 droplets, specific enzymatic lysis of Staphylococcus spp. and recombinase polymerase amplification (RPA) of species (vicK) – and resistance (mecA) -markers. LabDisk actuation, incubation and multi-channel fluorescence detection is demonstrated with a clinical isolate and spiked nasal swab samples down to a limit of detection (LOD) of 3 ± 0.3 CFU μl−1 for MRSA. The novel approach of the digital single cell detection is suggested to improve hospital admission screening, timely decision making, and goal-oriented antibiotic therapy. The implementation of a higher degree of multiplexing is required to translate the results into clinical practice.
S. Hin, D. Baumgartner, M. Specht, J. Lüddecke, E. M. Arjmand, B. Johannsen, L. Schiedel, M. Rombach, N. Paust, F. von Stetten, R. Zengerle, N. Wipf, P. Müller, K. Mavridis, J. Vontas, K. Mitsakakis, * Indicates equally contributing authors
VectorDisk: A Microfluidic Platform Integrating Diagnostic Markers for Evidence-Based Mosquito Control
2020 Processes, Band: 8, Nummer: 12, Seite: 1677
KurzfassungEffective mosquito monitoring relies on the accurate identification and characterization of the target population. Since this process requires specialist knowledge and equipment that is not widely available, automated field-deployable systems are highly desirable. We present a centrifugal microfluidic cartridge, the VectorDisk, which integrates TaqMan PCR assays in two feasibility studies, aiming to assess multiplexing capability, specificity, and reproducibility in detecting disk-integrated vector-related assays. In the first study, pools of 10 mosquitoes were used as samples. We tested 18 disks with 27 DNA and RNA assays each, using a combination of multiple microfluidic chambers and detection wavelengths (geometric and color multiplexing) to identify mosquito and malaria parasite species as well as insecticide resistance mechanisms. In the second study, purified nucleic acids served as samples to test arboviral and malaria infective mosquito assays. Nine disks were tested with 14 assays each. No false positive results were detected on any of the disks. The coeffcient of variation in reproducibility tests was <10%. The modular nature of the platform, the easy adaptation of the primer/probe panels, the cold chain independence, the rapid (2–3 h) analysis, and the assay multiplexing capacity are key features, rendering the VectorDisk a potential candidate for automated vector analysis.
M. Schulz, S. Probst, S. Calabrese, A. Homann, N. Borst, M. Weiss, F. von Stetten, R. Zengerle, N. Paust
Versatile Tool for Droplet Generation in Standard Reaction Tubes by Centrifugal Step Emulsification
2020 Molecules, Band: 25, Nummer: 8, Seite: 1914
KurzfassungWe present a versatile tool for the generation of monodisperse water-in-fluorinated-oil droplets in standard reaction tubes by centrifugal step emulsification. The microfluidic cartridge is designed as an insert into a standard 2 mL reaction tube and can be processed in standard laboratory centrifuges. It allows for droplet generation and subsequent transfer for any downstream analysis or further use, does not need any specialized device, and manufacturing is simple because it consists of two parts only: A structured substrate and a sealing foil. The design of the structured substrate is compatible to injection molding to allow manufacturing at large scale. Droplets are generated in fluorinated oil and collected in the reaction tube for subsequent analysis. For sample sizes up to 100 µL with a viscosity range of 1 mPa·s–4 mPa·s, we demonstrate stable droplet generation and transfer of more than 6 × 105 monodisperse droplets (droplet diameter 66 µm ± 3 µm, CV ≤ 4%) in less than 10 min. With two application examples, a digital droplet polymerase chain reaction (ddPCR) and digital droplet loop mediated isothermal amplification (ddLAMP), we demonstrate the compatibility of the droplet production for two main amplification techniques. Both applications show a high degree of linearity (ddPCR: R2 ≥ 0.994; ddLAMP: R2 ≥ 0.998), which demonstrates that the cartridge and the droplet generation method do not compromise assay performance.

2019

P. Juelg, M. Specht, E. Kipf, M. Lehnert, C. Eckert, M. Keller, T. Hutzenlaub, F. von Stetten, R. Zengerle, N. Paust
Automated serial dilutions for high-dynamic-range assays enabled by fill-level-coupled valving in centrifugal microfluidics
2019 Lab Chip, Band: 19, Seiten: 2205 - 2219
KurzfassungWe introduce a new concept for centrifugal microfluidics that enables fully automated serial dilution generation without any additional means besides temperature control. Key feature is time-independent, serial valving of mixing chambers by fill-level-coupled temperature change rate (FLC-TCR) actuated valving. The automated dilution is realized under continuous rotation which enables reliable control of wetting liquids without the need of any additional fabrication steps such as hydrophobic coatings. All fluidic features are implemented in a monolithic fashion and disks are manufactured by foil thermoforming for scalable manufacturing. The new valving concept is demonstrated to reliably prevent valving if the diluted sample is not added to the mixing chamber (n = 30) and ensures valving if the dilution stage is completed (n = 15). Accuracy and precision of the automated serial dilution are verified by on-disk generation of qPCR standard curve dilutions and compared with manually generated reference dilutions. In a first step, the 5-log-stages standard curves are evaluated in a commercial qPCR thermocycler revealing a linearity of R² ≥ 99.92 % for the proposed LabDisk method vs. R² ≥ 99.67 % in manual reference dilutions. In a second step, the disk automated serial dilution is combined with on-disk qPCR thermocycling and readout, both inside a LabDisk Player. A 4-log-stages linearity of R² ≥ 99.81 % and a sensitivity of one leukemia associated ETV6-RUNX1 mutant DNA copy in a background of 100,000 wild-type DNA copies is achieved.
M. Schulz, F. von Stetten, R. Zengerle, N. Paust
Centrifugal Step Emulsification: How Buoyancy Enables High Generation Rates of Monodisperse Droplets
2019 Langmuir : the ACS Journal of Surfaces and Colloids, Band: 35, Nummer: 30, Seiten: 9809 - 9815
KurzfassungWe demonstrate that buoyancy in centrifugal step emulsification enables substantially higher generation rates of monodisperse droplets compared to pressure driven set-ups. Step emulsification in general can produce droplets in comparatively simple systems (only one moving liquid) with a low CV of <5% in droplet diameter and with a minimum dead volume. If operated below a critical capillary number, the droplet diameter is defined by geometry and surface forces only. Above that critical capillary number, however, jetting occurs, leading to an increased droplet diameter and CV. Consequently, generation rates of monodisperse droplets are limited in pressure-driven systems. In this paper, we show that centrifugal step emulsification can overcome this limitation by applying sufficient buoyancy to the system. The buoyancy, induced by the centrifugal field and a density difference of the continuous and disperse phase, supports droplet necking by pulling the forming droplet away from the nozzle. The influence of buoyancy is studied using specific microfluidic designs that allow for supplying different buoyancies to the same droplet generation rates. For a droplet diameter of 100 μm, droplet generation at rates above 2.8k droplets per second and nozzle were reached, which is an increase of more than a factor of 8 in comparison to pressure-driven systems.

2018

M. Lehnert, E. Kipf, F. Schlenker, N. Borst, R. Zengerle, F. von Stetten
Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides
2018 Anal Methods-uk
KurzfassungIn this study we optimised the fluorescence signal generation of contact quenched universal reporter oligonucleotides. These are used as secondary probes in real-time Mediator Probe PCR to detect the sequence-specific cleavage of label-free primary mediator probes. Since the fluorescence signal generation of a universal reporter is not influenced by the target DNA sequence, optimisation of the fluorescence signal-to-noise ratio will improve the performance of all Mediator Probe PCRs that are based on this type of universal reporter. To determine the critical factors influencing signal-to-noise optimisation, we systematically analysed four parameters. These parameters were type of fluorophore, type of quencher molecule, intramolecular orientation of both residuals, and the number of quencher labels. In total, more than 30 different fluorogenic universal reporter structures were analysed, covering the whole fluorescence spectrum from green to crimson. From our results, we deduced a novel set of guidelines for signal-to-noise optimisation in the design of contact quenched, fluorogenic universal reporter oligonucleotides. We confirmed these guidelines in a different thermocycler, and by designing a second set of universal reporters, which were used for multiplex real-time PCR quantification of acute lymphoblastic leukaemia marker sequences. This optimised biplex Mediator Probe PCR showed an improved performance under clinical conditions, with a 10 times higher resolution regarding the limit of quantification. In addition to Mediator Probe PCR, these guidelines may also prove useful in signal-to-noise optimisation of other fluorescence-based assays where contact quenched oligonucleotides or secondary reporter molecules are used.
S. Hin, M. Loskyll, V. Klein, M. Keller, O. Strohmeier, F. von Stetten, R. Zengerle, K. Mitsakakis
Membrane-based sample inlet for centrifugal microfluidic cartridges
2018 Microelectron Eng, Band: 187-188, Seiten: 78 - 83
KurzfassungCentrifugal microfluidics enables the rapid execution of complex blood sample analyses in a fully automated manner at the point of care. However, during blood sample addition, the cartridge is at rest and centrifugal forces are not present to allocate the sample in a controlled way. We present a versatile approach for the user-friendly, well-controlled and safe sample addition into a centrifugal microfluidic cartridge for use in serology. It features a commercial (plasma separation) membrane stacked into the inlet chamber of the cartridge. This combination of sample inlet and plasma separation in one structural unit brings the advantage of reducing footprint in highly integrated point-of-care testing. By using a pipette the user may add a blood sample (90 μL) to the membrane holding the liquid by capillary forces. Flowing across the membrane, cells separate from blood plasma. The blood plasma releases into the downstream structure upon centrifugation. The mean plasma recovery rate was 57.3 (± 4.7) % and mean plasma purity 99.5 (± 0.6) % from samples of varying hematocrit (36%–59%). Furthermore, the analyte C-reactive protein (CRP) did not significantly adsorb to the membrane. This was concluded, since CRP immunoassay results with plasma from spiked whole blood, obtained from membrane-based plasma separation and from plasma separation on a standard laboratory centrifuge, did not significantly differ. Thus, the suggested approach is promising for simultaneous application as sample inlet holding the sample by capillary forces and as a plasma separation module for centrifugal microfluidics. Potential future applications may include other sample matrices. The use with further blood transfer devices (capillary, directly from fingertip) seems possible, yet requiring further evaluation.
L. Becherer, M. Bakheit, S. Frischmann, S. Stinco, N. Borst, R. Zengerle, F. von Stetten
Simplified real-time multiplex detection of loop-mediated isothermal amplification (LAMP) using novel mediator displacement probes with universal reporters
2018 Anal Chem, Band: 90, Seiten: 4741 - 4748
KurzfassungA variety of real-time detection techniques for LAMP based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel Mediator Displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1±0.1 min shorter than MB, n = 36), improved signal to noise fluorescence ratio (MD: 5.9±0.4, MB: 2.7±0.4; n = 15) and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 & HTLV-1 and a biplex LAMP of Haemophilus ducreyi & Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

2017

N. Borst, F. Schuler, S. Wadle, M. Schulz, M. Specht, J. Li, L. Becherer, M. Trotter, A. B. Rodríguez-Martínez, N. Paust, R. Zengerle, F. von Stetten
A technology platform for digital nucleic acid diagnostics at the point of care
2017 Laboratoriumsmedizin, Band: 41, Nummer: 5
KurzfassungThe combination of digital amplification and centrifugal microfluidics can enable quantitative and fast diagnostics at the point of care (PoC). The new unit operation of centrifugal step emulsification allows high throughput droplet generation. Different methods for digital nucleic acid analysis, including PCR, recombinase polymerase amplification (RPA) and loop mediated isothermal amplification (LAMP), have already been demonstrated. Our novel approach of integrated sample-to-answer analysis is introduced, and examples for the detection of HIV and single cell analysis of antibiotic resistant bacteria are presented. Next to these LabDisk based systems, a microfluidic cartridge termed DropChip allows for digital amplification using only commercially available laboratory devices.
S. Zehnle, M. Rombach, R. Zengerle, F. von Stetten, N. Paust
Network simulation-based optimization of centrifugo-pneumatic blood plasma separation
2017 Biomicrofluidics, Band: 11, Seite: 024114
KurzfassungAutomated and robust separation of 14 ll of plasma from 40 ll of whole blood at a purity of 99.81%60.11% within 43 s is demonstrated for the hematocrit range of 20%–60% in a centrifugal microfluidic polymer disk. At high rotational frequency, red blood cells (RBCs) within whole blood are concentrated in a radial outer RBC collection chamber. Simultaneously, plasma is concentrated in a radial inner pneumatic chamber, where a defined air volume is enclosed and compressed. Subsequent reduction of the rotational frequency to not lower than 25 Hz enables rapid transfer of supernatant plasma into a plasma collection chamber, with highly suppressed resuspension of red blood cells. Disk design and the rotational protocol are optimized to make the process fast, robust, and insusceptible for undesired cell resuspension. Numerical network simulation with lumped model elements is used to predict and optimize the fluidic characteristics. Lysis of the remaining red blood cells in the purified plasma, followed by measurement of the hemoglobin concentration, was used to determine plasma purity. Due to the pneumatic actuation, no surface treatment of the fluidic cartridge or any additional external means are required, offering the possibility for low-cost mass fabrication technologies, such as injection molding or thermoforming.
M. Keller, G. Czilwik, J. Schott, I. Schwarz, K. Dormanns, F. von Stetten, R. Zengerle, N. Paust
Robust temperature change rate actuated valving and switching for highly integrated centrifugal microfluidics
2017 Lab Chip, Band: 17, Seiten: 864 - 875
KurzfassungWe present new unit operations for valving and switching in centrifugal microfluidics that are actuated by a temperature change rate (TCR) and controlled by the rotational frequency. Implementation is realized simply by introducing a comparatively large fluidic resistance to an air vent of a fluidic structure downstream of a siphon channel. During temperature decrease at a given TCR, air pressure inside the downstream structure decreases and the fluidic resistance of the air vent slows down air pressure compensation allowing for a thermally induced underpressure to build up temporarily. Thereby the rate of temperature change determines the time course of the underpressure for a given geometry. The thermally induced underpressure pulls liquid against a centrifugal counterpressure above a siphon crest, which triggers the valve or switch. The centrifugal counterpressure (adjusted by rotation) serves as independent control parameter to allow or prevent valving or switching at any TCR. The unit operations are thus compatible to any temperature or centrifugation protocol prior to valving or switching. In contrast to existing methods, this compatibility is achieved at no additional costs: neither additional fabrication steps, nor additional disk space or external means are required besides global temperature control, which is per se needed for the assay. For layout, an analytical model is provided and verified. The TCR actuated unit operations are demonstrated, first, by a stand-alone switch that routes liquid to either one of the two collection chambers (n = 6) and, second, by studying robustness of TCR actuated valving within a microfluidic cartridge for highly integrated nucleic acid testing. Valving could safely be prevented during PCR by compensating the thermally induced underpressure of 3.52 kPa by centrifugal counterpressure at a rotational frequency of 30 Hz with a minimum safety range to valving of 2.03 kPa. Subsequently, a thermally induced underpressure of 2.55 kPa was utilized for robust siphon valving at 3 Hz with a minimum safety range of 2.32 kPa.

2016

F. Schuler, M. Trotter, M. Geltman, F. Schwemmer, S. Wadle, E. Domínguez-Garrido, M. López, C. Cervera-Acedo, P. Santibáñez, F. von Stetten, R. Zengerle, N. Paust
Digital Droplet PCR on Disk
2016 Lab Chip, Band: 16, Seiten: 208 - 216
KurzfassungExisting systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis.
F. Schuler, C. Siber, S. Hin, S. Wadle, N. Paust, R. Zengerle, F. von Stetten
Digital droplet LAMP as microfluidic App on standard laboratory devices
2016 Anal Methods-uk, Band: 8, Seiten: 2750 - 2755
KurzfassungDigital nucleic acid amplification methods are a growing research field that allows for absolute quantification of DNA making the need of standard curves redundant. However, most existing digital amplification systems require specialized laboratory devices and costly investments. The required disposable cartridges are device specific and not interchangeable. Here, we present digital droplet loopmediated isothermal amplification (ddLAMP) as a microfluidic App on standard laboratory devices. ddLAMP is implemented on a disposable polymer chip (DropChip) of the format of a standard microscope slide. After DNA denaturation off-chip the reaction mix is emulsified in the DropChip in a mini-centrifuge in 6 minutes. The DropChip is transferred to an in situ thermal cycler for 1 hour of incubation. Afterwards, a fluorescence scan in a microarray scanner is performed. The DropChip allows for absolute quantification with a dynamic range of 15-1500 DNA copies μl-1. Assay conditions were optimized for ddLAMP and comparison of ddPCR and ddLAMP for genomic E. coli DNA reveals very good concordance.
F. Schwemmer, C. E. Blanchet, A. Spilotros, D. Kosse, S. Zehnle, H. D. T. Mertens, M. A. Graewert, M. Rössle, N. Paust, D. I. Svergun, F. von Stetten, R. Zengerle, D. Mark
LabDisk for SAXS: a centrifugal microfluidic sample preparation platform for small-angle X-ray scattering
2016 Lab Chip, Band: 16, Seiten: 1161 - 1170
KurzfassungWe present a centrifugal microfluidic LabDisk for protein structure analysis via small-angle X-ray scattering (SAXS) on synchrotron beamlines. One LabDisk prepares 120 different measurement conditions, grouped into six dilution matrices. Each dilution matrix: (1) features automatic generation of 20 different measurement conditions from three input liquids and (2) requires only 2.5 μl of protein solution, which corresponds to a tenfold reduction in sample volume in comparison to the state of the art. 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). We demonstrate: (1) aliquoting of 40 nl aliquots for five different liquids typically used in SAXS and (2) confirm fluidic performance of aliquoting, merging, mixing and read-out from SAXS experiments (2.7–4.4% CV of protein concentration). We apply the LabDisk for SAXS for basic analysis methods, such as measurement of the radius of gyration, and advanced analysis methods, such as the ab initio calculation of 3D models. The suitability of the LabDisk for SAXS for protein structure analysis under different environmental conditions is demonstrated for glucose isomerase under varying protein and NaCl concentrations. We show that the apparent radius of gyration of the negatively charged glucose isomerase decreases with increasing protein concentration at low salt concentration. At high salt concentration the radius of gyration (Rg) does not change with protein concentrations. Such experiments can be performed by a non-expert, since the LabDisk for SAXS does not require attachment of tubings or pumps and can be filled 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.
F. Stumpf, F. Schwemmer, T. Hutzenlaub, D. Baumann, O. Strohmeier, G. Dingemanns, G. Simons, C. Sager, L. Plobner, F. von Stetten, R. Zengerle, D. Mark
LabDisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza A H3N2 virus
2016 Lab Chip, Band: 16, Seiten: 199 - 207
KurzfassungPortable point-of-care devices for pathogen detection require easy, minimal and user-friendly handling steps and need to have the same diagnostic performance compared to centralized laboratories. In this work we present a fully automated sample-to-answer detection of influenza A H3N2 virus in a centrifugal LabDisk with complete prestorage of reagents. Thus, the initial supply of the sample 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 polymerase chain reaction (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. We demonstrate successful detection of the respiratory pathogen influenza A H3N2 virus 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. Furthermore, we detected RNA bacteriophage MS2 acting as internal control in 3 LabDisks with a sample concentration down to 75 plaque forming units (pfu) per ml. All 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.
F. Schuler, M. Trotter, R. Zengerle, F. von Stetten
Monochrome Multiplexing in Polymerase Chain Reaction by Photobleaching of Fluorogenic Hydrolysis Probes
2016 Anal Chem, Band: 88, Nummer: 5, Seiten: 2590 - 2595
KurzfassungMultiplexing in polymerase chain reaction (PCR) is a technique widely used to save cost and sample material and to increase sensitivity compared to distributing a sample to several singleplex reactions. One of the most common methods to detect the different amplification products is the use of fluorogenic probes that emit at different wavelengths (colors). To reduce the number of detection channels, several methods for monochrome multiplexing have been suggested. However, they pose restrictions to the amplifiable target length, the sequence, or the melting temperature. To circumvent these limitations, we suggest a novel approach that uses different fluorophores with the same emission maximum. Discrimination is achieved by their different fluorescence stability during photobleaching. Atto488 (emitting at the same wavelength as 6-carboxyfluorescein, FAM) and Atto467N (emitting at the same wavelength as cyanine 5, Cy5) were found to bleach significantly less than FAM and Cy5; i.e., the final fluorescence of Atto dyes was more than tripled compared to FAM and Cy5. We successfully applied this method by performing a 4-plex PCR targeting antibiotic resistance genes in S. aureus using only 2 color channels. Confidence of discrimination between the targets was >99.9% at high copy initial copy numbers of 100 000 copies. Cases where both targets were present could be discriminated with equal confidence for Cy5 channel and reduced levels of confidence (>68%) for FAM channel. Moreover, a 2- plex digital PCR reaction in 1 color channel was shown. In the future, the degree of multiplexing may be increased by adding fluorogenic probe pairs with other emission wavelengths. The method may also be applied to other probe and assay formats, such as Förster resonance energy transfer (FRET) probes and immunoassays.
S. Wadle, M. Lehnert, S. Rubenwolf, R. Zengerle, F. von Stetten
Real-time PCR probe optimization using design of experiments approach
2016 Biomolecular Detection and Quantification, Band: 7, Seiten: 1 - 8
KurzfassungPrimer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3–14 target copies/10 μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7–11 copies/10 μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times.
S. Burger, M. Schulz, F. von Stetten, R. Zengerle, N. Paust
Rigorous buoyancy driven bubble mixing for centrifugal microfluidics
2016 Lab Chip, Seiten: 261 - 268
KurzfassungWe present batch-mode mixing for centrifugal microfluidics operated at fixed rotational frequency. Gas is generated by the disk integrated decomposition of hydrogen peroxide (H2O2) to liquid water (H2O) and gaseous oxygen (O2) and inserted into a mixing chamber. There, bubbles are formed that ascent through the liquid in the artificial gravity field and lead to drag flow. Additionaly, strong buoyancy causes deformation and rupture of the gas bubbles and induces strong mixing flows in the liquids. Buoyancy driven bubble mixing is quantitatively compared to shake mode mixing, mixing by reciprocation and vortex mixing. To determine mixing efficiencies in a meaningful way, the different mixers are employed for mixing of a lysis reagent and human whole blood. Subsequently, DNA is extracted from the lysate and the amount of DNA recovered is taken as a measure for mixing efficiency. Relative to standard vortex mixing, DNA extraction based on buoyancy driven bubble mixing resulted in yields of 92 ± 8% (100 s mixing time) and 100 ± 8% (600 s) at 130g centrifugal acceleration. Shake mode mixing yields 96 ± 11% and is thus equal to buoyancy driven bubble mixing. An advantage of buoyancy driven bubble mixing is that it can be operated at fixed rotational frequency, however. The additional costs of implementing buoyancy driven bubble mixing are low since both the activation liquid and the catalyst are very low cost and no external means are required in the processing device. Furthermore, buoyancy driven bubble mixing can easily be integrated in a monolithic manner and is compatible to scalable manufacturing technologies such as injection moulding or thermoforming. We consider buoyancy driven bubble mixing an excellent alternative to shake mode mixing, in particular if the processing device is not capable of providing fast changes of rotational frequency or if the low average rotational frequency is challenging for the other integrated fluidic operations.
S. Wadle, M. Lehnert, F. Schuler, R. Köppel, A. Serr, R. Zengerle, F. von Stetten
Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters
2016 BioTechniques, Band: 61, Seiten: 123 - 128
KurzfassungMediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2–5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength.

2015

S.K. Vashist, T. van Oordt, E. M. Schneider, R. Zengerle, F. von Stetten, J.H.T. Luong
A smartphone-based colorimetric reader for bioanalytical applications using the screen-based bottom illumination provided by gadgets
2015 Biosens Bioelectron, Band: 67, Seiten: 248 - 255
KurzfassungA smartphone-based colorimetric reader (SBCR) was developed using a Samsung Galaxy SIII mini, a gadget (iPAD mini, iPAD4 or iPhone 5s), integrated with a custom-made dark hood and base holder assembly. The smartphone equipped with a back camera (5 megapixels resolution) was used for colorimetric imaging via the hood and base-holder assembly. A 96- or 24-well microtiter plate (MTP) was positioned on the gadget's screensaver that provides white light-based bottom illumination only in the specific regions corresponding to the bottom of MTP's wells. The pixel intensity of the captured images was determined by an image processing algorithm. The developed SBCR was evaluated and compared with a commercial MTP reader (MTPR) for three model assays: our recently developed human C-reactive protein sandwich enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase direct ELISA, and bicinchoninic acid protein estimation assay. SBCR had the same precision, dynamic range, detection limit and sensitivity as MTPR for all three assays. With advanced microfabrication and data processing, SBCR will become more compact, lighter, inexpensive and enriched with more features. Therefore, SBCR with a remarkable computing power could be an ideal point-of-care (POC) colorimetric detection device for the next-generation of cost-effective POC diagnostics, immunoassays and diversified bioanalytical applications.
M. Keller, J. Naue, R. Zengerle, F. von Stetten, U. Schmidt
Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment
2015 Plos One, Band: 10, Nummer: 7, Seite: e0131845
KurzfassungNested PCR remains a labor-intensive and error-prone biomolecular analysis. Laboratory workflow automation by precise control of minute liquid volumes in centrifugal microfluidic Lab-on-a-Chip systems holds great potential for such applications. However, the majority of these systems require costly custom-made processing devices. Our idea is to augment a standard laboratory device, here a centrifugal real-time PCR thermocycler, with inbuilt liquid handling capabilities for automation. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards. For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler. It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. 92.2% of the performed tests were recognized as fluidically failure-free sample handling and used for evaluation. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.
O. Strohmeier, S. Keil, B. Kanat, P. Patel, M. Niedrig, M. Weidmann, F. Hufert, J. Drexler, R. Zengerle, F. von Stetten
Automated nucleic acid extraction from whole blood, B . subtilis, E. coli, and Rift Valley fever virus on a centrifugal microfluidic LabDisk
2015 Rsc Adv, Band: 5, Seite: 32144
KurzfassungWe present total nucleic acid extraction from whole blood, Gram‐positive Bacillus subtilis, Gram‐negative Escherichia coli, and Rift Valley fever RNA virus on a low‐cost, centrifugal microfluidic LabDisk cartridge processed in a light‐weight (< 2 kg) and portable processing device. Compared to earlier work in disk based centrifugal microfluidics, this includes the following advances: combined lysis and nucleic acid purification on one cartridge and handling of sample volumes as large as 200 μL. The presented system has been validated for logarithmic dilutions of aforementioned bacteria and viruses from various sample matrices including blood plasma and culture media and extraction of human DNA from whole blood. Recovered DNA and RNA concentrations in the eluate were characterized by quantitative PCR to: 58.2 % ‐ 98.5 %, 45.3 % ‐ 102.1 % and 29.5 % ‐ 34.2 % versus manual reference for Bacillus subtilis, Escherichia coli and Rift Valley fever virus, respectively. For extraction of human DNA from whole blood, similar results for on‐disk ((10.1 ± 7.6) x 104 DNA copies) and manual reference extraction ((10.2 ± 6.3) x 104 DNA copies)) could be achieved. Eluates from on‐disk extraction show slightly increased ethanol concentrations of 4.1 ± 0.3 % to 5.5 ± 0.2 % compared to manual reference (2.0 ± 0.5 % to 3.6 ± 0.6 %). The complete process chain for sample preparation is automatically performed within ~30 minutes, including ~15 minutes lysis time. It is amenable to concatenation with downstream modules for multiplex nucleic acid amplification as recently demonstrated for panel testing of various pathogens at the point of care.
F. Schuler, N. Paust, R. Zengerle, F. von Stetten
Centrifugal Step Emulsification can Produce Water in Oil Emulsions with Extremely High Internal Volume Fractions
2015 Micromachines, Band: 6, Nummer: 8, Seiten: 1180 - 1188
KurzfassungThe high throughput preparation of emulsions with high internal volume fractions is important for many different applications, e.g., drug delivery. However, most emulsification techniques reach only low internal volume fractions and need stable flow rates that are often difficult to control. Here, we present a centrifugal high throughput step emulsification disk for the fast and easy production of emulsions with high internal volume fractions above 95%. The disk produces droplets at generation rates of up to 3700 droplets/s and, for the first time, enables the generation of emulsions with internal volume fractions of >97%. The coefficient of variation between droplet sizes is very good (4%). We apply our system to show the in situ generation of gel emulsion. In the future, the recently introduced unit operation of centrifugal step emulsification may be used for the high throughput production of droplets as reaction compartments for clinical diagnostics or as starting material for micromaterial synthesis.
F. Schuler, F. Schwemmer, M. Trotter, S. Wadle, R. Zengerle, F. von Stetten, N. Paust
Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA
2015 Lab Chip, Band: 15, Seiten: 2759 - 2766
KurzfassungAqueous microdroplets provide miniaturized reaction compartments for numerous chemical, biochemical or pharmaceutical applications. We introduce centrifugal step emulsification for the fast and easy production of monodispers droplets. Homogenous droplets with preselectable diameters in a range from 120 μm to 170 μm were generated with coefficients of variation of 2-4% and zero run-in time or dead volume. The droplet diameter depends on the nozzle geometry (depth, width, and step size) and interfacial tensions, only. Droplet size is demonstrated to be independent of the dispersed phase flow rate between 0.01-1 μl/s, proving the robustness of the centrifugal approach. Centrifugal step emulsification can easily be combined with existing centrifugal microfluidic unit operations, is compatible to scalable manufacturing technologies such as thermoforming or injection moulding and enables fast emulsification (> 500 droplets per second and nozzle) with minimal handling effort (2-3 pipetting steps). The centrifugal microfluidic droplet generation was used to perform the first digital droplet recombinase polymerase amplification (ddRPA). It was used for absolute quantification of Listerias monocytogenes DNA concentration standards with a total analysis time below 30 min. Compared to digital droplet polymerase chain reaction (ddPCR), with processing times of about 2 hours, the overall processing time of digital analysis was reduced by more than a factor of 4.
F. Schwemmer, T. Hutzenlaub, D. Buselmeier, N. Paust, F. von Stetten, D. Mark, R. Zengerle, D. Kosse
Centrifugo-pneumatic multi-liquid aliquoting – parallel aliquoting and combination of multiple liquids in centrifugal microfluidics
2015 Lab Chip, Band: 15, Seiten: 3250 - 3258
KurzfassungThe generation of mixtures with precisely metered volumes is essential for reproducible automation of laboratory workflows. Splitting a given liquid into well-defined metered sub-volumes, the so-called aliquoting, has been frequently demonstrated on centrifugal microfluidics. However, so far no solution exists for assays that require simultaneous aliquoting of multiple, different liquids and the subsequent pairwise combination of aliquots with full fluidic separation before combination. Here, we introduce 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, we demonstrate simultaneous aliquoting of 1.) a common assay reagent into twenty 5 µl aliquots and 2.) five different sample liquids, each into four aliquots of 5 µl. Subsequently, the reagent and sample aliquots are simultaneously transported and combined into twenty collection chambers. All coefficients of variation for metered volumes were between 0.4% - 1.0% for intra-run variations and 0.5% - 1.2% for inter-run variations. The aliquoting structure is compatible to 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 / m3 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. Fabrication is compatible to scalable fabrication technologies such as injection molding or thermoforming and does not require any additional fabrication steps such as hydrophilic or hydrophobic coatings or integration of active valves.
Y. Zhao, F. Schwemmer, S. Zehnle, F. von Stetten, R. Zengerle, N. Paust
Centrifugo-pneumatic sedimentation, re-suspension and transport of microparticles
2015 Lab Chip, Seiten: 4133 - 4137
KurzfassungMicroparticles are widely used as solid phase for affinity based separation. Here, we introduce a new method for automated handling of microparticles in centrifugal microfluidics that is not restricted by the particle size and requires neither auxiliary means such as magnets nor coating of microfluidic structures. All steps are initiated and controlled by the speed of rotation, only. It is based on storage and “on demand” release of pneumatic energy within tuneable time frames: A slow release of the pneumatic energy triggers a first fluidic path through which the supernatant above the sedimented particles is removed. An abrupt release triggers a second path which allows for liquid routing and transport of the re-suspended particles. Re-suspension of particles is thereby achieved by quickly changing the speed of rotation. We demonstrate the exchange of the particle carrier medium with supernatant removal efficiency of more than 99.5% and particle loss below 4%. Re-suspension and subsequent transport of suspended particles shows particle loss below 7%. The method targets for the automation of particle based assays e.g. DNA extractions and immunoassays. It is compatible to monolithic integration and suitable for mass production technologies e.g. thermoforming or injection moulding.
M. Keller, S. Wadle, N. Paust, L. Dreesen, C. Nuese, O. Strohmeier, R. Zengerle, F. von Stetten
Centrifugo-thermopneumatic fluid control for valving and aliquoting applied to multiplex real-time PCR on off-the-shelf centrifugal thermocycler
2015 Rsc Adv, Band: 5, Seiten: 89603 - 89611
KurzfassungWe introduce microfluidic automation of geometrically multiplexed real-time PCR to off-the-shelf Rotor-Gene Q thermocyclers (RGQ, QIAGEN GmbH, Hilden, Germany). For centrifugal fluid control the RGQ provides low and constant rotation of 400 rpm, only. Compatibility to this very limited flexibility of centrifugal actuation is achieved by using thermal gas compression and expansion for valving and aliquoting. In contrast to existing thermo-pneumatic actuation, centrifugo-thermopneumatic (CTP) fluid control employs the induced change of partial vapor pressure by global temperature control as actuation parameter for two new unit operations: CTP siphon valving and CTP two-stage aliquoting. CTP siphon valving was demonstrated to reliably transfer sample liquid in all cases (n = 35) and CTP two-step aliquoting transfers metered aliquots of 18.2 ± 1.2 μl (CV 6.7%, n = 8) into reaction cavities within 5 s (n = 24). Thermal characteristics of CTP two-stage aliquoting were found to be in good agreement with an introduced analytical model (R2 = 0.9876, n = 3). A microfluidic disk segment comprising both new unit operations was used for automation of real-time PCR amplification of Escherichia coli DNA. Required primers and probes were pre-stored in the reaction cavities and a comparison with reference reactions in conventional PCR tubes yielded the same PCR efficiency, repeatability, and reproducibility.
L. Drechsel, M. Schulz, F. von Stetten, C. Moldovanc, R. Zengerle, N. Paust
Electrochemical pesticide detection with AutoDip – a portable platform for automation of crude sample analyses
2015 Lab Chip, Band: 15, Seiten: 704 - 710
KurzfassungLab-on-a-chip devices hold promise for automation of complex workflows from sample to answer with minimal consumption of reagents in portable devices. However, complex, inhomogeneous samples as they occur in environmental or food analysis may block microchannels and thus often cause malfunction of the system. Here we present the novel AutoDip platform which is based on the movement of a solid phase through the reagents and sample instead of transporting a sequence of reagents through a fixed solid phase. A ball-pen mechanism operated by an external actuator automates unit operations such as incubation and washing by consecutively dipping the solid phase into the corresponding liquids. The platform is applied to electrochemical detection of organophosphorus pesticides in real food samples using an acetylcholinesterase (AChE) biosensor. Minimal sample preparation and an integrated reagent pre-storage module hold promise for easy handling of the assay. Detection of the pesticide chlorpyrifos-oxon (CPO) spiked into apple samples at concentrations of 10−7 M has been demonstrated. This concentration is below the maximum residue level for chlorpyrifos in apples defined by the European Commission.
A.G. Venkatesh, S. K. Vashist, E. M. Schneider, R. Zengerle, F. von Stetten, J. H.T. Luong
Graphene-based C-reactive protein immunoassay with smartphone readout
2015 Protocol Exchange
KurzfassungA highly-sensitive immunoassay (IA) procedure with minimal process steps has been developed for the detection of human C-reactive protein (CRP) in less than 30 min. Graphene nanoplatelets (GNP) was admixed with 3-aminopropyltriethoxysilane (APTES) and EDC-activated anti-human CRP antibody (Ab) to form a stable complex that was then covalently attached to a KOH-pretreated polystyrene microtiter plate (MTP). The developed one-step kinetics-based IA involves the formation of a sandwich immune complex followed by two washings and an enzyme-based colorimetric reaction that was read by a smartphone-based colorimetric reader (SBCR). The detection range of CRP was 0.03-81 ng mL-1 with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.07 ng mL-1 and 0.9 ng mL-1, respectively. Moreover, the developed IA enabled the detection of CRP spiked in diluted human whole blood and plasma as well as CRP present in clinical plasma samples with high analytical precision, thereby demonstrating its immense utility for biomedical diagnostics.
G.Czilwik, S.K.Vashist, V.Klein, G.Roth, A.Buderer, F. von Stetten, R. Zengerle, D.Mark
Magnetic chemiluminescent immunoassay for human C-reactive protein on the centrifugal microfluidics platform
2015 Rsc Adv, Band: 5, Seiten: 61906 - 61912
KurzfassungHuman C-reactive protein (CRP) has been reported as an inflammatory biomarker with the highest reference for use in clinical practice. However, the existing analytical techniques are lacking automation and simplicity, as desired for a prospective immunoassay format for point-of-care (PoC) analysis. We have developed an automated magnetic chemiluminescent immunoassay (MCIA) on a mobile analyser for rapid PoC determination of CRP. The MCIA is fully automated after the initial loading of sample and immunoreagents at the inlet ports. The automated protocol involves the transportation of magnetic capture microparticles between adjacent reaction compartments using a set of stationary magnets, a microfluidic polymer disposable and a specific centrifugal protocol. The developed MCIA has a sample-to-answer time of 25 min and hands-on time of approximately 5 min. It detects the entire pathophysiological range of CRP, as desired for clinically-relevant high sensitivity CRP immunoassay format, i.e. 3–81 ng mL−1 in diluted human serum with a limit of detection (LOD) and limit of quantification (LOQ) of 1.5 ng mL−1 and 1.8 ng mL−1, respectively.
F. Schwemmer, S. Zehnle, D. Mark, F. von Stetten, R. Zengerle, N. Paust
Microfluidic timer for timed valving and pumping in centrifugal microfluidics
2015 Lab Chip, Band: 15, Seiten: 1545 - 1553
KurzfassungAccurate timing of microfluidic operations is essential for the automation of complex laboratory workflows, in particular for the supply of sample and reagents. Here we present a new unit operation for timed valving and pumping in centrifugal microfluidics. It is based on temporary storage of pneumatic energy and 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 releases after a pre-defined delay time. We demonstrate timing for 1.) the sequential release of 4 liquids at times of 2.7 s ± 0.2 s, 14.0 s ± 0.5 s, 43.4 s ± 1.0 s and 133.8 s ± 2.3 s, 2.) timed valving of typical assay reagents (contact angles 36° - 78°, viscosities 0.9 mPa s - 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 to all wetting properties and viscosities of common assay reagents and does neither require assistive equipment, nor coatings. It can be monolithically integrated into a microfluidic test carrier and is compatible to scalable fabrication technologies such as thermoforming or injection molding.
G. Czilwik, I. Schwarz, M. Keller, S. Wadle, S. Zehnle, F. von Stetten, D. Mark, R. Zengerle, N. Paust
Microfluidic vapor-diffusion barrier for pressure reduction in fully closed PCR modules
2015 Lab Chip, Band: 15, Seiten: 1084 - 1091
KurzfassungMicrofluidic systems for polymerase chain reaction (PCR) should be fully closed to avoid vapor loss and to exclude the risk of contaminating the test environment. In closed systems however, the high temperatures of up to 95°C associated with PCR cause high overpressures up to 100 kPa, dominated by the increase of vapor partial pressure upon evaporation. Such high overpressures pose challenges to the mechanical stability of microfluidic chips as well as to the liquid handling in integrated sample-to-answer systems. In this work, we drastically reduce the pressure increase in fully closed PCR systems by integrating a microchannel that serves as a vapor-diffusion barrier (VDB), separating the liquid-filled PCR chamber from an auxiliary air chamber. In such configurations, propagation of vapor from the PCR chamber into the auxiliary air chamber and as a consequence the increase of pressure is limited by the slow diffusion process of vapor through the VDB. At temperature increase from 23°C to 95°C, we demonstrate the reduction of overpressure from more than 80 kPa without the VDB to only 35 kPa with the VDB. We further demonstrate proper function of VDB and its easy integration with downstream processes for PCR based nucleic acid amplification within centrifugal microfluidics. Without integration of the VDB, malfunction due to pressure-induced delamination of the microfluidic chip occurred.
G. Czilwik, D. Baumann, D. Mark, R. Zengerle, F. von Stetten
Mikrofluidische Automatisierung in der Molekulardiagnostik
2015 BIOspektrum, Band: 21, Seiten: 622 - 624
KurzfassungEasy-to-use point-of-care (POC) tests provide rapid diagnostic results near the patient, enabling fast decision-making in patient management. However, the implementation of automated molecular diagnostic POC tests still faces technical challenges. We demonstrate a highly sensitive POC platform for automated molecular diagnostics of neonatal sepsis that uses a mobile analyzer and unit-use test carriers.
S. Zehnle, F. Schwemmer, R. Bergmann, F. von Stetten, R. Zengerle, N. Paust
Pneumatic siphon valving and switching in centrifugal microfluidics controlled by rotational frequency or rotational acceleration
2015 Microfluid Nanofluid, Band: 19, Seiten: 1259 - 1269
KurzfassungAir-pressure-mediated, pneumatic siphon valves employ temporary storage and subsequent release of pneumatic energy, exclusively controlled by rotation of the disk. Implementation is easy, and robust valves can be integrated in a monolithic way at minimum additional costs. However, so far, pneumatic siphon valving requires deceleration from high to low rotational frequencies. Valve opening is performed always when the rotation of the disk drops below a critical rotational frequency. To overcome this limitation, we introduce new concepts for pneumatic siphon valving which enable operation of the disk at any rotational frequency without unwanted bursts of the siphon valves. Thus, the design space for pneumatic siphon valves in centrifugal microfluidics is significantly extended. Three types of pneumatic siphon valves are presented with release control at (1) rotational frequencies between 25 and 48 Hz, (2) positive rotational accelerations between 1 and 22 Hz s−1, and (3) negative rotational accelerations between 5 and 20 Hz s−1. Finally, we combine two valve types to realize robust switching into two fluidic paths with flow rate ratios of 94/6 and 0/100.
G. Czilwik, T. Messinger, O. Strohmeier, S. Wadle, F. von Stetten, N. Paust, G. Roth, R. Zengerle, P. Saarinen, J. Niittymäki, K. McAllister, O. Sheils, J. O'Leary, D. Mark
Rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic LabDisk using highly sensitive nested PCR with integrated sample preparation
2015 Lab Chip, Band: 15, Seiten: 3749 - 3759
KurzfassungDiagnosis of infectious diseases suffers from long turnaround times for gold standard culture-based identification of bacterial pathogens, therefore impeding timely specific antimicrobial treatment based on laboratory evidence. Rapid molecular diagnostics-based technologies enable detection of microorganisms within hours however cumbersome workflows and complex equipment still prevent their widespread use in the routine clinical microbiology setting. We developed a centrifugal-microfluidic “LabDisk” system for rapid and highly-sensitive pathogen detection on a point-of-care analyser. The unit-use LabDisk with pre-stored reagents features fully automated and integrated DNA extraction, consensus multiplex PCR pre-amplification and geometrically-multiplexed species-specific real-time PCR. Processing merely requires loading of the sample and DNA extraction reagents with minimal hands-on time of approximately 5 min. We demonstrate detection of as few as 3 colony-forming-units (cfu) of Staphylococcus warneri, 200 cfu of Streptococcus agalactiae, 5 cfu of Escherichia coli and 2 cfu of Haemophilus influenzae in a 200 μL serum sample. The turnaround time of the complete analysis from “sample-to-result” was 3 h and 45 min. The LabDisk consequently provides an easy-to-use molecular diagnostic platform for rapid and highly-sensitive detection of bacterial pathogens without requiring major hands-on time and complex laboratory instrumentation.

2014

S.K. Vashist, G. Czilwik, T. van Oordt, F. von Stetten, R. Zengerle,, E.M. Schneider, J.H.T. Luong
A rapid one-step kinetics-based immunoassay procedure for the highly-sensitive detection of C-reactive protein
2014 Protocol Exchange
M. Hoehl, E. Schulte Bocholt, A. Kloke, N. Paust, F. von Stetten, R. Zengerle, J. Steigert, A. Slocum
A versatile-deployable bacterial detection system for food and environmental safety based on LabTube-automated DNA purification, LabReader-integrated amplification, readout and analysis
2014 Analyst, Band: 139, Seiten: 2788 - 2798
KurzfassungContamination of foods is a public health hazard that episodically causes thousands of deaths and sickens millions worldwide. To ensure food safety and quality, rapid, low-cost and easy-to-use detection methods 10 are desirable. Here, the LabSystem is introduced for integrated, automated DNA purification, amplification and detection. It consists of a disposable, centrifugally-driven DNA purification platform (LabTube) and the subsequent amplification and detection in a low-cost UV/vis-reader (LabReader). For demonstration of the LabSystem in the context of food safety, purification of Escherichia coli (nonpathogenic E. coli and pathogenic verotoxin-producing E. coli (VTEC)) in water and milk, and the 15 product-spoiler Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice was integrated and optimized in the LabTube. Inside the LabReader, the purified DNA was amplified, readout and analyzed using both qualitative isothermal loop-mediated DNA amplification (LAMP) and quantitative real-time PCR. For the LAMP-LabSystem, the combined detection limits for purification and amplification of externally lysed VTEC and A. acidoterrestris is 102-103 cell-equivalents. In the PCR-LabSystem for E. coli cells, the quantification limit is 102 20 cell-equivalents including LabTube-integrated lysis. The demonstrated LabSystem only requires a laboratory centrifuge (to operate the disposable, fully closed LabTube) and the low-cost LabReader for DNA amplification, readout and analysis. Compared with commercial DNA amplification devices, the LabReader improves sensitivity and specificity by the simultaneous readout of four wavelengths and the continuous readout during temperature cycling. The 25 use of a detachable eluate tube as an interface affords semi-automation of the LabSystem, which does not require specialized training. It reduces hands-on time from about 50 to 3 min with only two handling steps: sample input and transfer of the detachable detection tube.
M. Hoehl, M. Weissert, A. Dannenberg, T. Nesch, N. Paust, F. von Stetten, R. Zengerle, A. Slocum, J. Steigert
Centrifugal LabTube platform for fully automated DNA purification and LAMP amplification based on an integrated, low-cost heating-system
2014 Biomed Microdevices, Band: 16, Nummer: 3, Seiten: 375 - 385
KurzfassungThis paper introduces a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA purification platform (LabTube). We demonstrate LabTube-based fully automated DNA purification of as low as 100 cell-equivalents of verotoxin-producing Escherichia coli (VTEC) in water, milk and apple juice in a laboratory centrifuge, followed by integrated and automated LAMP amplification with a reduction of hands-on time from 45 to 1 min. The heating system consists of two parallel SMD thick film resistors and a NTC as heating and temperature sensing elements. They are driven by a 3 V battery and controlled by a microcontroller. The LAMP reagents are stored in the elution chamber and the amplification starts immediately after the eluate is purged into the chamber. The LabTube, including a microcontroller-based heating system, demonstrates contamination-free and automated sample-to-answer nucleic acid testing within a laboratory centrifuge. The heating system can be easily parallelized within one LabTube and it is deployable for a variety of heating and electrical applications.
S. K. Vashist, E.M. Schneider, R. Zengerle, F. von Stetten, J.H.T. Luong
Graphene-based rapid and highly-sensitive immunoassay for C-re active protein using a smartphone-based colorimetric reader
2014 Biosens Bioelectron, Band: 66, Seiten: 169 - 176
KurzfassungA novel immunoassay (IA) has been developed for human C-reactive protein (CRP), an important biomarker and tissue preserving factor for infection and inflammation. Graphene nanoplatelets (GNP) and 3-aminopropyltriethoxysilane (APTES) were admixed and covalently attached to a polystyrene based-microtiter plate (MTP), pretreated with KOH. The resulting surface served as a stable layer for the covalent attachment of the anti-human CRP antibody. The IA procedure was based on the one-step kinetics-based sandwich IA employing a minimum number of process steps, whereas the enzymatic reaction solution was monitored by a smartphone-based colorimetric reader. With a limit of detection and a limit of quantification of 0.07 ng mL−1 and 0.9 ng mL−1, it precisely detected CRP spiked in diluted human whole blood and plasma as well as the CRP levels in clinical plasma samples. The results obtained for “real-world” patient samples agreed well with those of the conventional immunosorbent assay and the clinically-accredited analyzer-based IA. The antibody-bound GNP-functionalized MTPs retained its original activity after 6 weeks of storage in 0.1 M PBS, pH 7.4 at 4 °C.
T. van Oordt, Y. Barb, R. Zengerle, F. von Stetten
Lamination of Polyethylene Composite Films by Ultrasonic Welding: Investigation of Peel Behavior and Identification of Optimum Welding Parameters
2014 J Appl Polym Sci, Band: 131, Seiten: 40291 - 40298
KurzfassungThe lamination of different polyethylene (PE) composite films by ultrasonic welding to fabricate peelable seals that open at defined burst pressures is investigated. The sealing time, pressure, and amplitude were varied within the range of 100–400 ms, 50– 250 kPa, and 12–24 mm, 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 will allow the systematic adjustment of ultrasonic welding parameters for PE films.
S.K. Vashist, G. Czilwik, T. van Oordt, F. von Stetten, R. Zengerle, E.M. Schneider, J.H.T. Luong
One-step kinetics-based immunoassay for the highly-sensitive detection of C-reactive protein in less than 30 minutes
2014 Anal Biochem, Band: 456, Seiten: 32 - 37
KurzfassungThe article reveals a rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the highly-sensitive detection of human C-reactive protein (CRP) in less than 30 min. It employs a one-step kinetics-based highly simplified and cost-effective sandwich ELISA procedure with minimum process steps. The procedure involves the formation of a sandwich immune complex on capture anti-human CRP antibody-bound Dynabeads® in 15 min followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3-81 ng mL-1 with a limit of detection and analytical sensitivity of 0.4 ng mL-1 and 0.7 ng mL-1, respectively. It detects CRP spiked in diluted human whole blood and serum with high analytical precision as confirmed by conventional sandwich ELISA. Moreover, the results of the developed ELISA for the determination of CRP in the ethylenediaminetetraacetic acid (EDTA) plasma samples of patients are in good agreement with those obtained by the conventional ELISA. The developed immunoassay has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.
C. Escadafal, O. Faye, A. A. Sall, O. Faye, M. Weidmann, O. Strohmeier, F. von Stetten, J. Drexler, M. Eberhard, M. Niedrig, P. Patel
Rapid molecular assys for the detection of yellow fever virus in low-resource settings
2014 Plos Neglect Trop D, Band: 8, Nummer: 3, Seite: e2730
KurzfassungDespite the use of a safe and effective vaccine, yellow fever virus is still causing hundreds of thousands of infections and tens of thousands of deaths every year. The disease is widespread in South America and Africa where several outbreaks have occurred in the past years. As the disease is difficult to distinguish from other illnesses during its early stage, it is necessary to develop reliable, rapid and simple diagnostic methods to confirm YF cases to be able to respond effectively to outbreaks through vaccination and vector control. In this study, we describe the development a diagnostic method for YFV, using an isothermal technology called recombinase polymerase amplification which allows detection of the virus within 20 minutes, using a portable and easy-to-use device. The YFV RPA assay proved to be a specific and sensitive detection method during testing in the laboratory and under field conditions in Senegal.
O. Strohmeier, N. Marquart, D. Mark, G. Roth, R. Zengerle, F. von Stetten
Real-time PCR based detection of a panel of foodborne pathogens on a centrifugal microfluidic “LabDisk” with on-disk quality controls and standards for quantification
2014 Anal Methods-uk, Band: 6, Seiten: 2038 - 2046
M. Rombach, D. Kosse, B. Faltin, S. Wadle, G. Roth, R. Zengerle, F. von Stetten
Real-time stability testing of air-dried primers and fluorogenic hydrolysis probes stabilized by trehalose and xanthan
2014 Biotechniques, Band: 57, Nummer: 3, Seiten: 151 - 155
KurzfassungA method for conserving primers and differently labeled fluorogenic hydrolysis (i.e., TaqMan) probes at ambient conditions is presented. 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 cyclo 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 polymerase chain reaction (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.
A. Kloke, A. R. Fiebach, S. Zhang, L. Drechsel, S. Niekrawietz, M. Hoehl, R. Kneusel, K. Panthel, J. Steigert, F. von Stetten, R. Zengerle, N. Paust
The LabTube – A novel microfluidic platform for assay automation in laboratory centrifuges
2014 Lab Chip, Band: 14, Seiten: 1527 - 1537
KurzfassungAssay automation is the key for successful transformation of modern biotechnology into routine workflows. Yet, it requires considerable investment in processing devices and auxiliary infrastructure, which is not cost-efficient for laboratories with low or medium sample throughput or point-of-care testing. To close this gap, we present the LabTube platform, which is based on assay specific disposable cartridges for processing in laboratory centrifuges. LabTube cartridges comprise interfaces for sample loading and downstream applications and fluidic unit operations for release of prestored reagents, mixing, and solid phase extraction. Process control is achieved by a centrifugally-actuated ballpen mechanism. To demonstrate the workflow and functionality of the LabTube platform, we show two LabTube automated sample preparation assays from laboratory routine: DNA extractions from whole blood and purification of His-tagged proteins. Equal DNA and protein yield were observed compared to manual reference runs, while LabTube automation could significantly reduce the hands-ontime to one minute per extraction.

2013

Y. Abbas, J. Miwa, R. Zengerle, F. von Stetten
Active Continuous-Flow Micromixer Using an External Braille Pin Actuator Array
2013 Micromachines, Band: 4, Seiten: 80 - 89
KurzfassungWe present a continuous-flow active micromixer based on channel-wall deflection in a polydimethylsiloxane (PDMS) chip for volume flows in the range up to 2 μL s−1 which is intended as a novel unit operation for the microfluidic Braille pin actuated platform. The chip design comprises a main microchannel connected to a series of side channels with dead ends aligned on the Braille pins. Computer-controlled deflection of the side-channel walls induces chaotic advection in the main-channel, which substantially accelerates mixing in low-Reynolds number flow. Sufficient mixing (mixing index MI below 0.1) of volume flows up to 0.5 μL s−1 could be achieved within residence times ~500 ms in the micromixer. As an application, continuous dilution of a yeast cell sample by a ratio down to 1:10 was successfully demonstrated. The mixer is intended to serve as a component of bio-analytical devices or as a unit operation in the microfluidic Braille pin actuated platform.
O. Strohmeier, A. Emperle, G. Roth, D. Mark, R. Zengerle, F. von Stetten
Centrifugal gas-phase transition magnetophoresis (GTM) – a generic method for automation of magnetic bead based assays on the centrifugal microfluidic platform and application to DNA purification
2013 Lab Chip (Lab On A Chip) 2013, Band: 13, Seiten: 146 - 155
KurzfassungTransportation of magnetic beads between different reagents plays a crucial role in many biological assays e.g. for purification of biomolecules or cells where the beads act as a mobile solid support. Therefore, usually a complex set-up either for fluidic processing or for manipulation of magnetic beads is required. To circumvent these drawbacks, we present a facile and automated method for the transportation of magnetic beads between multiple microfluidic chambers on a centrifugal microfluidic cartridge ‘‘LabDisk’’. The method excels by requiring only one stack of stationary permanent magnets, a specific microfluidic layout without actively controlled valves and a predefined frequency protocol for rotation of the LabDisk. Magnetic beads were transported through three fluidically separated chambers with a yield of 82.6% ¡ 3.6%. Bead based DNA purification from a dilution series of a Listeria innocua lysate and from a lambda phage DNA standard was demonstrated where the three chambers were used for binding, washing and elution of DNA. Recovery of L. innocua DNA was up to 68% ¡ 24% and for lambda phage DNA 43% ¡ 10% compared to manual reference purification in test tubes. Complete purification was conducted automatically within 12.5 min. Since all reagents can be preloaded onto the LabDisk prior to purification, no further hands-on steps are required during processing. Due to its modular and generic character, the presented method could also be adapted to other magnetic bead based assays e.g. to immunoassays or protein affinity purification, solely requiring the adjustment of number and volumes of the fluidic chambers.
D. Mark, F. von Stetten, R. Zengerle
Chips with everything: microfluidics and diagnostics
2013 Medical Device Developments
T. van Oordt, Y. Barb, J. Smetana, R. Zengerle, F. von Stetten
Miniature stick-packaging – an industrial technology for pre-storage and release of reagents in lab-on-a-chip systems
2013 Lab Chip, Band: 13, Nummer: 15, Seiten: 2888 - 2892
KurzfassungStick-packaging of goods in tubular-shaped composite-foil pouches has become a popular technology for food and drug packaging. We miniaturized stick-packaging for use in lab-on-a-chip (LOAC) systems to prestore and on-demand release the liquid and dry reagents in a volume range of 80–500 ml. 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 ml pre-stored liquid is released), long-term storage tests (where there is loss of only ,0.5% for simulated 2 years) and air transport simulation tests. The developed technology 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.
O. Strohmeier, S. Laßmann, B. Riedel, D. Mark, G. Roth, M. Werner, R. Zengerle, F. von Stetten
Multiplex genotyping of KRAS point mutations in tumor cell DNA by allele-specific real-time PCR on a centrifugal microfluidic disk segment
2013 Microchim Acta, Band: 181, Seiten: 1681 - 1688
KurzfassungPoint Mutations on the Kirsten rat sarcoma viral oncogene homolog (KRAS) have been identified as an important predictive biomarker for response to cancer therapy targeting the epidermal growth factor receptor. KRAS mutations are prevalent in up to 40 % of all colorectal carcinomas, and routinely conducted KRAS genotyping is becoming mandatory to predict therapy success and to reduce therapy costs. We report a low-cost, disposable and ready-to-use centrifugal microfluidic cartridge (termed GeneSlice) containing preloaded primers and probes. The GeneSlice cartridge enables the parallel detection of the seven most relevant KRAS point mutations by allele-specific real-time PCR. It represents a cost effective alternative to dideoxy-sequencing with a faster time-to-result (~ 2 h versus up to 20 h in case of dd-sequencing). Microfluidic processing of the GeneSlice along with allele-specific amplification and real-time detection are conducted in a slightly modified, commercially available PCR thermocycler. Intra-chip standard deviation of Cq values on the GeneSlices is negligible (GeneSlice 1: Cq,std.dev. = 0.13; GeneSlice 2: Cq,std.dev = 0.26). In 23 of 24 experiments, the data for genotyping 6 cancer cell lines (n = 4 per cell line) agreed with dd-sequencing. Additionally, DNA derived from microdissected formalin-fixed and paraffin embedded colorectal carcinomas of two cases was genotyped correctly and reproducibly (n = 3 per patient; one GeneSlice excluded from evaluation). The GeneSlice therefore clearly demonstrated the potential to become a valuable tool for routine diagnostics of KRAS mutations by reducing costs and hands-on time.
T. van Oordt, G. B. Stevens, S. K. Vashist, R. Zengerle, F. von Stetten
Rapid and highly sensitive luciferase reporter assay for the automated detection of botulinum toxin in the centrifugal microfluidic LabDisk platform
2013 Rsc Adv, Band: 3, Seiten: 22046 - 22052
KurzfassungWe developed a system for the detection of botulinum neurotoxin (BoNT) type A based on a highly sensitive luciferase reporter assay 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 due to the release of firefly luciferase, pre-bound to microbeads via a cleavable peptide linker, in response to BoNT. It detected purified BoNT, 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 to 6 nM with an analytical sensitivity and limit of detection of 10–39 pM and 6–14 pM, respectively. The intra-disk, intra-day and inter-day variability were in the range of 1–13%, 1–7% and 10–13%, respectively. The developed assay correlated well with the conventional microwell plate assay. It is superior to the conventional BoNT assays in terms of portability, cost-effectiveness, lesser sample requirement, lesser number of steps and detection of a broad spectrum of BoNT serotypes and subtypes. It takes only 30 minutes, which is ideal for point-of-need BoNT detection in the case of security threats and food monitoring.

2012

S. Zehnle, F. Schwemmer, G. Roth, F. von Stetten, R. Zengerle, N. Paust
Centrifugo-dynamic inward pumping of liquids on a centrifugal microfluidic platform
2012 Lab Chip (Lab on a Chip) 2012, Band: 12, Seiten: 5142 - 5145
KurzfassungWe present a method to pump liquids in a centrifugal microfluidic spinning disk from a radial outward position to a radial inward position. Centrifugal forces are applied to compress air in a cavity, this way storing pneumatic energy. The cavity is connected to an outlet channel having a lower hydraulic resistance compared to the inlet channel. The stored pneumatic energy is quickly released by fast reduction of rotational frequency. This way liquid is transported mainly through the channel with lower resistance, directing the liquid radially inwards. Pump efficiencies of .75% per pump cycle have been demonstrated for water, ethanol, a highly viscous lysis buffer and whole blood. By employing three pump cycles, water has been pumped radially inwards with an efficiency of .90%. The inward pumping requires centrifugation only, which is intrinsically available on every centrifugal microfluidic platform.

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B. Faltin, S. Wadle, G. Roth, R. Zengerle, F. von Stetten
Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free Primary Probes and Standardized Secondary Universal Fluorogenic Reporters
2012 Clin Chem, Band: 58, Nummer: 11, Seiten: 1546 - 1556
Daniel Mark, Felix von Stetten, Roland Zengerle
Microfluidic Apps for off-the-shelf instruments
2012 Lab Chip, Band: 12, Seiten: 2464 - 2468
S.K. Vashist, A. G. Venkatesh, K. Mitsakakis, G. Czilwik, G. Roth, F. von Stetten, R. Zengerle
Nanotechnology-Based Biosensors and Diagnostics: Technology Push versus Industrial/Healthcare Requirements
2012 BioNanoSci., Band: 2, Seiten: 115 - 126
KurzfassungThere have been considerable advances in the field of nanotechnology-based biosensors and diagnostics (NBBD) during the last two decades. These include the production of nanomaterials (NMs), employing them for new biosensing and diagnostic applications, their extensive characterization for in vitro and in vivo applications, and toxicity analysis. All these developments have led to tremendous technology push and successful demonstrations of several promising NBBD. However, there has been a significant lag in their commercialization, especially due to the lack of international regulatory guidelines for evaluating the safety of NMs and the growing public concerns about their toxicity. Despite these numerous advances and the recent regulatory approval of several NMs, it still remains to be seen if NBBD are superior to conventional ones (not based on NMs), reliable, reproducible, cost effective, and robust enough to meet the requirements of industries and healthcare. This manuscript provides a critical review of NBBD, the technology push, and the industrial/healthcare requirements.
S. Rubenwolf, S. Sané, L. Hussein, J. Kestel, F. von Stetten, G. Urban, M. Krueger, R. Zengerle, S. Kerzenmacher
Prolongation of electrode lifetime in biofuel cells by periodic enzyme renewal
2012 Appl Microbiol Biotechnol, Band: 96, Seiten: 841 - 849
KurzfassungAbstract Enzymatically catalyzed biofuel cells show unique specificity and promise high power densities, but suffer from a limited lifetime due to enzyme deactivation. In the present work, we demonstrate a novel concept to extend the lifetime of a laccase-catalyzed oxygen reduction cathode in which we decouple the electrode lifetime from the limited enzyme lifetime by a regular resupply of fresh enzymes. Thereto, the adsorption behavior of laccase from Trametes versicolor to buckypaper electrode material, as well as its time-dependent deactivation characteristics, has been investigated. Laccase shows a Langmuir-type adsorption to the carbon nanotube-based buckypaper electrodes, with a mean residence time of 2 days per molecule. In a citrate buffer of pH 5, laccase does not show any deactivation at room temperature for 2 days and exhibits a half-life of 9 days. In a long-term experiment, the laccase electrodes were operated at a constant galvanostatic load. The laccase-containing catholyte was periodically exchanged against a freshly prepared one every second day to provide sufficient active enzymes in the catholyte for the replacement of desorbed inactive enzymes. Compared to a corresponding control experiment without catholyte exchange, this procedure resulted in a 2.5 times longer cathode lifetime of 19±9 days in which the electrode showed a potential above 0.744 V vs. normal hydrogen electrode at 110 μAcm−2. This clearly indicates the successful exchange of molecules by desorption and re-adsorption and is a first step toward the realization of a selfregenerating enzymatic biofuel cell in which enzyme-producing microorganisms are integrated into the electrode to continuously resupply fresh enzymes.
Jochen Hoffmann, Martin Trotter, Felix von Stetten, Roland Zengerle, Günter Roth
Solid-phase PCR in a picowell array for immobilizing and arraying 100,000 PCR products to a microscope slide
2012 Lab Chip, Band: 12, Nummer: 17, Seiten: 3049 - 3054
KurzfassungWe present a method for performing highly parallel PCR reactions in a picowell array (PWA) simultaneously immobilizing generated PCR products covalent and spatially-resolved onto a microscope slide via solid-phase PCR (SP-PCR). This so called PWA-SP-PCR is performed in picowell arrays featuring 100,000 wells∙cm^-2 of 19 pL reaction volumes with a surface-to-volume-ratio of 0.2 µm-1. Positive signals are obtained in 97.2 % of the 110,000 wells on an area of 110 mm^2. Immobilized DNA is either detected sequence-unspecific via streptavidin-Cy5 or sequence-specific by Cy3 labeled hybridization probes. Amplification and immobilization is demonstrated for template DNA ranging from 100 bp up to 1513 bp length. Compared to widely established emulsion based PCR (emPCR) approaches, leading to PCR products immobilized onto bead surfaces in highly parallel manner, the novel technique results in direct spatial registration of immobilized PCR products in a microarray format. This enables the subsequent use for massively parallel analysis similar to standard microarrays.
Jochen Hoffmann, Sebastian Hin, Felix von Stetten, Roland Zengerle, Günter Roth
Universal protocol for grafting PCR primers onto various lab-on-a-chip substrates for solid-phase PCR
2012 RSC Advances, Band: 2, Nummer: 9, Seiten: 3885 - 3889
KurzfassungA universal protocol for grafting PCR primers onto glass, PDMS, COP, COC, and PP is developed and evaluated by solid-phase PCR (SP-PCR). Primers are immobilized in a PCR compatible way featuring spots with high homogeneity and integrity. Furthermore, we show a protocol for binding a PCR product to immobilized PCR primers via solid-phase PCR (SP-PCR). Previously reported ‘‘enhanced SP-PCR’’ (Z. Kahn et al. Anal. Biochem., 2008, 375, 391–393) is improved in terms of factorial signal increase from 9.9 to 86.8 and specificity from 11.7 to 45.9. The presented immobilization- and SP-PCR protocols may enable integration of DNA microarrays directly into microfluidic lab-on-a-chip cartridges of various materials for analysis via SP-PCR. Beside planar microarrays, another interesting application could be to coat the inner surfaces of a chip with PCR primers to recover generated PCR products in digital PCR systems.

2011

A. Kloke, B. Biller, U. Kräling, S. Kerzenmacher, R. Zengerle, F. von Stetten
A Single Layer Glucose Fuel Cell Intended as Power Supplying Coating for Medical Implants
2011 Fuel Cells, Band: 11, Nummer: 2, Seiten: 316 - 326
KurzfassungWe present a novel type of abiotically catalysed implantable glucose fuel cell with anode and cathode placed side by side,using a Raney-platinum glucose oxidation anode with high tolerance towards oxygen. In contrast to conventional assembly designs used for implantable glucose fuel cells, no permeable cathode mounted in front of the anode to effect oxygen depletion is required. At 2.2 ± 0.1 lW cm–2 the single layer fuel cell exhibits only half the maximum power density of the conventional fuel cell, which solely stems from the doubled total fuel cell area demand. Nevertheless, the novel single layer design is advantageous in terms of simplified fabrication and reduced overall thickness, facilitating implementation of the fuel cell as a power supplying coating directly on the surface of medical implants. Furthermore,the single layer design offers an attractive possibility to diminish the reduction of power density by limited oxygen mass transfer to the cathode by increasing the cathode to anode area proportion. With doubled cathode area proportion a by 36% higher power density can be reached. To calculate the optimum cathode to anode area proportions,we introduced an analytical model based on the experimentally determined polarisation resistances of the individual electrodes. Keywords: Biomedical Devices, Energy Harvesting, Implantable Glucose Fuel Cell, Oxygen Tolerant Platinum Electrodes, Power Supply
L. Hussein, S. Rubenwolf, F. von Stetten, G. Urban, R. Zengerle, M. Krueger, S. Kerzenmacher
A highly efficient buckypaper-based electrode material for mediatorless laccase-catalyzed dioxygen reduction
2011 Biosens Bioelectron, Band: 26, Seiten: 4133 - 4138
KurzfassungThe redox enzyme laccase from Trametes versicolor efficiently catalyzes the oxygen reduction reaction (ORR) in mediatorless biofuel cell cathodes when adsorbed onto multi-walled carbon nanotubes (MWCNTs). In this work we demonstrate that the fabrication of MWCNTs in form of buckypaper (BP) results in an excellent electrode material for laccase-catalyzed cathodes. BPs are mechanically stable, self-entangling mats with high dispersion of MWCNTs resulting in easy to handle homogeneous layers with highly mesoporous structures and excellent electrical conductivities. All biocathodes have been electrochemically investigated in oxygen-saturated buffer at pH 5 by galvanostatic polarization and potentiodynamic linear sweep voltammetry. Both methods confirm an efficient direct interaction of laccase with BP with a high open circuit potential of 0.882V vs. normal hydrogen electrode (NHE). The high oxygen reduction performance leads to high current densities of 422±71 µAcm−2 at a typical cathode potential of 0.744V vs. NHE. When the current density is normalized to the mass of the electrode material (mass activity), the BPbased film electrodes exhibit a 68-fold higher current density at 0.744Vvs.NHEthan electrodes fabricated from the same MWCNTs in a non-dispersed agglomerated form as packed electrodes. This clearly shows that MWCNTs can act more efficiently as cathode when prepared in form of BP. This can be attributed to reduced diffusional mass transfer limitations and enhanced electrical conductivity. BP is thus a very promising material for the construction of mediatorless laccase cathodes for ORR in biofuel cells. In addition we demonstrated that these electrodes exhibit a high tolerance towards glucose, the most common bioanode fuel.
S. Kerzenmacher, U. Kräling, T. Metz, R. Zengerle, F. von Stetten
A potentially implantable glucose fuel cell with Raney-platinum film electrodes for improved hydrolytic and oxidative stability
2011 J Power Sources, Band: 196, Seiten: 1264 - 1272
KurzfassungWe present an improved abiotically catalyzed glucose fuel cell, intended as energy harvesting tissue implantable power supply for medical implants. The fuel cell is constructed from a Raney-platinum film cathode deposited on a silicon substrate with micro-machined feedholes for glucose permeability, arranged in front of a Raney-platinum film anode. A novelty is the application of platinum for both electrodes and the complete abdication of hydrogel binders. This overcomes the limited stability against hydrolytic and oxidative attack encountered with previous glucose fuel cells fabricated from activated carbon particles dispersed in a hydrogel matrix. During performance characterization in phosphate buffered saline under physiological concentrations of glucose and oxygen the diffusion resistance to be expected from tissue capsule formation was taken into account. Despite the resulting limited oxygen supply, the Raney-platinum fuel cells exhibit a power density of up to (4.4±0.2)µWcm−2 at 7.0% oxygen saturation. This exceeds the performance of our previous carbon-based prototypes, and can be attributed to the higher catalytic activity of platinum cathodes and in particular the increased oxygen tolerance of the Raney-platinum film anodes.
Daniel Mark, Patrick Weber, Sascha Lutz, Maximilian Focke, Roland Zengerle, Felix von Stetten
Aliquoting on the centrifugal microfluidic platform based on centrifugo-pneumatic valves
2011 Microfluid Nanofluid, Band: 10, Seiten: 1279 - 1288
KurzfassungWe present a new method for aliquoting liquids on the centrifugal microfluidic platform. Aliquoting is an essential unit operation to perform multiple parallel assays (‘‘geometric multiplexing’’) from one individual sample, such as genotyping by real-time polymerase chain reactions (PCR), or homogeneous immunoassay panels. Our method is a two-stage process with an initial metering phase and a subsequent transport phase initiated by switching a centrifugo- pneumatic valve. The method enables aliquoting liquids into completely separated reaction cavities. It includes precise metering that is independent on the volume of prestored reagents in the receiving cavities. It further excludes any cross-contamination between the receiving cavities. We characterized the performance for prototypes fabricated by three different technologies: micro-milling, thermoforming of foils, and injection molding. An initial volume of*90 ll was split into 8 aliquots of 10 ll volume each plus a waste reservoir on a thermoformed foil disk resulting in a coefficient of variation (CV) of the metered volumes of 3.6%. A similar volume of*105 ll was split into 16 aliquots of 6 ll volume each on micro-milled and injection-molded disks and the corresponding CVs were 2.8 and 2.2%, respectively. Thus, the compatibility of the novel aliquoting structure to the aforementioned prototyping and production technologies is demonstrated. Additionally, the important question of achievable volume precision of the aliquoting structure with respect to the production tolerances inherent to each of these production technologies is addressed experimentally and theoretically. The new method is amenable to low cost mass production, since it does not require any post-replication surface modifications like hydrophobic patches.
Marc Karle, Johannes Wöhrle, Junichi Miwa, Nils Paust, Günter Roth, Roland Zengerle, Felix von Stetten
Controlled counter-flow motion of magnetic bead chains rolling along microchannels
2011 Microfluid Nanofluid, Band: 10, Nummer: 4, Seiten: 935 - 939
KurzfassungWe demonstrate controlled transport of superparamagnetic beads in the opposite direction of a laminar flow. A permanent magnet assembles 200 nm magnetic particles into about 200 lm long bead chains that are aligned in parallel to the magnetic field lines. Due to a magnetic field gradient, the bead chains are attracted towards the wall of a microfluidic channel. A rotation of the permanent magnet results in a rotation of the bead chains in the opposite direction to the magnet. Due to friction on the surface, the bead chains roll along the channel wall, even in counter-flow direction, up to at a maximum counter-flow velocity of 8 mm s(-1). Based on this approach, magnetic beads can be accurately manoeuvred within microfluidic channels. This counter-flow motion can be efficiently be used in Lab-on-a-Chip systems, e.g. for implementing washing steps in DNA purification.
M. Focke, D. Kosse, D. Al-Bamerni, S. Lutz, C. Mueller, H. Reinecke, R. Zengerle, F. von Stetten
Microthermoforming of microfluidic substrates by soft lithography (µTSL): optimization using design of experiments
2011 J Micromech Microeng, Band: 21, Nummer: 11, Seiten: 115001 - 115012
KurzfassungWe present a detailed analysis of microthermoforming by soft lithography (µTSL) for replication of foil-based microfluidic substrates. The process was systematically optimized by design of experiments (DOE) enabling fabrication of defect-free lab-on-a-chip devices. After the assessment of typical error patterns we optimized the process toward the minimum deviation between mold and thermoformed foil substrates. The following process parameters have most significant impact on the dimensional responses (p < 0.05): critical temperature before start of evacuation, molding temperature, pressure of pre-stretching and duration of pre-stretching as well as duration of molding pressure. The most relevant parameter is molding temperature with >40% relative impact. The DOE results in an empirical process model with a maximum deviation between the prediction and experimental proof of 2% for the optimum parameter set. Finally, process optimization is validated by the fabrication and testing of a microfluidic structure for blood plasma separation from human whole blood. The optimized process enabled metering of a nominal volume of 4.0 mu l of blood plasma with an accuracy deviation of 3% and a metering precision of +/- 7.0%. The µTSL process takes about 30 min and easily enables the replication of 300 mu m wide microchannels having vertical sidewalls without any draft angles in a well-controllable way. It proves to be suitable for multiple applications in the field of microfluidic devices.
Stefanie Rubenwolf, Sven Kerzenmacher, Roland Zengerle, Felix von Stetten
Strategies to extend the lifetime of bioelectrochemical enzyme electrodes for biosensing and biofuel cell applications
2011 Appl Microbiol Biot, Band: 89, Nummer: 5, Seiten: 1315 - 1322
KurzfassungEnzymes are powerful catalysts for biosensor and biofuel cell electrodes due to their unique substrate specificity. This specificity is defined by the amino acid chain's complex three-dimensional structure based on noncovalent forces, being also responsible for the very limited enzyme lifetime of days to weeks. Many electrochemical applications, however, would benefit from lifetimes over months to years. This mini-review provides a critical overview of strategies and ideas dealing with the problem of short enzyme lifetime, which limits the overall lifetime of bioelectrochemical electrodes. The most common approaches aim to stabilize the enzyme itself. Various immobilization techniques have been used to reduce flexibility of the amino acid chain by introducing covalent or non-covalent binding forces to external molecules. The enzyme can also be stabilized using genetic engineering methods to increase the binding forces within the protein or by optimizing the environment in order to reduce destabilizing interactions. In contrast, renewing the inactivated catalyst decouples overall system lifetime from the limited enzyme lifetime and thereby promises theoretically unlimited electrode lifetimes. Active catalyst can be supplied by exchanging the electrolyte repeatedly. Alternatively, integrated microorganisms can display the enzymes on their surface or secrete them to the electrolyte, allowing unattended power supply for long-term applications. Keywords Enzyme inactivation . Biofuel cell . Biosensor . Amino acid replacement . Immobilization . Self-regeneration

2010

A. Kloke, S. Rubenwolf, C. Bücking, J. Gescher, S. Kerzenmacher, R. Zengerle, F. von Stetten
A versatile miniature bioreactor and its application to bioelectrochemistry studies
2010 Biosens Bioelectron, Band: 25, Seiten: 2559 - 2565
KurzfassungOften, reproducible investigations on bio-microsystems essentially require a flexible but well-defined experimental setup, which in its features corresponds to a bioreactor. We therefore developed a miniature bioreactor with a volume in the range of a few millilitre that is assembled by alternate stacking of individual polycarbonate elements and silicone gaskets. All the necessary supply pipes are incorporated as bore holes or cavities within the individual elements. Their combination allows for a bioreactor assembly that is easily adaptable in size and functionality to experimental demands. It allows for controlling oxygen transfer as well as the monitoring of dissolved oxygen concentration and pH-value. The system provides access for media exchange or sterile sampling. A mass transfer coefficient for oxygen (kLa) of 4.3×10−3 s−1 at aflowrate of only 15 ml min−1 and a mixing time of 1.5 s at aflowrate of 11 ml min−1 were observed for the modular bioreactor. Single reactor chambers can be interconnected via ion-conductive membranes to form a two-chamber test setup for investigations on electrochemical systems such as fuel cells or sensors. The versatile applicability of this modular and flexible bioreactor was demonstrated by recording a growth curve of Escherichia coli (including monitoring of pH and oxygen) saturation, and also as by two bioelectrochemical experiments. In the first electrochemical experiment the use of the bioreactor enabled a direct comparison of electrode materials for a laccase-catalyzed oxygen reduction electrode. In a second experiment, the bioreactor was utilized to characterize the influence of outer membrane cytochromes on the performance of Shewanella oneidensis in a microbial fuel cell.
Stefanie Rubenwolf, Oliver Strohmeier, Arne Kloke, Sven Kerzenmacher, Roland Zengerle, Felix von Stetten
Carbon electrodes for direct electron transfer type laccase cathodes investigated by current density–cathode potential behaviour
2010 Biosens Bioelectron, Band: 26, Seiten: 841 - 845
KurzfassungDirect electron transfer from carbon electrodes to adsorbed laccase (EC 1.10.3.2) from Trametes versicolor is widely used to enable mediatorless enzymatic biofuel cell cathodes. However, data published so far are poorly comparable in terms of oxygen reduction performance. We thus present a comparative characterization of carbon-based electrode materials as cathode in half-cell configuration, employing adsorbed laccase as oxygen reduction catalyst. Open circuit potentials and performances were significantly increased by laccase adsorption, indicating the occurrence of direct electron transfer. At a potential of 0.5V vs. SCE volume-normalized current densities of approximately 10, 37, 40, 70, and 77 µAcm−3 were measured for cathodes nanotubes, carbon nanofibers and multi-walled carbon nanotubes, respectively. In addition, we could show that both, carbon nanotubes and porous carbon tubes exhibit dramatically lower current densities compared to graphite felt and carbon nanofibers when normalized to BET surface instead of electrode volume. Further work will be required to clarify whether this stems from material-dependent interaction of enzyme and electrode surface or constricted enzyme adsorption due to agglomeration of the nanotubes. In case of the latter, an improved dispersion of the nanotubes upon electrode fabrication may greatly enhance their performance.
Maximilian Focke, Fabian Stumpf, Günter Roth, Roland Zengerle, Felix von Stetten
Centrifugal microfluidic system für primary amplification and secondary real-time PCR
2010 Lab Chip, Band: 10, Seiten: 3210 - 3212
KurzfassungPre-amplification is a basis for numerous polymerase chain reaction (PCR) protocols but bears severe contamination risks due to handling of high-copy DNA samples. Therefore we developed a selfcontained centrifugal microfluidic system comprising pre-stored reagents; it enables pre-amplification of specific DNA sequences prior to automated aliquoting and real-time PCR in a modified commercial thermocycler.
Marc Karle, Junichi Miwa, Gregor Czilwik, Volker Auwärter, Günter Roth, Roland Zengerle, Felix von Stetten
Continuous microfluidic DNA extraction using phase-transfer magnetophoresis
2010 Lab Chip, Band: 10, Seiten: 3284 - 3290
KurzfassungThis paper reports a novel microfluidic-chip based platform using "phase-transfer magnetophoresis" enabling continuous biomolecule processing. As an example we demonstrate for the first time continuous DNA extraction from cell lysate on a microfluidic chip. After mixing bacterial Escherichia coli culture with superparamagnetic bead suspension, lysis and binding buffers, DNA is released from cells and captured by the beads. These DNA carrying beads are continuously transported across the interfaces between co-flowing laminar streams of sample mixture, washing and elution buffer. Bead actuation is achieved by applying a time-varying magnetic field generated by a rotating permanent magnet. Flagella-like chains of magnetic beads are formed and transported along the microfluidic channels by an interplay of fluid drag and periodic magnetic entrapment. The turnover time for DNA extraction was approximately 2 minutes with a sample flow rate of 0.75 µl/s and an eluate flow rate of 0.35 µl/s. DNA recovery was 147% (on average) compared to bead based batch-wise extraction in reference tubes within a dilution series experiment over 7 orders of magnitude. The novel platform is suggested for automation of various magnetic bead based applications that require continuous sample processing, e.g. continuous DNA extraction for flow-through PCR, capture and analysis of cells and continuous immunoassays. Potential applications are seen in the field of biological safety monitoring, bioprocess control, environmental monitoring, or epidemiological studies such as monitoring the load of antibiotic resistant bacteria in waste water from hospitals.
Sascha Lutz, Patrick Weber, Max Focke, Bernd Faltin, Jochen Hoffmann, Claas Müller, Daniel Mark, Günter Roth, Peter Munday, Niall Armes, Olaf Piepenburg, Roland Zengerle, Felix von Stetten
Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA)
2010 Lab Chip, Band: 10, Seiten: 887 - 893
KurzfassungFor the first time we demonstrate a self-sufficient lab-on-a-foil system for the fully automated analysis of nucleic acids which is based on the recently available isothermal recombinase polymerase amplification (RPA). The system consists of a novel, foil-based centrifugal microfluidic cartridge including prestored liquid and dry reagents, and a commercially available centrifugal analyzer for incubation at 37 C and real-time fluorescence detection. The system was characterized with an assay for the detection of the antibiotic resistance gene mecA of Staphylococcus aureus. The limit of detection was <10 copies and time-to-result was <20 min. Microfluidic unit operations comprise storage and release of liquid reagents, reconstitution of lyophilized reagents, aliquoting the sample into #30 independent reaction cavities, and mixing of reagents with the DNA samples. The foil-based cartridge was produced by blow-molding and sealed with a self-adhesive tape. The demonstrated system excels existing PCR based lab-on-a-chip platforms in terms of energy efficiency and time-to-result. Applications are suggested in the field of mobile point-of-care analysis, B-detection, or in combination with continuous monitoring systems.
M. Focke, F. Stumpf, B. Faltin, P. Reith, D. Bamarni, S. Wadle, C. Müller, H. Reinecke, J. Schrenzel, P. Francois, D. Mark, G. Roth, R. Zengerle, F. von Stetten
Microstructuring of polymer films for sensitive genotyping by real-time PCR on a centrifugal microfluidic platform
2010 Lab Chip, Band: 10, Seiten: 2519 - 2526
KurzfassungWe present a novel process flow enabling prototyping of microfluidic cartridges made out of polymer films. Its high performance is proven by implementation of a microfluidic genotyping assay testing 22 DNA samples including clinical isolates from patients infected by methicilin-resistant Staphylococcus aureus (MRSA). The microfluidic cartridges (disks) are fabricated by a novel process called microthermoforming by soft lithography (mTSL). Positive moulds are applied allowing for higher moulding precision and very easy demoulding when compared to conventional microthermoforming. High replication accuracies with geometric disk-to-disk variations of less than 1% are typical. We describe and characterise fabrication and application of microfluidic cartridges with wall thicknesses <188 mm thus enabling efficient thermocycling during real-time polymerase chain reaction (PCR). The microfluidic cartridges are designed for operation in a slightly modified commercial thermocycling instrument. This approach demonstrates new opportunities for both microfluidic developments and well-established laboratory instruments. The microfluidic protocol is controlled by centrifugal forces and divides the liquid sample parallely into independent aliquots of 9.8 ml (CV 3.4%, N ¼ 32 wells). The genotyping assays are performed with pre-stored primers and probes for real-time PCR showing a limit of detection well below 10 copies of DNA per reaction well (N ¼ 24 wells in 3 independent disks). The system was evaluated by 44 genotyping assays comprising 22 DNA samples plus duplicates in a total of 11 disks. The samples contained clinical samples of seven different genotypes of MRSA as well as positive and negative controls. The results are in excellent agreement with the reference in microtubes.
Jochen Hoffmann, Daniel Mark, Sascha Lutz, Roland Zengerle, Felix von Stetten
Pre-storage of liquid reagents in glass ampoules for DNA extraction on a fully integrated lab-on-a-chip cartridge
2010 Lab Chip, Band: 10, Seiten: 1480 - 1484
KurzfassungSelf-containing, ready-to-use cartridges are essential for mobile Lab-on-a-Chip (LoaC) systems intended for Point-of-Care (POC) use. Up to now, a common weak point in many LoaC developments is the need to dispense liquid reagents into the test cartridge before or during processing of the assay. To address this issue we have developed an efficient method for fusing liquid reagents into glass ampoules, which are then sealed into a centrifugally operated cartridge. For on-demand reagent release, the ampoules are disrupted through the flexible lid of the cartridge. Upon centrifugation, 98.7 mL out of 100 mL (CV ¼ 2.5%) of the pre-stored contents are released into the microfluidic system. No liquid loss is observed for ethanol and H2O stored for 300 days at room temperature. Frozen storage is possible without damage to the ampoules. Applicability of this concept is demonstrated by performing a LoaC integrated DNA extraction after 140 days of reagent pre-storage. DNA yield from 32 mL of whole blood was up to 199 ng, which is 77% of an off-chip reference extraction. The presented approach allows the improvement of existing LoaC cartridges where pre-storage of liquid reagents was not implemented yet.
S. Kerzenmacher, U. Kräling, M. Schroeder, R. Brämer, R. Zengerle, F. von Stetten
Raney-platinum film electrodes for potentially implantable glucose fuel cells. Part 1: Nickel-free glucose oxidation anodes
2010 J Power Sources, Band: 195, Seiten: 6516 - 6523
KurzfassungWe present a novel fabrication route yielding Raney-platinum film electrodes intended as glucose oxidation anodes for potentially implantable fuel cells. Fabrication roots on thermal alloying of an extractable metal with bulk platinum at 200 ◦C for 48 h. In contrast to earlier works using carcinogenic nickel, we employ zinc as potentially biocompatible alloying partner. Microstructure analysis indicates that after removal of extractable zinc the porous Raney-platinum film (roughness factor ∼2700) consists predominantly of the Pt3Zn phase. Release of zinc during electrode operation can be expected to have no significant effect on physiological normal levels in blood and serum, which promises good biocompatibility. In contrast to previous anodes based on hydrogel-bound catalyst particles the novel anodes exhibit excellent resistance against hydrolytic and oxidative attack. Furthermore, they exhibit significantly lower polarization with up to approximately 100mV more negative electrode potentials in the current density range relevant for fuel cell operation. The anodes’ amenability to surface modification with protective polymers is demonstrated by the exemplary application of an approximately 300nm thin Nafion coating. This had only a marginal effect on the anode long-term stability and amino acid tolerance. While in physiological glucose solution after approximately 100 h of operation gradually increasing performance degradation occurs, rapid electrode polarization within 24 h is observed in artificial tissue fluid. Optimization approaches may include catalyst enhancement by adatom surface modification and the application of specifically designed protective polymers with controlled charge and mesh size.
S. Kerzenmacher, U. Kräling, M. Schroeder, R. Brämer, R. Zengerle, F. von Stetten
Raney-platinum film electrodes for potentially implantable glucose fuel cells. Part 2: Glucose-tolerant oxygen reduction cathodes
2010 J Power Sources, Band: 195, Seiten: 6524 - 6531
KurzfassungWe report the fabrication and characterization of glucose-tolerant Raney-platinum cathodes for oxygen reduction in potentially implantable glucose fuel. Fabricated by extraction of aluminum from 1µm thin platinum–aluminum bi-layers annealed at 300 °C, the novel cathodes show excellent resistance against hydrolytic and oxidative attack. This renders them superior over previous cathodes fabricated from hydrogel-bound catalyst particles. Annealing times of 60, 120, and 240 min result in approximately 400–550nm thin porous films (roughness factors ∼100–150), which contain platinum and aluminum in a ratio of ∼9:1. Aluminum release during electrode operation can be expected to have no significant effect on physiological normal levels, which promises good biocompatibility. Annealing time has a distinct influence on the density of trenches formed in the cathode. Higher trench densities lead to lower electrode potentials in the presence of glucose. This suggests that glucose sensitivity is governed by mixed potential formation resulting from oxygen depletion within the trenches. During performance characterization the diffusion resistance to be expected from tissue capsule formation upon electrode implantation was taken into account by placing a membrane in front of the cathode. Despite the resulting limited oxygen supply, cathodes prepared by annealing for 60 min show more positive electrode potentials than previous cathodes fabricated from hydrogel-bound activated carbon. Compared to operation in phosphate buffered saline containing 3.0mMglucose, a potential loss of approximately 120mV occurs in artificial tissue fluid. This can be reduced to approximately 90mV with a protective Nafion layer that is easily electro-coated onto the Raney-platinum film.

2009

S. Kerzenmacher, K. Mutschler, U. Kräling, H. Baumer, J. Ducrée, R. Zengerle, F. von Stetten
A complete testing environment for the automated parallel performance characterization of biofuel cells: design, validation, and application
2009 J Appl Electrochem, Band: 39, Nummer: 9, Seiten: 1477 - 1485
KurzfassungWe present a complete testing environment for the parallel performance characterization of biofuel cells. Besides rapid-assembly electrode fixtures and an aseptic electrochemical reactor, it comprises a 24-channel electrical testing system that bridges the gap between simple load resistors and costly multi-channel potentiostats. The computer- controlled testing system features active current control to enable the forced operation of half-cell electrodes, whereas galvanic isolation between individual channels ensures interference-free operation of multiple fuel cells immersed in a common testing solution. Implemented into the control software is an automated procedure for the step-wise recording of polarization curves. This way, performance overestimation due to a too fast increase in load current can be circumvented. As an applicational example, three abiotically catalyzed glucose fuel cells are characterized simultaneously in a common testing solution. Complete disclosure of the electrical system (incl. printed circuit board layout, control software, and circuit diagrams) in the online supplementary material accompanying this paper allows researchers to replicate our setup in their lab and can serve as inspiration for the design of similar systems adapted to specific requirements.
Daniel Mark, Tobias Metz, Stefan Haeberle, Sascha Lutz, Jens Ducrée, Roland Zengerle, Felix von Stetten
Centrifugo-Pneumatic Valve for Metering of Highly Wetting Liquids on Centrifugal Microfluidic Platforms
2009 Lab Chip, Band: 9, Seiten: 3599 - 3603
KurzfassungWe designed and experimentally validated a new type of passive valve for centrifugal microfluidic platforms. A liquid column entering an unvented receiving chamber is stopped by the counter-pressure of compressed air. This valve opens under defined conditions at high centrifugal frequencies at which the interface between liquid and air becomes unstable and enables a phase exchange, forwarding the liquid. Burst frequencies of the valve were determined for liquids typically used in biochemical assays: pure water, water with detergent concentrations between 0.01 % and 10 %, and pure ethanol. Burst frequencies between 8.5 ± 0.6 Hz and 27.9 ± 2.0 Hz were measured for different surface tensions. The burst frequencies can be tuned by simple geometrical changes in the valving structure. The valve does not require ultra-precise structures or local surface modifications and is therefore ideal for low-cost microfluidic polymer disks. Potential applications are in the field of mulitparameter- and panel analysis, such as PCR-genotyping.

2008

S. Haeberle, L. Naegele, R. Burger, F. von Stetten, R. Zengerle, J. Ducrée
Alginate bead fabrication and encapsulation of living cells under centrifugally induced artificial gravity conditions
2008 J Microencapsul, Band: 25, Nummer: 4, Seiten: 267 - 274
KurzfassungWe present a novel method for the direct, centrifugally induced fabrication of small, Ca2+-hardened alginate beads at polymer-tube micronozzles. The bead diameter can arbitrarily be adjusted between 180 µm and 800 µm by the nozzle geometry and spinning frequencies between 5 Hz and 28 Hz. The size distribution of the main peak features a CV of 7 – 16%, only. Up to 600 beads per second and channel are issued from the micronozzle through an air gap towards the curing agent contained in a standard lab tube (“Eppi”). Several tubes can be mounted on a “flying bucket” rotor where they align horizontally under rotation and return to a vertical position as soon as the rotor is at rest. The centrifugally induced, ultra-high artificial gravity conditions (up to 180 g) even allow the micro-encapsulation of alginate solutions displaying viscosities up to 50 Pa s, i.e. about 50,000 times the viscosity of water! With this low cost technology for micro encapsulation, HN25 and PC12 cells have successfully been encapsulated while maintaining vitality.
Kerzenmacher S, Ducree J, Zengerle R, von Stetten F
An Abiotically Catalyzed Glucose Fuel Cell for Powering Medical Implants: Reconstructed Manufacturing Protocol and Analysis of Performance
2008 J Power Sources, Band: 182, Nummer: 1, Seiten: 66 - 75
KurzfassungAlthough the first abiotically catalyzed glucose fuel cells have already been developed as sustainable power supply for medical implants in the 1970s, no detailed information concerning the fabrication of these devices has been published so far. Here we present a comprehensive manufacturing protocol for such a fuel cell, together with a detailed analysis of long-term stability performance in neutral buffer containing physiological amounts of glucose and oxygen. In air saturated solution a power density of (3.3 +/- 0.2) µW cm-2 is displayed after 10 days of operation, that gradually decreases to a value of (1.0 +/- 0.05) µW cm-2 in the course of 224 days. A novelty of this work is the characterization of fuel cell performance with individually resolved electrode potentials. Using this technique, we can show that the major part of performance degradation originates from a positive shift of the anode potential, indicating that a more poisoning resistant glucose oxidation catalyst would improve the degradation behavior of the fuel cell. As further factors influencing performance an incomplete reactant separation and a mass transfer governed cathode reaction under the relatively low oxygen partial pressures of body tissue have been identified. Consequently we propose an oxygen depleting electrode interlayer and the application of more effective oxygen reduction catalysts as promising strategies to further improve the fuel cell performance under physiological concentrations of glucose and oxygen.
Diana Hodgins, Arnaud Bertsch, Nils Post, Manfred Frischholz, Bart Volckaerts, John Spensley, J.M. Wasikiewicz, Henry Higgins, Felix von Stetten, Laurence Kenney
Healthy Aims: Developing New Medical Implants and Diagnostic Equipment
2008 Pervasive Computing, Band: 7, Nummer: 1, Seiten: 14 - 21

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2021

H. C. Ates, A. Brunauer, F. von Stetten, G. A. Urban, F. Güder, A. Merkoçi, S. M. Früh, C. Dincer
Integrated Devices for Non‐Invasive Diagnostics
2021 Advanced Functional Materials, Band: 31, Nummer: 15, Seite: 2010388
Kurzfassung“Sample‐in‐answer‐out” type integrated diagnostic devices have been widely recognized as the ultimate solution to simplify testing across healthcare systems. Such systems are equipped with advanced fluidic, mechanical, chemical, biological, and electronic components to handle patient samples without any manual steps therefore have the potential to accelerate intervention and improve patient outcomes. In this regard, the combination of integrated devices and non‐invasive sampling has gained a substantial interest to further improve the comfort and safety of patients. In this Review, the pioneering developments in integrated diagnostics are covered and their potential in non‐invasive sampling is discussed. The key properties of possible sample types are highlighted by addressing their relevance for the clinical practice. Last, the factors affecting the transition of integrated devices from academia to the market are identified by analyzing the technology readiness levels of selected examples and alternative remedies are explored to increase the rate of survival during this transition.

2020

M. Trotter, N. Borst, R. Thewes, F. von Stetten
Electrochemical DNA sensing – Principles, commercial systems, and applications
2020 Biosens Bioelectron, Band: 154, Seite: 112069
KurzfassungDriven by the vision of robust and portable, yet sensitive DNA detection systems for point-of-need applications, the development of electrochemical DNA sensing principles has been of high interest. Many different principles have been developed and these are regularly reviewed. However, the maturity of electrochemical principles and their ability to produce competitive real-world applications is rarely assessed. In this review, general electrochemical DNA sensing principles are briefly introduced and categorized into heterogeneous vs. homogeneous approaches, and then the subcategories label-free vs. labeled and reagent-less vs. reagent-dependent principles. We then focus on reviewing the electrochemical sensing principles implemented in DNA detection systems, which are commercially available or close to market entry, considering the complete analysis process, automation and the field of application. This allows us to outline and discuss which principles have proved suitable for which kinds of applications, as well as the stage of integration and automation. Examples from all the identified categories of electrochemical DNA sensing principles have found application in commercial detection systems or advanced prototypes. Various applications have already been demonstrated, ranging from on-site skin care testing, to food safety to the most frequent in vitro diagnostic tests, partially conducted in automated sample-to-answer devices. Our review is intended to enable researchers in areas related to electrochemistry, biochemistry or microfluidics to assess the commercial state of the art of electrochemical nucleic acid testing, and the interdisciplinary challenges for further improvements.

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2019

J. Li, J. Macdonald, F. von Stetten
Correction: Review: a comprehensive summary of a decade development of the recombinase polymerase amplification
2019 Analyst, Band: 144, Nummer: 31

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J. Li, J. Macdonald, F. von Stetten
Review: a comprehensive summary of a decade development of the recombinase polymerase amplification
2019 Analyst, Band: 144, Nummer: 31, Seiten: 31 - 67
KurzfassungNucleic acid amplification has permeated every field in the life sciences since the introduction of the classic polymerase chain reaction (PCR) method in 1983. Yet, despite its fundamental reach, PCR has been constrained within the walls of a laboratory, due to its requirement for a sophisticated thermocycling machine, limiting external application in low-resource settings. New isothermal amplification strategies are seeking to break through traditional laboratory boundaries by providing nucleic acid replication at constant temperatures. Of these methods, recombinase polymerase amplification (RPA) is one of the fastest developing, experiencing rapid uptake and market, even though it was introduced comparatively late. Critically, RPA’s technology potentiates highly accessible and sensitive nucleic acid amplification outside of laboratory, and even self-testing. Here we provide a comprehensive review of the equipmentfree simplicity of RPA over its first decade of development. Our review includes key knowledge of RPA technology, such as its reaction components, mechanism, sensitivities and specificities, and distinctive detection methods. The review also provides know-how for developing RPA assays, and information about commercially available RPA reaction kits and accessories. We summarise critical RPA experimental tips and issues available through data mining the published literature, to assist researchers in mastering the RPA reaction. We also outline influential hotspots of RPA development, and conclude with outlooks for future development and implications for eclipsing PCR and further revolutionising the life sciences.

2018

K. Mitsakakis / V. D’Acremont, S. Hin, F. von Stetten, R. Zengerle
Diagnostic tools for tackling febrile illness and enhancing patient management
2018 Microelectron Eng, Band: 201, Seiten: 26 - 59
KurzfassungMost patients with acute infectious diseases develop fever, which is frequently a reason to visit health facilities in resource-limited settings. The symptomatic overlap between febrile diseases impedes their diagnosis on clinical grounds. Therefore, the World Health Organization promotes an integrated management of febrile illness. Along this line, we present an overview of endemic and epidemic etiologies of fever and state-of-the-art diagnostic tools used in the field. It becomes evident that there is an urgent need for the development of novel technologies to fulfill end-users’ requirements. This need can be met with Point-of-Care and near-patient diagnostic platforms, as well as e-Health clinical algorithms, which co-assess test results with key clinical elements and biosensors, assisting clinicians in patient triage and management, thus enhancing disease surveillance and outbreak alerts. This review gives an overview of diagnostic technologies featuring a platform based approach: (i) assay (nucleic acid amplification technologies are examined); (ii) cartridge (microfluidic technologies are presented); (iii) instrument (various detection technologies are discussed); and at the end proposes a way that such technologies can be interfaced with electronic clinical decision-making algorithms towards a broad and complete diagnostic ecosystem.

2016

M. Karle, S. K. Vashist, R. Zengerle, F. von Stetten
Microfluidic solutions enabling continuous processing and monitoring of biological samples: A Review
2016 Anal Chim Acta, Band: 929, Seiten: 1 - 22
KurzfassungThe last decade has witnessed tremendous advances in employing microfluidic solutions enabling Continuous Processing and Monitoring of Biological Samples (CPMBS), which is an essential requirement for the control of bio-processes. The microfluidic systems are superior to the traditional inline sensors due to their ability to implement complex analytical procedures, such as multi-step sample preparation, and enabling the online measurement of parameters. This manuscript provides a backgound review of microfluidic approaches employing laminar flow, hydrodynamic separation, acoustophoresis, electrophoresis, dielectrophoresis, magnetophoresis and segmented flow for the continuous processing and monitoring of biological samples. The principles, advantages and limitations of each microfluidic approach are described along with its potential applications. The challenges in the field and the future directions are also provided.

2015

O. Strohmeier, M. Keller, F. Schwemmer, S. Zehnle, D. Mark, F. von Stetten, R. Zengerle, N. Paust
Centrifugal microfluidic platforms: advanced unit operations and applications
2015 Chem Soc Rev, Band: 44, Seiten: 6187 - 6229
KurzfassungCentrifugal microfluidics has evolved into a mature technology. Several major diagnostic companies either have products on the market or are currently evaluating centrifugal microfluidics for product development. The fields of application are widespread and include clinical chemistry, immunodiagnostics and protein analysis, cell handling, molecular diagnostics, as well as food, water, and soil analysis. Nevertheless, new fluidic functions and applications that expand the possibilities of centrifugal microfluidics are being introduced at a high pace. In this review, we first present an up-to-date comprehensive overview of centrifugal microfluidic unit operations. Then, we introduce the term “process chain” to review how these unit operations can be combined for the automation of laboratory workflows. Such aggregation of basic functionalities enables efficient fluidic design at a higher level of integration. Furthermore, we analyze how novel, ground-breaking unit operations may foster the integration of more complex applications. Among these are the storage of pneumatic energy to realize complex switching sequences or to pump liquids radially inward, as well as the complete pre-storage and release of reagents. In this context, centrifugal microfluidics provides major advantages over other microfluidic actuation principles: the pulse-free inertial liquid propulsion provided by centrifugal microfluidics allows for closed fluidic systems that are free of any interfaces to external pumps. Processed volumes are easily scalable from nanoliters to milliliters. Volume forces can be adjusted by rotation and thus, even for very small volumes, surface forces may easily be overcome in the centrifugal gravity field which enables the efficient separation of nanoliter volumes from channels, chambers or sensor matrixes as well as the removal of any disturbing bubbles. In summary, centrifugal microfluidics takes advantage of a comprehensive set of fluidic unit operations such as liquid transport, metering, mixing and valving. The available unit operations cover the entire range of automated liquid handling requirements and enable efficient miniaturization, parallelization, and integration of assays.

2013

B. Faltin, R. Zengerle, F. von Stetten
Current Methods for Fluorescence-Based Universal Sequence-Dependent Detection of Nucleic Acids in Homogenous Assays and Clinical Applications
2013 Clin Chem, Band: 59, Nummer: 11, Seiten: 1567 - 1582
KurzfassungSpecific and sensitive nucleic acid (NA) testing in research and clinical diagnostics is usually performed by use of labeled oligonucleotide probes. However, the use of target-specific fluorogenic probes increases the cost of analysis. Therefore, universal sequence-dependent (USD) NA detection methods have been developed to facilitate cost-effective target detection using standardized reagents.

2011

A. Kloke, F. von Stetten, R. Zengerle, S. Kerzenmacher
Strategies for the Fabrication of Porous Platinum Electrodes
2011 Adv Mater, Band: 23, Seiten: 4976 - 5008
KurzfassungPorous platinum is of high technological importance due to its various applications in fuel cells, sensors, stimulation electrodes, mechanical actuators and catalysis in general. Based on a discussion of the general principles behind the reduction of platinum salts and corresponding deposition processes this article discusses techniques available for platinum electrode fabrication. The numerous, different strategies available to fabricate platinum electrodes are reviewed and discussed in the context of their tuning parameters, strengths and weaknesses. These strategies comprise bottomup approaches as well as top-down approaches. In bottom-up approaches nanoparticles are synthesized in a fi rst step by chemical, photochemical or sonochemical means followed by an electrode formation step by e.g. thin fi lm technology or network formation to create a contiguous and conducting solid electrode structure. In top-down approaches fabrication starts with an already conductive electrode substrate. Corresponding strategies enable the fabrication of substrate-based electrodes by e.g. electrodeposition or the fabrication of self-supporting electrodes by dealloying. As a further top-down strategy, this review describes methods to decorate porous metals other than platinum with a surface layer of platinum. This way, fabrication methods not performable with platinum can be applied to the fabrication of platinum electrodes with the special benefi t of low platinum consumption.

2010

M. Focke, D. Kosse, C. Müller, H. Reinecke, R. Zengerle, F. von Stetten
Lab-on-a-Foil: microfluidics on thin and flexible films
2010 Lab Chip, Band: 10, Seiten: 1365 - 1386
KurzfassungFor the first time we demonstrate a self-sufficient lab-on-a-foil system for the fully automated analysis of nucleic acids which is based on the recently available isothermal recombinase polymerase amplification (RPA). The system consists of a novel, foil-based centrifugal microfluidic cartridge including prestored liquid and dry reagents, and a commercially available centrifugal analyzer for incubation at 37°C and real-time fluorescence detection. The system was characterized with an assay for the detection of the antibiotic resistance gene mecA of Staphylococcus aureus. The limit of detection was <10 copies and time-to-result was <20 min. Microfluidic unit operations comprise storage and release of liquid reagents, reconstitution of lyophilized reagents, aliquoting the sample into #30 independent reaction cavities, and mixing of reagents with the DNA samples. The foil-based cartridge was produced by blow-molding and sealed with a self-adhesive tape. The demonstrated system excels existing PCR based lab-on-a-chip platforms in terms of energy efficiency and time-to-result. Applications are suggested in the field of mobile point-of-care analysis, B-detection, or in combination with continuous monitoring systems.
D. Mark, S. Häberle, G. Roth, F. von Stetten, R. Zengerle
Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications
2010 Chem Soc Rev, Band: 39, Seiten: 1153 - 1182
KurzfassungThis critical review summarizes developments in microfluidic platforms that enable the miniaturization, integration,automation and parallelization of (bio-)chemical assays (see S. Haeberle and R. Zengerle, Lab Chip, 2007, 7, 1094–1110, for an earlier review). In contrast to isolated application-specific solutions, a microfluidic platform provides a set of fluidic unit operations, which are designed for easy combination within a well-defined fabrication technology. This allows the easy, fast, and cost-efficient implementation of different application-specific (bio-)chemical processes. In our review we focus on recent developments from the last decade (2000s). We start with a brief introduction into technical advances, major market segments and promising applications. We continue with a detailed characterization of different microfluidic platforms, comprising a short definition, the functional principle, microfluidic unit operations,application examples as well as strengths and limitations of every platform. The microfluidic platforms in focus are lateral flow tests, linear actuated devices, pressure driven laminar flow, microfluidic large scale integration, segmented flow microfluidics, centrifugal microfluidics, electrokinetics, electrowetting, surface acoustic waves, and dedicated systems for massively parallel analysis. This review concludes with the attempt to provide a selection scheme for microfluidic platforms which is based on their characteristics according to key requirements of different applications and market segments. Applied selection criteria comprise portability, costs of instrument and disposability, sample throughput, number of parameters per sample, reagent consumption, precision, diversity of microfluidic unit operations and the flexibility in programming different liquid handling protocols (295 references).

2008

S. Kerzenmacher, J. Ducrée, R. Zengerle, F. von Stetten
Energy Harvesting by Implantable Abiotically Catalyzed Glucose Fuel Cells
2008 J Power Sources, Band: 182, Nummer: 1, Seiten: 1 - 17
KurzfassungAbiotically catalyzed glucose fuel cells are a newly re-discovered approach to realize an autonomous energy supply for low power medical implants that solely relies on the electrochemical reaction of oxygen and glucose available from body fluids. The key advantage of a fuel cell driven power supply over conventional battery based approaches is the abundant availability of both reactants in body fluids, rendering the need for regular battery replacement or external charging mechanisms obsolete. Abiotic electrode catalysts, such as noble metals or activated carbon, exhibit favorable characteristics for long-term application in an implantable glucose fuel cell. In contrast to enzymes they are long-term stable and amenable to heat sterilization and corresponding fuel cells have already been developed in the late 1960s. However, the concept has drawn only little attention over the last decades, and research has mainly been focused on the development of enzymatic glucose fuel cells. This review therefore covers the development of implantable glucose fuel cells based on abiotic catalysts since the 1960s, with special regard to their applicability as sustainable micro power generators for implantable devices. The historical achievements of various academic and industrial research groups are critically reviewed, and different embodiment concepts are presented. While encouraging results have been achieved both in in-vitro and in preliminary in-vivo experiments, no further developments have been reported since the introduction of implantable lithium iodine batteries in the mid 1970s. In terms of design and fabrication of operational devices only limited information is documented in the literature, and further effort will be necessary to develop an understanding of the parameters influencing device performance and stability under in-vivo conditions.

2007

Ducree,J., Haeberle,S., Lutz,S., Pausch,S., von Stetten,F., Zengerle,R.
The centrifugal microfluidic Bio-Disk platform
2007 Journal of Micromechanics and Microengineering, Band: 17, Seiten: S103 - S115

Buchbeiträge

Jahre: 2017 | 2016 | 2015 | 2014 | 2012

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2017

A.G. Venkatesh, T. van Oordt, E. M. Schneider, R. Zengerle, F. von Stetten, J.H.T. Luong, S.K. Vashist
A Smartphone-Based Colorimetric Reader for Human C-Reactive Protein Immunoassay
In: Biosensors and Biodetection: Methods and Protocols Volume 1: Optical-Based Detectors, Methods in Molecular Biology, vol. 1571
2017, Springer, Avraham Rasooly and Ben Prickril, Seiten: 343 - 356, Avraham Rasooly and Ben Prickril,
KurzfassungA smartphone-based colorimetric reader (SBCR), comprising a Samsung Galaxy SIII mini, a gadget (iPAD mini, iPAD4, or iPhone 5s) and a custom-made dark hood and base holder assembly, is used for human Creactive protein (CRP) immunoassay. A 96-well microtiter plate (MTP) is positioned on the gadget’s screensaver to provide white light-based bottom illumination only in the specific regions corresponding to the well’s bottom. The images captured by the smartphone’s back camera are analyzed by a novel image processing algorithm. Based on one-step kinetics-based human C-reactive protein immunoassay (IA), SBCR is evaluated and compared with a commercial MTP reader (MTPR). For analysis of CRP spiked in diluted human whole blood and plasma as well as CRP in clinical plasma samples, SBCR exhibits the same precision, dynamic range, detection limit, and sensitivity as MTPR for the developed IA (DIA). Considering its compactness, low cost, advanced features and a remarkable computing power, SBCR is an ideal point-ofcare (POC) colorimetric detection device for the next-generation of cost-effective POC testing (POCT).

2016

S. K. Vashist, A.G. Venkatesh, R. Zengerle, F. von Stetten, J.H.T. Luong
Smartphone-based in vitro diagnostic technologies for personalized healthcare monitoring and management
In: Nanobiosensors for personalized and onsite biomedical diagnosis
2016, The Institution of Engineering and Technology, UK, Pranjal Chandra, Seiten: 231 - 251, Pranjal Chandra, ISBN: 978-1-84919-950-6

2015

S.K. Vashist, A.G. Venkatesh, R. Zengerle, F. von Stetten, J.H.T. Luong
Smart-phone based Point-of-Care for mobile healthcare
In: Handbook of Biotechnology, Bioengineering and Biomedical Applications
2015, National Design and Research Forum, Gundu H. R. Rao and Dr. L.V. Muralikrishna Reddy, Seiten: 313 - 332, Gundu H. R. Rao and Dr. L.V. Muralikrishna Reddy,

2014

D. Mark, F. von Stetten, R. Zengerle
Lab on a Chip: The Vision Becomes Reality
In: Smart Systems Integration for Micro- and Nanotechnologies
2014, goldenbogen, Seiten: 73 - 81, ISBN: 9783932434785
KurzfassungThis chapter elucidates the current socio-economical status of Lab-on-a-Chip (LOAC) technology. It starts from the original vision of LOAC to miniaturize and automate assays by microfluidic integration and then focusses on microfluidic Lab-on-a-Chip products in the field of point-of-care (POC)diagnostics. These are discussed in the light of their market impact, approval by the FDA (Food and Drug Administration), and their status of process integration and automation as categorized by the CLIA (Clinical Laboratory Improvement Amendments). Future requirements to strenghten the competiveness of microfluidic based POC products are seen in the implementation of a microfluidic platform based development approach, and foundry based manufacturing process.
S. Wadle, S. Rubenwolf, M. Lehnert, B. Faltin, M. Weidmann, F. Hufert, R. Zengerle, F. von Stetten
Mediator Probe PCR: Detection of Real-Time PCR by Label-Free Probes and a Universal Fluorogenic Reporter
In: Quantitative Real-Time PCR: Methods and Protocols
2014, Springer, R. Biassoni, A. Raso, HUMANA PRESS INC., Seiten: 55 - 73, R. Biassoni, A. Raso, HUMANA PRESS INC.,
KurzfassungMediator probe PCR (MP PCR) is a novel detection format for real-time nucleic acid analysis. Label-free mediator probes (MP) and fluorogenic universal reporter (UR) oligonucleotides are combined to accomplish signal generation. Compared to conventional hydrolysis probe PCRs costs can thus be saved by using the same fluorogenic UR for signal generation in different assays. This tutorial provides a practical guideline to MP and UR design. MP design rules are very similar to those of hydrolysis probes. The major difference is in the replacement of the fluorophore and quencher by one UR-specific sequence tag, the mediator. Further protocols for the setup of reactions, to detect either DNA or RNA targets with clinical diagnostic target detection as models, are explained. Ready to use designs for URs are suggested and guidelines for their de novo design are provided as well, including a protocol for UR signal generation characterization.

2012

S. Haeberle, D. Mark, F. von Stetten, R. Zengerle
Microfluidic Platforms for Lab-on-a-Chip Applications
In: Microsystems and Nanotechnology
2012, Springer, Z. Zhou, Z. Wang, L. Lin, Seiten: 853 - 895, Z. Zhou, Z. Wang, L. Lin, ISBN: 978-3-642-18293-8

Kurzbeiträge

Jahre: 2017 | 2015 | 2012 | 2011 | 2005

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2017

N. Borst, F. von Stetten
Eine Technologieplattform für die digitale Nukleinsäureanalytik
2017 Management & Krankenhaus, Band: 2017-04, Seite: 37

2015

F. Schuler, F. von Stetten
Tröpfchenschleuder
2015 Laborjournal, Band: 7-8, Seiten: 52 - 53

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2012

B. Faltin, S. Wadle, G. Roth, S. Rubenwolf, M. Lehnert, R. Zengerle, F. von Stetten
Neuer Echtzeit-Nachweis von Nukleinsäuren
2012 Laborwelt, Band: 4, Seiten: 10 - 11

2011

Daniel Mark, Günter Roth, Dominique Kosse, Nils Paust, Roland Zengerle, Felix von Stetten
Assay-Automatisierung als Dienstleistung - Mikrofluidische Integration von Laborprozessen durch den Lab-on-a-Chip Design- & Foundry-Service
2011 Labo, Band: April, Seiten: 10 - 13
D. Mark, G. Roth, R. Zengerle, F. von Stetten
Laborautomation - Die zentrifugal-mikrofluidische Plattform
2011 BIOspektrum, Nummer: 6, Seiten: 1 - 4

2005

Woias P, Manoli Y, Nann T, von Stetten F
Energy Harvesting for Autonomous Microsystems
2005 mst-news, Band: 4, Seiten: 42 - 45

Vorträge

Jahre: 2024 | 2017 | 2016 | 2015 | 2014 | 2013 | 2012 | 2011 | 2009

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2024

E. Kipf, P. Jülg, F. Schlenker, M. Fillies, C. Kranig, M. Specht, S. Groeneveld-Krentz, S. Neumann, R. Zengerle, F. von Stetten, N. Borst, R. Kirschner-Schwabe, T. Hutzenlaub, M. Lehnert, C. Eckert
Messung des individuellen Therapieansprechens von akuten lymphoblastischen Leukämien (IRMA-4-ALL)
2024 Translationale Forschung in der Personalisierten Medizin – Chancen und Hürden, Netzwerkveranstaltung für geförderte Projekte der „Innovationen für die individualisierte Medizin“ und der „Translationsp

2017

F. Schuler, S. Wadle, N. Borst, M. Schulz, L. Becherer, J. Li, M. Specht, T. Hutzenlaub, N. Paust, R. Zengerle, F. von Stetten
A Technology Platform for Digital Nucleic Acid Diagnostics at the Point of Care (invited talk)
2017 3. Münchner point-of-care testing symposium, Deutsche Gesellschaft für Klinische Chemie und Laboratoriumsmedizin (DGKL), München, 13.-15. 3. 2017
F. von Stetten
A technology platform for digital nucleic acid testing at the point of need (invited talk)
2017 Potsdam Days on Bioanalysis 2017, Potsdam, 23.-24.11.2017
F. Schuler, N. Borst, S. Wadle, M. Schulz, M. Specht, J. Li, L. Becherer, T. Hutzenlaub, N. Paust, R. Zengerle, F. von Stetten
Centrifugal Step Emulsification Allows Miniaturized Digital Droplet-RPA,-LAMP and -PCR on the Centrifugal Microfluidic Platform (invited talk)
2017 9th Annual lab-on-a-chip & microfluidics Conference, Selectbio, Munich, 10.-11.5. 2017

2016

F. von Stetten
Accelerated Development of Multiplex Real-Time PCR by Preloaded Fluorogenic Reporters and Label-Free Mediator Probes (talk)
2016 4th Point-of-Care Diagnostics & Global Health World Congress 2016, San Diego USA, 26.-28.09.2016
F. von Stetten
Accelerated development of multiplex real-time PCR by a fluorogenic mastermix (invited talk)
2016 4th qPCR and Digital PCR Congress, London / UK 20.-21.10.2016
F. von Stetten
Centrifugal Step Emulsification Allows Miniaturized Digital Droplet-RPA, -LAMP and -PCR on the Centrifugal Microfluidic Platform (talk)
2016 8th Lab-on-a-Chip, Microfluidics & Microarrays World Congress 2016, San Diego USA, 26.-28.09.2016
F. von Stetten
Neue Ansätze zur nukleinsäurebasierten Vor-Ort-Diagnostik von Infektionskrankheiten (invited talk)
2016 Fachtagung Bio Medizin Technik: Innovationen in Diagnostik und Therapie von Infektionen, Hannover, 06.4.2016

2015

F. von Stetten
Mediator probe PCR – label free probes and universal fluorogenic reporters for the detection of different RNA and DNA sequences (talk)
2015 Spring meeting, European Materials Research Society, Lille, France, 11.-15.05.2015

2014

F. von Stetten
LabTube - a novel centrifugal microfluidic lab-on-a-chip platform for operation in standard laboratory centrifuges (invited key-note presentation)
2014 Biosensors 2014, Melbourne, Australia, 27.-30.05.2014
F. von Stetten
LabTube and LabDisk - prospective centrifugal microfluidic platforms for sample-to-answer point-of-care nucleic acid testing (invited key-note presentation)
2014 Lab‐on‐a‐Chip World Congress, San Diego/USA, 18. - 19. September 2014
F. von Stetten
LabTube – a novel "microfluidic app" for sample preparation and diagnostics operated in a laboratory centrifuge (invited talk)
2014 Lab-on-a-Chip European Congress, SelectBio, Berlin, 10.-11.03.2014
F. von Stetten
Mediator Probe PCR - Principle, Applications and Guidelines for Primer and Probe Design (invited talk)
2014 Advances in Biodetection & Biosensors, Berlin, 10.-11. 03.2014
S.K. Vashist, T. van Oordt, A. Kloke, F. Von Stetten, R. Zengerle
Smartphone-based colorimetric readers (SBCR) for bioanalytical applications and SBCR-based in vitro diagnostics for mobile healthcare
2014 Clusterkonferenz MicroTEC Südwest, Freiburg, 05.-06.05.2014
F. von Stetten
Universal reporters of mediator probe PCR as target-independent biosensors for detection of five different RNA and DNA sequences (invited key-note presentation)
2014 Biosensors 2014, Melbourne, Australia, 27.-30.05.2014

2013

R. Zengerle, D. Mark, O. Strohmeier, D. Kosse, N. Paust, F. von Stetten, G. Czilwik, T. van Oordt, M. Keller, J. Drexler
LabDisk - a set of novel centrifugal microfluidc unit operations enables point of care sample-to-answer nucleic acid testing
2013 Gordon Research Conference on ”Physics and Chemistry of Microfluidics: Challenges, Advances and New Technologies for Diagnostics” in Lucca, Tuscany, Italy , 09. – 14.06.2013
F. von Stetten
LabDisk - a set of novel centrifugal microfluidc unit operations enables point of care sample-to-answer nucleic acid testing (talk)
2013 MicroTAS 2013, Freiburg, 27.-31.10.2013
F. von Stetten, A. Kloke, A. Fiebach, L. Drechsel, S. Zhang, N. Paust, J. Steigert, R. Zengerle
LabTube - a novel centrifugal microfluidic lab-on-a-chip platform for operation in standard laboratory centrifuges
2013 Gordon Research Conference on ”Physics and Chemistry of Microfluidics: Challenges, Advances and New Technologies for Diagnostics” in Lucca, Tuscany, Italy , 09. – 14.06.2013
F. von Stetten, B. Punnamoottil
Labor im Zentrifugenröhrchen zur dezentralen patientennahen Diagnostik (invited talk)
2013 Mobile Diagnostik am Point-of-Care, Deutsche Gesellschaft für Biomedizinische Technik, Düsseldorf, 13.11.2013
F. von Stetten
Physikalische und biologische Sensoren, fluidische Integration, und Fertigungstechnologie mit einem Anwendungsbeispiel zur Wasseranalytik (invited talk)
2013 Fachsymposium des Vereins HybridSensorNet, Karlsruhe, 29.11.2013

2012

F. von Stetten
A functional blister-pack LabDisk system for point of care testing (invited talk)
2012 6th Workshop “Chemical and Biological Micro Laboratory Technology“, Ilmenau/Elgersburg, Germany, 2012
R. Zengerle, J. Hoffmann, G. Roth, D. Mark, F. von Stetten
Microfluidic Apps for off-the-shelf Instruments
2012 1st International POSTECH Biomedical Device Workshop 2012; Pohang, Korea , 19.07.2012- 20.07.2012

2011

F. von Stetten
A functional blister-pack LabDisk system for rapid field testing of biological threats (invited talk)
2011 18th Annual Biodetection Technologies, Washington, 23.06.2011
F. von Stetten
Centrifugal Microfluidic Platform for Molecular Diagnostics (talk)
2011 19th IFCC-EFCC European Congress of Clinical Chemistry and Laboratory Medicine, Berlin, 15- 19.05.2011

2009

F. von Stetten, M. Focke, S. Lutz, B. Faltin, J. Burger, P. Weber, C. Mueller, T. Metz, G. Roth, D. Mark, R. Zengerle
Miniaturisation, automation and integration of biochemical assays based on centrifugal microfluidics (talk)
2009 6. Deutschen BioSensor Symposiums, Freiburg, 2009

Konferenzbeiträge

Jahre: 2025 | 2024 | 2023 | 2022 | 2021 | 2020 | 2019 | 2018 | 2017 | 2016 | 2015 | 2014 | 2013 | 2012 | 2011 | 2010 | 2009 | 2008 | 2007 | 2006

zurück zur Übersicht aller Publikationen

2025

C. Hensel, C. Kleber, J. N. Klatt, T. Hutzenlaub, F. von Stetten, J. C. Behrends, A. Schreiber
Protein Sample Preparation for Nanopore Recording
2025 Single Molecule Protein Sequencing (SMPS), Bolzano/Italy, January 18-24, 2025

2024

M. Neugebauer, S. Calabrese, A. Markl, L. Schumacher, T. Wolper, T-T. Truong, P. Juelg, N. Borst, T. Hutzenlaub, F. Scherer, F. von Stetten, M. Lehnert
Increasing digital PCR performance in liquid biopsy using reporter emission multiplexing
2024 13th CNAPS International Symposium on Circulating Nucleic Acids in Plasma and Serum, Graz/Austria, 4-6 March 2024
S. Calabrese, A. Markl, M. Neugebauer, L. Schumacher, N. Borst, F. Scherer, F. von Stetten, M. Lehnert
Reporter emission multiplexing (REM) Increases detection capacities of digital PCR devices leading to sensitive, precise and specific liquid biopsy assays
2024 EUDIP 2024 - European Digital PCR Symposium 2024, Gent/Belgium, 6-8 February 2024

2023

B. Breiner, D. Kainz, S. Wagner, M. Gavage, S. Sahakalkan, R. Marega, F. von Stetten, A. Klebes
Automated allergen sample preparation and detection via centrifugal microfluidic lateral flow assay
2023 Eurosensors 2023, Lecce, Italy, 10. - 13.09.2023
Z. Shu, S. Hennig, P. Koltay, S. Kartmann, F. von Stetten, R. Zengerle
Automated sample preparation platform for protein diagnostics based on open microfluidics electrophoresis
2023 Black Forest Nanopore Meeting, Freiburg, 06.-09.11.2023

2022

M. Trotter, A. Schreiber, D. Kleinknecht, N. Borst, M. Kuderer, K. Gläser, F. von Stetten
Mobile elektrochemische Plattform zur molekularen Diagnostik in der häuslichen Pflege
2022 5. Müchner Poit-of-Care Testing Symposium, Munich, September,27-29, 2022
F. von Stetten
Novel German innovation network “nanodiag BW” - nanopore diagnostics of epigenetic markers”
2022 SMPS3 (Single-molecule protein sequencing), Delft, The Netherlands, October, 27 - November, 3, 2022
T. Lange, T. Groß, Á. Jeney, J. Scherzinger, E. S. C. Niemöller, S. Zimmermann, P. Koltay, F. von Stetten, R. Zengerle, C. Jeney
Validation of scRNA-seq fold changes by single-cell reverse transcription digital PCR
2022 Single-Cell Genomics, Gordon Research Conference, Les Diablerets, VD, Switzerland, May 1- 6, 2022
S. Hennig, Z. Shu, L. Gutzweiler, P. Koltay, F. von Stetten, R. Zengerle, S. M. Früh
“Paper-based open microfluidics platform for automatic protein analysis
2022 SLAS Europe 2022 Conference and Exhibition, Dublin, Ireland, May 26, 2022

2021

M. Schulz, J. Ruediger, E. Landmann, M. Bakheit, S. Frischmann, D. Rassler, A. R. Homann, F. von Stetten, R. Zengerle, N.Paust
High dynamic range digital assay enabled by dual volume centrifugal step emulsification
2021 MST-Kongress, Ludwigsburg, 08.-10.11.2021
M. Trotter, D. Juric, Z. Bagherian, N. Borst, K. Gläser, F. von Stetten, A. Zimmermann
Integration of nanoporous gold electrodes in microfluidic chambers by inkjet-printing for biosensing applications
2021 MST-Kongress, Ludwigsburg, 08.-10.11.2021
D. Baumgartner, B. Johannsen, N. Paust, F. von Stetten, R. Zengerle, K. Mavridis, J. Vontas, K. Mitsakakis
Microfluidic-based molecular analysis of plant psts for insecticide resistance management (Superpests-Disk)
2021
A. Brunauer, A.-S. Kittel, R. D. Verboket, F. von Stetten, S. M. Früh
Multianalyte-Assays: Simultaneous detection of protein and nucleic acid biomarkers
2021 European Biosensor Symposium (EBS), online, 09.-12.03.2021
A.-S. Kittel, R. D. Verboket, F. von Stetten, S. M. Früh
Multianalyte-Assays: Simultaneous detection of protein and nucleic acid biomarkers
2021 European Biosensor Symposium (EBS), online, 09.-12.03.2021
Y.-T. Kao, S. Calabrese, N. Borst, M. Lehnert, Y.-K. Lai, F. Schlenker, R. Zengerle, P. Garstecki, F. von Stetten
One-step amplification-free bacteria detection by optimizes LNA/DNA molecular beacons in droplets
2021 MicroTAS 2021, Palm Springs/USA, 10.-14.10.2021, online
L. Becherer, J.F. Hess, S. Frischmann, M. Bakheit, H. Nitschko, S. Stinco, F. Zitz, H. Hofer, G. Porro, F. Hausladen, K. Stock, D. Drossart, H. Wurm, H. Kuhn, D. Huber, T. Hutzenlaub, N. Paust, M. Keller, O. Strohmeier, S. Wadle, N. Borst, R. Zengerle, F. von Stetten
Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP
2021 MST-Kongress, Ludwigsburg, 08.-10.11.2021
A. Brunauer, R. D. Verboket, D. M. Kainz, F. von Stetten, S. M. Früh
Rapid nucleic acid lateral flow immunoassay for the detection of Pseudomonas aeruginosa
2021 Biosensors 2021, Haeundae-gu, South Korea, (online), 26. - 29.07.2021
A. Klebes, A.-S. Kittel, R. D. Verboket, F. von Stetten, S. M. Früh
Simultaneous detection of protein and nucleic acid biomarkers via paper-based multianalyte sensor
2021 MicroTAS 2021, Palm Springs/USA, 10.-14.10.2021, online
D. Baumgartner, B. Johannsen, N. Paust, F. von Stetten, R. Zengerle, K. Mavridis, J. Vontas, K. Mitsakakis
SuperPests LabDisk: a microfluidic-based molecular diagnostic platform for detection of biotypes, resistance mutations and plant pathogens P3.021
2021 Biosensors, Haeundae-gu, South Korea (online), 26. – 29.07.2021
T. Härpfer, L. Becherer, M. Bakheit, S. Frischmann, S. Stinco, N. Borst, R. Zengerle, F. von Stetten
Universal multiplex mediator displacement LAMP Nr. P3.062 analytical and clinical validation on the example of HIV, Haemophilus ducreyi and Treponema pallidum
2021 Biosensors, , Haeundae-gu, South Korea (online) , 26. – 29.07.2021

2020

A. Brunauer, B. Breiner, S. Hennig, D. Kainz, R. Verboket, B. Johannsen, D. Baumgartner, K. Mitsakakis, L. Gutzweiler, Z. Shu, P. Koltay, T. Hutzenlaub, N. Paust, R. Zengerle, F. von Stetten, S. M. Früh
Actuation principles for bioanalytical platforms to combat infectious diseases
2020 Virtual EMBL Conference: Microfluidics: Designing the Next Wave of Biological Inquiry 2020, 13.-15.07.2020
F. Schlenker, E. Kipf, N. Borst, T. Hutzenlaub, N. Paust, R. Zengerle, F. von Stetten, P. Juelg
Centrifugal microfluidic 4 Plex digital droplet PCR for quantification of circulating tumor DNA
2020 MicroTAS 2020, 04.-09.10.2020, virtual
F. Schlenker, E. Kipf, M. Deuter, I. Hoeffkes, N. Borst, T. Hutzenlaub, R. Zengerle, F. von Stetten, N. von Bubnoff, P. Juelg
Low optimization effort for highly sensitive assays for point mutation quantification in CTDNA: the Mediator Probe digital PCR
2020 Liquid Biopsy Symposium, 30.10.2020, virtuell

2019

M. Lehnert, E. Kipf, F. Schlenker, R. Zengerle, N. Borst, F. von Stetten
Multiplex Mediator Probe Real-Time PCR - Optimization and Guideline Development through Systematic Characterisation of Label Free Mediator Probes and Fluorogenic Universal Reporters
2019 9th Gene Quantification Event qPCR dPCR & NGS 2019, 18 - 22 March 2019, in Freising-Weihenstephan, Germany
KurzfassungMediator Probe PCR is a powerful and robust real-time PCR technology for multiplex DNA detection and quantification. It uses label free mediator probes, for molecular detection of nucleic acids during DNA amplification, in combination with fluorogenic universal reporters for signal generation. During PCR, target sequence specific mediator probes are cleaved by the polymerase and a generic sequence, the mediator, is set free. In the second step the mediator binds to the universal reporter, where it is extended by the polymerase. This generates a strong fluorescence signal increase. Due to the separation of DNA detection and signal generation many advantages arise. Mediator probes are not limited in their design by properties of the target sequence and a standard set of highly optimised fluorogenic universal reporters can be used for multiplex Mediator Probe PCR, right from the start.1 In the last years Mediator Probe PCR evolved from an innovative new method to an optimised and robust multiplexing technology. This was achieved by systematic characterisation of its molecular processes, which again was advantaged by the separation of DNA detection and signal generation. A DoE approach was used for the optimisation of Mediator Probes, focusing on their binding strengths.2 In parallel, a set of universal reporters with improved signal-to-noise ratios was established by successive testing over 40 molecular structures with different fluorophore-quencher labels and configurations.1 As a result, distinct guidelines exist, which enable fast adaption of new DNA targets and facilitate multiplex Mediator Probe PCR design. The capability of the technology was shown by highly sensitive, precise and specific multiplex Mediator Probe real-time PCRs in different areas of molecular diagnostics. These fields include monitoring of oncological disease, detection of pathogens or analysis of food samples.1,3
F. Schlenker, E. Kipf, S. Jenne, N. Borst, J. Lüddecke, K. Dormanns, T. Hutzenlaub, J. Steinert, N. Paust, R. Zengerle, F. von Stetten, P. Juelg
Doubling the order of color-multiplexing by photobleaching in Mediator Probe droplet digital PCR
2019 EACR-ESMO Joint Conference on Liquid Biopsies, Bergamo/Italien, 15. – 17. 05. 2019
J. Weisemann, G. B. Stevens, T. van Oordt, N. Krez, K. Dormanns, M. Karle, F. von Stetten, A. Rummel
Fast automatic detection of Botulinum neurotoxin by the BoNT LabDisk assay: results of a trans-european performance study
2019 Toxins 2019, Copenhagen/Denmark, 16. - 19.01.2019
KurzfassungBotulinum Neurotoxins (BoNT) are the most toxic naturally occurring substances for humans. After potential intoxication and occurrence of botulism symptoms, urgent detection of causative BoNT is highly desirable for successful medical treatment. The mouse bioassay (MBA) as gold standard requires a large number of mice, takes several days and is not suitable for point-of-care. Here, we developed an automatic in vitro assay to detect BoNT in complex biological matrices. The assay detects specific cleavage of SNARE proteins by BoNTs by fluorescent read out in a centrifugal microfluidic cartridge, termed BoNT LabDisk (BLD). The generic LabDisk comprises two different peptides able to distinguish between BoNT/ACE and BDF, respectively. The BLD prototype system was tested in a trans-European performance study by five naïve laboratories with expertise in BoNT detection to demonstrate practical applicability and performance of the BLD system. Eight blinded samples containing three complex biological matrices (human serum, bean juice, carrot juice) or buffer spiked with 10-1000 pM of BoNT/A and D were provided. Stability of samples was confirmed by mouse hemidiaphragm assay prior to shipment. The laboratories detected absence or presence of BoNT/A in 34 of the 40 samples correctly (85% accuracy). In three of ten bean juice or serum samples problems occurred due to high viscosity which requires improvement of sample preparation. In another three samples, signals close to background led to misinterpretation by experimentators. Altogether, the performance study showed that handling of the BLD assay is very easy, fast and practicable. The current detection limit is close to that of MBA. In conclusion, the BLD system allows the detection of functionally active BoNT in short assay times with low limits of detection within different matrices, without the need of animals or animal based reagents. The next step will be the adaption of the BLD assay for a direct serotyping of BoNT/A-F.
P. Juelg, M. Specht, M. Meyer, E. Kipf, F. Schlenker, F. Baensch, S. Neumann, F. von Stetten, R. Zengerle, N. Paust, M. Fillies, R. Kirschner-Schwabe, S. Groeneveld-Krentz, M. Lehnert, C. Eckert, T. Hutzenlaub
Individual Response Monitoring Assay (IRMA) – Standardization Of Personalized Multiplex Biomarker Quantification
2019 EACR-ESMO Joint Conference on Liquid Biopsies, Bergamo/Italien, 15. – 17.05. 2019
S. Burger, L. Drechsel, A. Homann, F. von Stetten, R. Zengerle, N. Paust
LabSlice XL – A centrifugal microfluidic cartridge for the automated bio-chemical processing of industrial process water
2019 Transducers, Berlin, 23. - 27. 06. 2019
M. Specht, J. Schemberg, T. Förster, S. Burger, M. Rombach, N. Paust, R. Zengerle, F. von Stetten, G. Gastrock, M. Karle
Microfluidic App for centrifugal separation and purification of lymphatic cancer cells from whole blood
2019 MST-Kongress, 28. - 30.Oktober 2019, Berlin
A. Brunauer, F. von Stetten, S.M. Früh
Sensitive detection of Pseudomonas aeruginosa based on recombinase polymerase amplification combined with an immunological assay
2019 Molecular Diagnostics Europe, Lissabon/Portugal, 06. - 09.05.2019
P. Juelg, M. Specht, M. Meyer, E. Kipf, F. Schlenker, F. Bänsch, S. Neumann, F. von Stetten, R. Zengerle, N. Paust, M. Fillies, R. Kirschner-Schwabe, S. Groeneveld-Krentz, M. Lehnert, C. Eckert, T. Hutzenlaub
Standardization of Personalized Multiplex Biomarker Quantification: Individual Response Monitoring Assay (IRMA)
2019 MST-Kongress, 28. - 30.Oktober 2019, Berlin
A. Brunauer, B. Breiner, D. Kainz, R. Verboket, F. von Stetten, R. Zengerle, N. Paust, T. Hutzenlaub, S. M. Früh
Towards digital diagnostic devices - From smart membrane cartridges to highly integrated test stripes
2019 Diagnostics-4-Future, 27. – 28. 11. 2019, Konstanz
F. von Stetten
Universal microarray for voltammetric real-time detection of DNA-amplification by mediator displacement loop mediated isothermal amplification (MP LAMP)
2019 4th “NanoBio Surfaces and Interfaces in Healthcare and Science Workshop” 16.-17.5.2019, Potsdam

2018

Z. Bagheryan, M. Trotter, L. Becherer, N. Borst, F. von Stetten
A universal approach for electrochemically DNA detection based on mediator displacement LAMP
2018 9th Euro Biosensors & Bioelectronics Congress November 29-30, 2018 Dublin, Ireland
P. Juelg, M. Specht, E. Kipf, M. Lehnert, C. Eckert, N. Paust, F. von Stetten, R. Zengerle, T. Hutzenlaub
Automation of qPCR based Minimal Residual Disease Monitoring by Centrifugal Microfluidics
2018 11th Symposium on minimal residual cancer, 03. – 05.05.2018, Montpellier, Frankreich
L. Becherer, M. Bakheit, S. Frischmann, S. Stinco, N. Borst, R. Zengerle, F. von Stetten
Fluorogenic real-time detection of loop-mediated isothermal amplification by novel mediator displacement probes demonstrated for HIV-1 and HTLV-1
2018 International Biotech Innovation Days, 23 - 25 May 2018 / Senftenberg, Germany
M. Schulz, N. Borst, M. Specht, S. Calabrese, F. von Stetten, R. Zengerle, N. Paust
From nasal swab to digital answer: unit operations for antibiotic resistance screening on a single cell level
2018 MicroTAS 2018, 11. -15. November 2018, Kaohsiung / Taiwan
L. Becherer, M. Schulz, H. Kuhn, M. Bakheit, S. Frischmann, F. Zitz, N. Borst, R. Zengerle, F. von Stetten
HIV-1 and HTLV-1 multiplex detection by digital mediator displacement LAMP
2018 MicroTAS 2018, 11. -15. November 2018, Kaohsiung / Taiwan
M. Trotter, N. Borst, F. von Stetten
Mediator probes for electrochemical DNA detection: Universal electrode functionalization for specific detection of different targets
2018 9th Euro Biosensors & Bioelectronics Congress November 29-30, 2018 Dublin, Ireland
L. Becherer, N. Borst, R. Zengerle, F. von Stetten
Simultaneous detection of HIV and HTLV by mediator displacement loop-mediated isothermal amplification
2018 6th World Congress on Control and Prevention of HIV/AIDS, STDs & STIs, Zürich/Switzerland, 27. – 28.08.2018
Kurzfassung6th World Congress on Control and Prevention of HIV/AIDS, STDs & STIs, Zürich/Switzerland, 27. – 28.08.2018
P. Juelg, M. Specht, E. Kipf, M. Lehnert, C. Eckert, N. Paust, F. von Stetten, R. Zengerle, T. Hutzenlaub
Towards Standardization of Molecular Diagnostic Workflows: Centrifugal Microfluidic Automation of qPCR for Cancer Monitoring
2018 6th International Molecular Diagnostics Europe, Lissabon / Portugal, 22. - 24. 05.2018

2017

M. Specht, M. Keller, J. Naue, U. Schmidt, R. Zengerle, F. von Stetten
Centrifugal Microfluidic Tools for Forensic Nucleic Analysis
2017 1st International Caparica Conference in Translational Forensics, Caparica / Portugal, 20. - 23.11.2017
KurzfassungThe analysis of DNA by polymerase chain reaction (PCR) is a routinely applied method within most molecular biology laboratories, including forensic laboratories [1]. Especially within the fields of investigative genetics as forensic science and food analysis, the analysis of human and animal DNA is required and samples with degraded or a low amount of DNA are a commonly occurring problem to overcome. [1]. The different DNA analytic techniques require a lot of manual handling steps, which give rise to a high contamination risk. We develop centrifugal microfluidic Lab-on-a-Chip systems, which are able to automate the laboratory processing steps without the need of investing in expensive robots. One of our platforms, the LabDisk is able to perform DNA extraction with subsequent Realtime PCR [2]. It is processed in a small “Point of Care” device, the LabDiskPlayer. A second aproach is to design microfluidic disk slices for a standard PCR thermocycler. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards [3]. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.
N. Borst, S. Wadle, K. Stock, M. Röder, E. Held-Föhn, A. Heiß, U. Götz, F. von Stetten
Innovative Diagnostikplattform zur Zuordnung von Krankenhauskeimen und deren Antibiotikaresistenzen
2017 microTEC Südwest Clusterkonferenz 2017, 27.-28. März 2017, Freiburg
N. Borst, M. Specht, M. Schulz, N. Paust, J. Li, S. Wadle, U. Götz, E. Held-Föhn, A. Heiß, F. Hausladen, R. Mader, K. Stock, M. Röder, A. Serr, G. Häcker, R. Zengerle, F. von Stetten
Integriertes System zur simultanen Detektion von Krankenhauskeimen und deren Antibiotikaresistenzen auf Einzelzellebene
2017 MST Kongress, München, 23. - 25.10.2017
KurzfassungBei Krankenhausinfektionen mit mehreren beteiligten Erregern ist die Zuordnung von Antibiotikaresistenzen zu den Erregern besonders wichtig um eine spezifische Therapie zu ermöglichen. Ein Projektkonsortium aus 6 Instituten der Innovationsallianz Baden Württemberg entwickelt im Projekt IDAK ein integriertes System das es künftig ermöglichen soll auf Einzelzellebene simultan die Spezies und die Resistenz aller Erreger in einer Probe zu bestimmen. Wir berichten von den aktuellen Forschungsarbeiten und ersten Ergebnissen. Diese umfassen: Entwicklung eines mikrofluidischen Testträgers zur Integration aller Prozessschritte; Überführung einzelner Bakterien eines Nasalabstriches in Tröpfchen; Bakterienlyse; digitaler multiplex-DNA Nachweis auf Basis einer isothermen Amplifikation; Multispektrale Fluoreszenzdetektion der Genotypen.
M. Rombach, M. Keller, N. Paust, F. von Stetten, D. Mark, R. Zengerle, M. Karle
The LabCard – A new approach for centrifugal assay automation
2017 MST Kongress, München, 23. - 25.10.2017
KurzfassungWe present the LabCard, a new centrifugal microfluidic approach for simultaneous processing of multiple test carriers by arranging them in the vertical plane. As a proof-of-concept, we fluidically integrated an assay for isothermal nucleic acid analyses of respiratory pathogens with the microfluidic network comprising unit operations and processing chains[1] for thermal lysis of a patient sample (25–75 μL), release of pre-stored reagents, mixing of reagents, aliquoting and distribution into amplification wells, which was successfully demonstrated in 5/5 runs with an overall fluidic processing time of 10 min.
S. Hin, M. Loskyll, V. Klein, M. Keller, O. Strohmeier, F. von Stetten, R.Zengerle, K. Mitsakakis
User-friendly membrane-based sample inlet for the centrifugal microfluidic LabDisk platform
2017 MNE 2017, Braga / Portugal, 18. - 22.09.2017
KurzfassungWe present a sample inlet for whole blood consisting of a plasma separation membrane placed into the inlet chamber of a centrifugal-microfluidic LabDisk to take up the sample via capillary forces. This approach offers advantages regarding several aspects: • Compatibility with a vast range of blood transfer device • Safe, well-controlled and easy application of blood sample without the danger of spilling • Combination of sample inlet with state-of-the art plasma separation leading to reduced footprint of the cartridge

2016

K. Mitsakakis, F. Stumpf, O. Strohmeier, V. Klein, D. Mark, F. von Stetten, J. R. Peham, C. Herz, P.N. Tawakoli, F. Wegehaupt, T. Attin, N. Bostanci, K. Bao, G.N. Belibasakis, J.P. Hays, G. Elshout, R.C. Huisman, S. Klein, A.P. Stubbs, L. Doms, A. Wolf, V. Rusu, S. Goethel, T. Binsl, A. Michie, J. Jancovicova, V. Kolar, M. Kostka, J. Smutny, M. Karpisek, C. Estephan, C. Cocaud, R. Zengerle
Chair/bedside diagnosis of oral and respiratory tract infections, and identification of antibiotic resistances for personalised monitoring and treatment
2016 pHealth 2016, 13th International Conference on Wearable, Micro & Nano Technologies for Personalized Health, 29-31 May, FORTH, Heraklion, Crete, Greece Studies in Health Technology and Informatics, Band: 224, Seiten: 61 - 66
KurzfassungGlobal healthcare systems are struggling with the enormous burden associated with infectious diseases, as well as the incessant rise of antimicrobial resistance. In order to adequately address these issues, there is an urgent need for rapid and accurate infectious disease diagnostics. The H2020 project DIAGORAS aims at diagnosing oral and respiratory tract infections using a fully integrated, automated and user-friendly platform for physicians’ offices, schools, elderly care units, community settings, etc. Oral diseases (periodontitis, dental caries) will be detected via multiplexed, quantitative analysis of salivary markers (bacterial DNA and host response proteins) for early prevention and personalised monitoring. Respiratory Tract Infections will be diagnosed by means of DNA/RNA differentiation so as to identify their bacterial or viral nature. Together with antibiotic resistance screening on the same platform, a more efficient treatment management is expected at the point-of-care. At the heart of DIAGORAS lies a centrifugal microfluidic platform (LabDisk and associated processing device) integrating all components and assays for a fully automated analysis. The project involves an interface with a clinical algorithm for the comprehensive presentation of results to end-users, thereby increasing the platform’s clinical utility. DIAGORAS’ performance will be validated at clinical settings and compared with gold standards.
F. Schuler, C. Siber, S. Hin, S. Wadle, N. Paust, R. Zengerle, F. von Stetten
Digital droplet loop-mediated isothermal amplification (ddLAMP) on a microscope slide
2016 Biosensors 2016, Goeteborg, 25. - 27.05.2016
S. Hin, M. Loskyll, V. Klein, O. Strohmeier, F. von Stetten, R. Zengerle, K. Mitsakakis
Membrane-based sample inlet for pathogen-containing whole blood on a centrifugal microfluidic platform (LabDisk)
2016 Biosensors 2016, Goeteborg, 25. - 27.05.2016
T. van Oordt, O. Strohmeier, K. Mitsakakis, S. Hin, R. Zengerle, F. von Stetten
Point-of-Need Detection of Biological Threats
2016 15th MEDICAL BIODEFENSE CONFERENCE, Munich, 26 - 29 April 2016 , Band: JP20, Seite: 58
KurzfassungThe state-of-the-art for detection of biological threats often requires complex stationary devices and experts, thus, limiting the capability for a fast and efficient response. A novel portable platform , designated "LabDisk" has been developed . The versatile analytical platform allows automated and easy-to-use detection of biological threats at the point-ofneed. lt consists of a centrifugal device and disposable disks for the detection of various toxins and pathogens.
M. Rombach, M. Keller, N. Paust, F. von Stetten, D. Mark, R. Zengerle, M. Karle
The LabCard – A new approach for centrifugal assay automation”
2016 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, µTAS 2016, Dublin / Irland, 09. – 13.10.2016

2015

F. Stumpf, F. Schwemmer, T. Hutzenlaub, O. Strohmeier, F. von Stetten, R. Zengerle, D. Mark
Automated sample-to-answer nucleic acid testing with frequency controlled reagent release from cartridge integrated stickpacks
2015 18th International Conference on Solid-State Sensors, Actuators and Microsystems (Transducers 2015), Anchorage, Alaska, 21. – 25.06.2015 IEEE, Seiten: 743 - 746
KurzfassungFor the first time we demonstrate an automated centrifugal Lab-on-a-Disk system for sample-to-answer point-of-care testing of multiple nucleic acid targets that features pre-storage of all required liquid reagents for nucleic acid extraction as well as primers and probes and magnetic beads. Highly wetting and thus hardly controllable liquid buffers were pre-stored in stickpacks with frequency controlled on-demand reagent release enabling automated addition of binding buffer after sample lysis. The self-contained Lab-on-a-Disk system automates all necessary assay steps for PCR-based pathogen detection: RNA extraction, aliquoting of the RNA and geometrically multiplexed real-time RT-PCR. As a proof-of-principle, we demonstrated detection of as little as 15 plaque forming units (pfu) of RNA bacteriophage MS2 in a 200 µL sample in 3.5 hours.
G. Czilwik, T. van Oordt, F. von Stetten, R. Zengerle, D. Mark
Automation of a magnetic immuno-PCR on a centrifugal point-of-care analyzer
2015 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA
F. Stumpf, F. Schwemmer, T. Hutzenlaub, O. Strohmeier, F. von Stetten, R. Zengerle, D. Mark
Automatisierte Nukleinsäurediagnostik in der LabDisk mittels frequenzgesteuerter Freisetzung vorgelagerter Reagenzien
2015 6. Mikrosystemtechnik Kongress (MST Kongress 2015), Karlsruhe, 26. – 28.10.2015
KurzfassungPräsentiert wird ein neues Verfahren zur automatisierten Sample-to-answer Point-of-Care Diagnostik auf der zentrifugal-mikrofluidischen LabDisk Plattform. Hochbenetzende und fluidisch schwer handhabbare Flüssigreagenzien für die Nukleinsäure-Extraktion werden in Stick-packs vorgelagert und können drehfrequenzgesteuert freigesetzt werden. Magnetische Beads, Primer und Fluoreszenzsonden werden durch Eintrocknung vorgelagert. Die LabDisk Plattform ermöglicht die Automatisierung aller erforderlichen Assayschritte der Pathogendetektion: RNA Extraktion und Aufreinigung, Aliquotieren der RNA sowie multiplex real-time RT-PCR. 15 PFU (plaque forming units) RNA-phage MS2 in einem 200 μL Sample werden in weniger als 3 Stunden detektiert.
F. Schuler, M. Trotter, S. Wadle, F. Schwemmer, R. Zengerle, F. von Stetten, N. Paust
Centrifugal microfluidic step emulsification for digital droplet recombinase polymerase amplification
2015 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA
KurzfassungFor the first time we show centrifugal step emulsification. It enables the fast and easy production of monodispers w/o droplets with minimal handling effort (3 pipetting steps). In contrast to previously presented centrifugal emulsification systems [1], homogenous droplets with pre-selectable diameters were generated with zero run-in time and zero dead volume. The centrifugal microfluidic step emulsification was used to perform the first digital droplet recombinase polymerase amplification (ddRPA). Compared to digital droplet PCR, the amplification time was reduced by a factor of 4 from 2 hours to 30 minutes.
M. Keller, A. Drzyzga, F. Schwemmer, R. Zengerle, F. von Stetten
Centrifugo-thermopneumatic wax valve for centrifugal microfluidic platforms
2015 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA
D. Baumann, S. Hin, F. Stumpf, V. Klein, K. Mitsakakis, D. Kosse, F. von Stetten, R. Zengerle, D. Mark
Fully automated stick-packaging for precise liquid reagent pre-storage and release in lab-on-a-chip disposables
2015 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA
S.K. Vashist, F. von Stetten, R. Zengerle, J.H.T. Luong
One-step antibody immobilization-based high sensitivity immunoassay procedure for potential in vitro diagnostics
2015 6. Mikrosystemtechnik Kongress (MST Kongress 2015), Karlsruhe, 26. – 28.10.2015
F. von Stetten
Risk and cost reduction by outsourcing design and fabrication of microfluidic chips – A service provider’s perspective
2015 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Gyeongju, KOREA, 25.-29.10.2015
A.G. Venkatesh, F. von Stetten, R. Zengerle, S.K. Vashist
Smartphone EasyELISA – A point-of-care platform for cost-effective and easy-to-use in vitro diagnostics
2015 6. Mikrosystemtechnik Kongress (MST Kongress 2015), Karlsruhe, 26. – 28.10.2015
A.G. Venkatesh, T. van Oordt, F. von Stetten, R. Zengerle, S.K. Vashist
Smartphone-based colorimetric readers for cost-effective in vitro diagnostics
2015 6. Mikrosystemtechnik Kongress (MST Kongress 2015), Karlsruhe, 26. – 28.10.2015
A. G. Venkatesh, F. von Stetten, R. Zengerle, S. K. Vashist
Smartphone-based immunoassay for human fetuin A
2015 6. Mikrosystemtechnik Kongress (MST Kongress 2015), Karlsruhe, 26. – 28.10.2015
S. Wadle, M. Lehnert, F. Schuler, R. Zengerle, F. von Stetten
Universelle Reporter der Mediatorsonden PCR als Zielsequenz-unabhängige Biosensoren zur Detektion fünf verschiedener RNA- und DNA-Sequenzen
2015 9. Deutsches BioSensor Symposium 11.–13. März 2015, München
KurzfassungIn der Molekulardiagnostik werden meist sogenannte Hydrolysesonden (hydrolysis probe, HP) zur Echtzeitdetektion von Nukleinsäuren verwendet. Hierbei ist für jede zu detektierende Zielsequenz die Synthese einer spezifischen dual-markierten HP notwendig. Deren Anschaffung ist sehr kostenintensiv, vor allem beim Bedarf nur geringer Bestellmengen. Zudem müssen HP bei jeder neu zu detektierenden Zielsequenz individuell bezüglich der Signalgenerierungseffizienz optimiert werden. Ein von uns publiziertes neuartiges Verfahren, die Mediatorsonden PCR (mediator probe PCR, MP PCR) [1, Abb.1] löst diese Probleme durch Verwendung eines dual-markierten Universellen Reporter Oligonukleotids (UR). Dieser Biosensor erzeugt unabhängig der zu detektierenden Zielsequenz ein Signal, initiiert durch die Interaktion mit nicht-markierten und somit kostengünstigen sequenzspezifischen Mediatorsonden. Im Vergleich zu [1] wurde die Fluoreszenz-Quenchingeffizienz am UR und die Reaktionszusammensetzung der MP (RT-)PCR Assays verbessert, um fünf verschiedene DNA und auch RNA Sequenzen von Atemwegsinfektion-verursachenden Viren zu detekieren. Die Detektionsreaktionen wurden mit HP-basierten Assays als Referenz verglichen, bei denen fünf verschiedene dual-markierte Sonden benötigt werden.
T. van Oordt, G.B. Stevens, A. Rummel, O. Strohmeier, G. Urban, R. Zengerle, F. von Stetten
Vollintegrierte zentrifugale Mikrofluidik für die Vor-Ort-Analyse
2015 9. Deutsches BioSensor Symposium 2015, München, 11. - 13. 03. 2015 , Seite: P71
O. Strohmeier, D. Mark, D. Kosse, R. Zengerle, F. von Stetten
Zentrifugale Mikrofluidik für die DNA Analytik
2015 Forschungstag der Baden-Württemberg Stiftung, Stuttgart, 22.07.2015
KurzfassungIm Rahmen des von der Baden-Württembergstiftung geförderten Projektes „Amplidisk“ wurden verschiedene Applikationen aus dem Bereich der molekularbiologischen Diagnostik mit Hilfe zentrifugal-mikrofluidischer Testträger „LabDisks“ automatisiert. Diese können in tragbaren Geräten prozessiert werden und bieten durch den hohen Automatisierungsgrad eine ideale Lösung für die zukünftige Diagnostik am Point-of-Care. Zunächst wurde im Rahmen des Projektes eine Methode zum Transport magnetischer Partikel zwischen verschiedenen, flüssigkeitsgefüllten Kammern auf einer LabDisk entwickelt [1]. Mit Hilfe dieser Methode konnte ein Prozess zur automatisierten und disk-integrierten DNA Extraktion implementiert werden wobei silica beschichtete Magnetpartikel als Festphase dienten [2]. In weiteren Arbeiten wurden LabDisks für den Nachweis von KRAS -Mutationen aus Tumorzell-DNA entwickelt die bis zu 7 gängige DNA - Mutationen in etwa 2 Stunden detektieren können während das Goldstandardverfahren, die Sequenzierung, hierzu bis zu 20 Stunden benötigt. Kenntnisse über den Mutationsstatus sind zwingend zur Einleitung einer spezifischen Therapie erforderlich [3]. Für ein zweites Anwendungsfeld konnte eine Disk zum qualitativen und quantitativen Nachweis von sechs typischen Lebensmittelpathogenen wie Listerien, Salmonellen oder EHEC entwickelt werden. [4] Die entwickelten Module wurden in weiteren Projekten erfolgreich gemeinsam auf einer Disk kombiniert um die gesamte Analyse zu integrieren.

2014

S.K. Vashist, F. von Stetten, R. Zengerle
A highly-sensitive rapid sandwich immunoassay for human fetuin A using the one/step antibody immobilization procedure
2014 Biosensors 2014, Melbourne, Australia, 27.05.2014 – 30.05.2014
R. Zengerle, D. Mark, N. Paust, F. von Stetten
Advanced centrifugal microfluidics and Microfluidic Apps
2014 EMBL Conference Series Microfluidics, Heidelberg, 23. - 25.07.2014
G. Czilwik, V. Klein, O. Strohmeier, G. Roth, F. von Stetten, R. Zengerle, S.K. Vashist, D. Mark
Automated detection of human C-reactive protein on centrifugal microfluidics-based LabDisk platform
2014 Biosensors 2014, Melbourne, Australia, 27.05.2014 – 30.05.2014
G. Czilwik, T. Messinger, O. Strohmeier, F. von Stetten, R. Zengerle, P. Saarinen, J. Niittymäki, K. McAllister, J. O´Leary, D. Mark
Automated point-of-care diagnosis of neonatal sepsis from a 200 µL serum sample based on a LabDisk with integrated DNA extraction, pre-amplification, and real-time PCR detection
2014 SepsEast 2014, Budapest, Ungarn, 18-20 September, 2014
KurzfassungSepsis remains among the major causes of infant death worldwide and management of patient sepsis is challenged by short-comings in current diagnostics. The diagnostic gold standard is blood culture that requires 1-3 days to attain a conclusive result. The immune system of a neonate is relatively immature compared to adults and progression of neonatal sepsis disease can be extremely fast. As a consequence intensive-care units administer broad-spectrum antibiotics when neonatal sepsis is only suspected and before the causative pathogen has been identified. Therefore, a rapid diagnostic tool for identification of causative pathogens is of major interest to give clinicians facts-based treatment options as early as possible. In this context, we developed a fully automated centrifugal microfluidic `LabDisk` system for highly sensitive detection of neonatal sepsis pathogens in human serum samples within 4 hours. All necessary assay steps are integrated on a single test carrier: DNA extraction, PCR pre-amplification and geometrically multiplexed realtime PCR. The reagents for the PCRs are already prestored in a dry format on the test carrier. Therefore, processing solely requires loading of the sample and DNA extraction reagents. Afterwards the entire process is automated using a rotational protocol in a mobile processing device. We demonstrate detection down to 3 colony-forming-units (cfu) of Staphylococcus warneri, 150 cfu of Streptococcus agalactiae, 5 cfu of Escherichia coli and 18 cfu of Haemophilus influenzae in 200 µL of serum. The system has the potential to reduce neonatal mortality and ensure appropriate treatment of newborn sepsis patients. Further development will include an adaptation of the LabDisk to process whole blood instead of serum, reduction of the analysis time and adding additional bacterial targets to the panel.
Y. Zhao, F. Schwemmer, S. Zehnle, F. von Stetten, R. Zengerle, N. Paust
Centrifugo-pneumatic handling of microparticles without external actuation as a new unit operation for centrifugal microfluidics
2014 MicroTAS 2014, San Antonio, USA, 26. – 30.10.2014 , Seiten: 21 - 23
KurzfassungFor the first time we present a microfluidic method for handling of microparticles that requires neither surface treatment of chambers and channels nor external actuators such as magnets. Thus it is not limited to handling of magnetic particles. Using centrifugal forces and temporary storage of pneumatic energy, only, we demonstrate 1) liquid mediated microparticle loading, 2) re-suspension of microparticles by shake mode at low centrifugation; sedimentation of microparticles and afterwards exchange of liquids with particle loss below 2% and supernatant removal efficiency of more than 99.5%, 3) re-suspension and subsequent transport of microparticles together with liquid reagent with particle loss of 6% or less.
K. Mitsakakis, S. Hin, V. Klein, O. Strohmeier, D. Mark, F. von Stetten, R. Zengerle
Disc-shaped Point-of-Care platform for infectious disease diagnosis
2014 NN14 Nanotexnology, 08.-11.07.2014, Thessaloniki, Greece
M. Keller, C. Nuese, P. Papireddy Vinayaka, R. Zengerle, F. von Stetten
Fluidic structure for temperature measurement under rotation in centrifugal microfluidics
2014 MicroTAS 2014, San Antonio, USA, 26. – 30.10.2014 , Seiten: 1416 - 1418
KurzfassungWe present a novel fluidic temperature measurement system (FTMS), which allows measurement under rotation at arbitrary positions. Temperature determination and control is of high importance for many biochemical applications integrated into centrifugal microfluidic (CM) cartridges while the integration of electronic temperature sensors into processing devices remains a challenge. By enclosing a well-defined air volume inside a fluidic chamber, temperature-induced volume expansion is utilized to displace fluorescent liquid into a detection chamber. Results of a proof-of-concept are in good accordance with temperatures of a commercially available thermocycler (Rotor-Gene Q, QIAGEN GmbH, Germany).
G. Czilwik, T. Messinger, O. Strohmeier, F. von Stetten, R. Zengerle, P. Saarinen, J. Niittymäki, K. McAllister, O.Sheils, D. Mark
Fully integrated PCR detection of pathogens for fast diagnosis of neonatal sepsis on LabDisk
2014 MicroTAS 2014, San Antonio, USA, 26. – 30.10.2014 , Seiten: 2528 - 2529
KurzfassungWe developed a fully automated centrifugal microfluidic `LabDisk` system for highly sensitive detection of neonatal sepsis pathogens in human serum samples within 4 hours. All necessary assay steps are integrated on a single test carrier: DNA extraction, pre-amplification and geometrically multiplexed realtime PCR. Processing solely requires loading of the sample and extraction reagents. Afterwards the entire process is automated using a rotational protocol in a mobile processing device. We demonstrate detection down to 3 colony-forming-units (cfu) of Staphylococcus warneri, 150 cfu of Streptococcus agalactiae, 5 cfu of Escherichia coli and 18 cfu of Haemophilus influenzae in 200 μL of serum.
O. Strohmeier, G. Czilwik, T. van Oordt, M. Keller, D. Mark, R. Zengerle, F. von Stetten
LabDisk: Zentrifugale Mikrofluidik zur voll-integrierten Automatisierung von Laborabläufen
2014 17. Heiligenstädter Kolloquium, Heiligenstadt, 22. - 24.09.2014 , Seiten: 139 - 146
KurzfassungZentrifugale Mikrofluidik auf preiswerten, CD-ähnlichen Einweg-Testträgern, sog. „LabDisks“, kann erfolgreich zur vollintegrierten Automatisierung von Laborabläufen in einem portablen Analysegerät, dem „LabDisk Player“ eingesetzt werden. Das Potential der LabDisk wird anhand von zwei Anwendungen demonstriert: (1) dem integrierten Nachweis von Biokampfstoffen auf DNA Ebene und (2) dem Nachweis von Botulinum Neurotoxin.
N. Paust, A. R. Fiebach, L. Drechsel, S. Zhang, R. Zengerle, F. von Stetten
LabTube - a novel centrifugal microfluidic lab-on-a-chip platform for operation in standard laboratory centrifuges
2014 IFCC WorldLab Istanbul, 22.-26-06-2014, Istanbul, Türkei
KurzfassungThe “LabTube” is a novel centrifugal lab-on-a-chip platform that uses a standard laboratory centrifuge as processing device. It automates multistep protocols such as nucleic acid extraction or protein purification, decreasing the hands-on-time from > 6 to 1 minute per extraction. Future applications will include point-ofcare sample-to-answer nucleic acid analysis. The LabTube approach lowers the market entry barrier for microfluidics as the user only requires to purchase disposable parts without the need to invest in specialized processing devices.
F. von Stetten, A. Kloke, A.R. Fiebach, J. Steigert, M. Hoehl, R. Zengerle, N. Paust
LabTube - a novel centrifugal microfluidic lab-on-a-chip platform for operation in standard laboratory centrifuges
2014 Biosensors 2014, Melbourne, Australia, 27.05.2014 – 30.05.2014
F. von Stetten
LabTube and LabDisk - prospective centrifugal microfluidic platforms for sample-to-answer point-of-care nucleic acid testing
2014 Lab‐on‐a‐Chip World Congress, San Diego/USA, 18. - 19. September 2014
F. von Stetten
LabTube – a novel "microfluidic app" for sample preparation and diagnostics operated in a laboratory centrifuge
2014 Lab-on-a-Chip European Congress, Berlin, 10.-11.03.2014
KurzfassungThe LabTube is a novel lab-on-a-chip cartridge processed by a laboratory centrifuge. Not requiring specialized processing devices this "microfluidic app" has low market entry barriers. Fully automated DNA-extraction and protein purification are demonstrated. The LabTube is amenable to sample-to-answer diagnostics.
F. von Stetten
Mediator Probe PCR - Principle, Applications and Guidelines for Primer and Probe Design
2014 Advances in Biodetection & Biosensors, Berlin, 10. - 11. 03.2014
KurzfassungMediator probe PCR is an alternative to establish sequence-specific fluorogenic probe based real-time PCRs. Detection relies on label-free primary probes and secondary universal fluorogenic reporters that can be synthesized at drecreased cost. Guidelines for probe design and applications for detection of HPV18, influenza B virus, and HAdV B7 are presented.
K. Mitsakakis, S. Hin, V. Klein, O. Strohmeier, D. Mark, F. von Stetten, R. Zengerle
Multi-pathogen identification on a centrifugal microfluidic platform
2014 CLINAM - European Foundation for Clinical Nanomedicine, 23. - 25. 06.2014, Basel/CH
S.K. Vashist, F. von Stetten, R. Zengerle
One-step kinetics based immunoassay for the detection of human fetuin A in 30 min
2014 Biosensors 2014, Melbourne, Australia, 27.05.2014 – 30.05.2014
M. Trotter, F. Stumpf, F. von Stetten, J. Hoffmann, R. Zengerle, G. Roth
One-step single cell solid-phase PCR
2014 5th annual conference for advances in qPCR & dPCR , Barcelona, Spain, 14. - 15. 05.2014
M. Rombach, S. Hin, O. Strohmeier, F. von Stetten, R. Zengerle, D. Mark
Pre-storage and release of purification reagents for full “hands-off” integration of DNA/RNA assays on the LabDisk platform
2014 MicroTAS 2014, San Antonio, USA, 26. – 30.10.2014 , Seiten: 1169 - 1171
KurzfassungFor the first time we demonstrate complete integration and pre-storage of all required compo-nents for nucleic acid extraction and purification on the centrifugal microfluidic LabDisk platform. On the one hand pre-storage of large volume liquid reagents from 200-–-550 μL is realized in stick-packs, that are stacked to decrease the footprint consumption on the LabDisk and are released au-tomatically during the run using centrifugal pressure (pcent-=-1 bar). On the other hand magnetic beads are mixed with PEG8000 (ratio 2:1) and air dried into the final cavity over 12 h. PEG8000 allows stable pre-storage and does not influence the extraction yield.
I. Schwarz, S. Zehnle, G. Czilwik, T. Hutzenlaub, F. von Stetten, D. Mark, R. Zengerle, N. Paust
Rapid development of centrifugal microfluidic assay automation by network-simulation based fluidic design
2014 Biosensors 2014, Melbourne, Australia, 27.05.2014 – 30.05.2014
S.K. Vashist, T. van Oordt, F. von Stetten, R. Zengerle
Rapid in vitro diagnostic procedures and platforms for point-of-care diagnostics
2014 MicroTAS 2014, San Antonio, USA, 26. – 30.10.2014
S.K. Vashist, T. van Oordt, E.M. Schneider, F. von Stetten, R. Zengerle
Smartphone and tablet-based point-of-care in vitro diagnostics and devices for mobile healthcare
2014 12th Psychoimmunology Expert Meeting, Guenzberg, Germany, 06.-09.03.2014 Neurology, Psychiatry and Brain Research (2014), Band: 2014, Seiten: 25 - 26
S.K. Vashist, T. van Oordt, F. von Stetten, R. Zengerle
Smartphone-based colorimetric reader for bioanalytical applications using tablet's/smartphone's screen-based bottom illumination
2014 Biosensors 2014, Melbourne, Australia, 27.05.2014 – 30.05.2014
V. Alagarswamy Govindaraj, J. Jin, F. von Stetten, R. Zengerle, S. K. Vashist
Smartphone-based immunoassay for the highly-sensitive point-of-care detection of human C-reactive protein in whole blood and serum
2014 Biosensors 2014, Melbourne, Australia, 27.05.2014 – 30.05.2014
F. von Stetten
Smartphone-based immunoassay for the highly-sensitive point-of-care detection of human C-reactive protein in whole blood and serum
2014 Biosensors 2014, Melbourne, Australia, 27-30.05.2014
S. Wadle, M. Follo, M. Lehnert, F. Schuler, N. von Bubnoff, R. Zengerle, F. von Stetten
Specific SNP detection by mediator probe digital droplet PCR
2014 Advances in dPCR & qPCR, Barcelona, Spain, 14. – 15. Mai 2014
KurzfassungMediator probe digital droplet PCR (MP ddPCR) was used to detect single nucleotide polymorphisms (SNP) at the cKit V559D gene locus, a gastrointestinal cancer biomarker [1]. In comparison to conventional PCR with dual-labelled hydrolysis probes (HP) the MP PCR combines label-free mediator probes with universal fluorogenic reporters [2]. In the presented experiment, specificity of the MP-based SNP detection was as high as SNP detection using HPs with locked-nucleic acid (LNA) modified nucleotides.
S. Hin, K. Mitsakakis, V. Klein, O. Strohmeier, M. Keller, D. Kosse, R. Zengerle, F. von Stetten, D. Mark
The Lab-on-a-Chip Design & Foundry Service
2014 2nd MFHS Conference 2014, Freiburg, 08 - 10.Okt. 2014 , Seiten: 195 - 198
KurzfassungThe Lab-on-a-Chip Design & Foundry Service offers the rapid development of point-of-care systems, which automate diagnostic assays delivered by the customer. The automation is done on a centrifugal microfluidic lab-on-a-chip platform, the LabDisk. The customer profits from the utilization of unit operations in the development process. Those unit operations apply standard rules for the design and production of the cartridges. Unit operations are combined to form process chains for the translation of a manual laboratory protocol into a fully automated analysis. Thus, risk, time, and costs are reduced during the development process, because a wellestablished production technology is used and existing developments can be re-used. To describe that development process, in this paper the example of a unit operation for liquid reagent release is implemented into a LabDisk for the EU-FP7 project DiscoGnosis. The liquid release is needed in a fluidic structure for nucleic acid extraction from pathogens out of a whole blood sample (50 μl). To implement this unit operation, five different buffer solutions are filled into stickpacks. For each buffer, a research-scale batch of 100 stickpacks is produced. It is demonstrated for all buffers that 4 out of 4 stickpacks release their content in the planned time in the protocol. The time effort for this first design iteration was two weeks, including specification phase, CAD, fabrication, and fluidic validation.
G. B. Stevens, T. Binz, W. Römer, T. van Oordt, G. A. Urban, R. Zengerle, F. von Stetten
Towards a full-function molecular-based assay for botulinum neurotoxin
2014 World Congress on Alternatives and Animal Use, Prague, 24. - 28. August 2014 Altex-altern Anim Ex, Band: 3, Nummer: 1/14, Seite: V-4-593
KurzfassungThousands of mice are used annually for potency testing of an ever growing number of products that use Botulinum Neurotoxin as the active ingredient. Here we present a first step in the development of a molecular-based assay to detect binding and translocation of Botulinum Neurotoxin type A (BoNT/A). It consists of a liposome (Giant Unilammelar Vesicles, GUVs) with a BoNT/A receptor (GT1b) on the surface. We demonstrate binding of BoNT/A to the GUVs using a fusion protein (eGFP-HcA) consisting of the BoNT/A binding domain (HcA) and Green Fluorescent Protein (GFP). Microscope images of prepared GUVs with HcA-eGFP show binding to vesicles containing GT1b. GUVs prepared without GT1b did not produce the same objects, so GT1b was necessary for binding of the eGFP-HcA. The next step is to demonstrate binding of a construct containing the BoNT/A translocation domain, in addition to the binding domain, to open the way for high-resolution confocal microscopy studies of translocation.When used with an automated sensitive luciferase reporter assay for the detection of BoNT/A proteolytic activity (van Oordt et al., 2013),the developed system has the potential to replace the mouse bioassay,measuring both the translocation and the enzymatic activity of the toxin.
S. Wadle, M. Lehnert, F. Schuler, R. Zengerle, F. von Stetten
Universal reporters of mediator probe PCR as target-independent biosensors
2014 IFCC WorldLab Istanbul, 22.-26.06.2014, Istanbul, Türkei
KurzfassungMolecular diagnostics often uses hydrolysis probes (HP) for real-time nucleic acid sensing. However, each target sequence requires synthesis of specific dual-labelled HPs, which are expensive, especially when used at low batch sizes. Also, HPs must be individually optimized for signal generation efficiencies for each target sequence to be detected. We have published a novel approach, the mediator probe PCR (MP PCR) [B. Faltin et al.: Clin Chem, vol. 58, pp. 1546-1556, 2012] which overcomes these issues by using a labelled but universal reporter oligonucleotide (UR) as a biosensor for target-independent signal generation. It is triggered by unlabelled and thus cost-effective sequence-specific mediator probes. Compared to [Faltin 2012] we improved UR quenching efficiencies and reaction setup of MP PCRs to detect 5 different DNA and also RNA target sequences of viruses causing respiratory tract infections. HP based assays, which required 5 different dual-labelled probes were run as references. MPs and the UR designs were adapted from [Faltin 2012] with the sequence-specific MP section equal to corresponding HP sequences. Nucleic acid standards from human adenovirus (hAdV), influenza virus A&B (InfA & B), human metapneumovirus (hMPV), and respiratory syncytial virus (RSV) were serially diluted enabling efficiency calculation and detection limit determination. Reaction efficiencies and the correlation of input- with back-calculated output concentrations were better for MP RT-PCRs than for HP RT-PCRs. 95 % detection limits were: hAdV 7 / 7 copies per reaction (MP / HP (RT-)PCR), InfA 4 / 18, InfB 10 / 14, hMPV 11 / 29, RSV 14 / 22. These correspond well to commercially available assays [L. Van Wesenbeeck et al.: J. Clin Microbiol., vol. 51, pp. 2977-2985, 2013]. As conclusion, one UR was used for sensing 5 different DNA and RNA targets by MP (RT-) PCR. Even higher reaction efficiencies and lower detection limits as with the more expensive HP (RT-) PCRs could be reached. The method is especially recommended if many different target-specific probes are required at low batch sizes. In future, multiplexing degrees shall be increased using UR-microarrays.
S. Wadle, M. Lehnert, R. Zengerle, F. von Stetten
Universal reporters of mediator probe PCR as target-independent biosensors for detection of five different RNA and DNA sequences
2014 Biosensors 2014, Melbourne, Australia, 27.05.2014 – 30.05.2014

2013

D. Kosse, F. Schwemmer, D. Buselmeier, R. Zengerle, F. von Stetten
3D microfluidic cartridges by gas pressure assisted thermal bonding of microthermoformed films
2013 Transducers 2013, Barcelona, Spain, 16. – 18.06.2013 , Seiten: 1318 - 1321
KurzfassungMicrothermoforming of film substrates is an emerging technology to form thin-walled microfluidic Lab-on-a-Chip cartridges. We present a process chain to fabricate thin-walled 3D microfluidic cartridges by thermally bonding a stack of microthermoformed polymer layers. The three layer bond stack comprises an upper and a lower microthermoformed film substrate separated by a flat intermediate film layer. For thermal bonding the films are pressed together by gas pressure, eliminating the need for any structure dependent tools. The capabilities of the process chain are demonstrated by bonding a complex microfluidic LabDisk demanding two stacked fluidic layers on top of each other.
G.Czilwik, O.Strohmeier, I. Schwarz, N.Paust, S. Zehnle, M.Kräft, F. von Stetten, R. Zengerle, D. Mark
An Integrated Centrifugal Lab-on-a-Chip System for fully automated detection of pathogens via Real-time PCR
2013 Lab-on-a-Chip European Congress, Barcelona 2013,05.03.2013 – 06.03.2013
G. Czilwik, O. Strohmeier, I. Schwarz, N. Paust, S. Zehnle, F. von Stetten, R. Zengerle, D. Mark
An integrated Lab-on-a-Chip system with DNA extraction, pre- and main PCR amplification for automated detection of low concentrated pathogens
2013 MicroTAS 2013, Freiburg, 27.-31.10.2013 , Seiten: 1607 - 1609
T. van Oordt, O. Strohmeier, S.K. Vashist, R. Zengerle, F. von Stetten
Automated detection of biological threats with a centrifugal lab-on-a-chip system
2013 2nd International Conference and Exhibition on Biosensors & Bioelectronics, Chicago, USA, 17 June 2013 , Band: 4, Nummer: 3, Seite: 56
L. Drechsel, M. Schulz, F. von Stetten, R. Zengerle, N. Paust
Automated on-site detection of organophosphorous pesticides in real food samples
2013 MicroTAS 2013, Freiburg, 27.-31.10.2013 , Seiten: 1923 - 1925
M. C. Weil, W. Hauser, D. Kosse, O. Strohmeier, F. von Stetten, R. Zengerle, D. Mark
Automatic Foodpathogen detection on a centrifugal microfluidic cartridge in a commercially available PCR thermocycler
2013 MicroTAS 2013, Freiburg, 27.-31.10.2013 , Seiten: 626 - 628
T. van Oordt, G. Stevens, S. Vashist, G. Urban, R. Zengerle, F. von Stetten
Automatisierte Vor-Ort-Detektion von Botulinum Toxin auf der LabDisk Plattform
2013 Microsystemtechnik (MST) Kongress 2013, Aachen, 14. - 16.10.2013 , Seiten: 376 - 379
KurzfassungDie LabDisk Plattform ist ein portables, universell einsetzbares System bestehend aus einem zentrifugalen Analysegerät und Einweg-Disks für die Detektion von diversen Pathogenen [1]. Erstmals konnte eine LabDisk zur vollautomatisierten Detektion von Botulinum Neurotoxin (BoNT) Typ A entwickelt und validiert werden. In einem Konzentrationsbereich von 8 – 2000 pM wurde sowohl die leichte Kette (LC) als auch das komplexe BoNT in Pufferlösung und Vollmilch nachgewiesen. Der Nachweis hat eine signifikante Bedeutung für schnelle Vor-Ort-Analysen im Falle eines terroristischen Anschlages.
S. Wadle, O. Strohmeier, M. Rombach, D. Mark, R. Zengerle, F. von Stetten
Automatisierung der DNA Extraktion aus Vollblut unter Verwendung magnetischer Partikel auf der LabDisk Plattform
2013 Microsystemtechnik (MST) Kongress 2013, Aachen, 14. - 16.10.2013 , Seiten: 403 - 405
KurzfassungDie Integration der Probenvorbereitung spielt eine Schlüsselrolle in der Entwicklung von point-of-care Diagnostik- Systemen. Wir demonstrieren die Integration einer Magnetpartikel-basierten DNA Extraktion aus 200 μl Vollblut in eine zentrifugal-mikrofluidische LabDisk. Die mithilfe der LabDisk extrahierte DNA (vier unabhängige Extraktionen aus einer Blutprobe) wurde auf ihre Qualität geprüft. Als Referenz wurden dabei kommerziell verfügbare Säulen mit Silicamembranen als Goldstandard der DNA-Extraktion verwendet. Es konnten gleich gute Ausbeuten einer Wachstumsfaktor-Gensequenz – bestimmt mithilfe qPCR – (c1:10 LabDisk = 4,6 +/- 0,7 ng/μl gg. c1:10 Aufreinigungssäulen = 4,1 +/- 0,4 ng/μl) sowie Reinheiten (A260/A280 ~ 1,8 +/- 0,1) erzielt werden. Im Fall der LabDisk-extrahierten DNA lag eine leicht erhöhte Ethanol-Konzentration (5,9 +/- 2,4) % im Vergleich zu (3,3 +/- 0,2) % bei Aufreinigungssäulen vor. Die integierte und vollautomatiserte LabDisk-basierte DNA-Extraktion kann für weitere Anwendungen empfohlen werden.
M. Karle, J. Wöhrle, F. von Stetten, R. Zengerle, D. Mark
Axial centrifugal filtration – A novel approach for rapid bacterial concentration from a large volume
2013 Transducers 2013, Barcelona, Spain, 16. – 18.06.2013 , Seiten: 1235 - 1238
KurzfassungA novel approach for filtration on a centrifugal microfluidic platform is presented for the first time. This approach is intended to concentrate bacteria from a large volume. In axial centrifugal filtration the filter is oriented perpendicular to the axis of rotation. This feature allows for integration of dead-end filtration while the filter cake is continuously removed from the filter by centrifugation. This prevents clogging of the filter. Furthermore, a continuous sample feed enables processing of large samples on one disk. Especially for analyzing drinking water large volumes have to be processed and solutions for rapid bacterial concentration are highly appreciated.
M. Keller, M. Focke, O. Strohmeier, P. Reith, G. Roth, D. Mark, R. Zengerle, F. von Stetten
Centrifugo-thermopneumatic aliquoting on LabDisk for DNA-based detection of different bacteria
2013 Microsystemtechnik (MST) Kongress 2013, Aachen, 14. - 16.10.2013 , Seiten: 31 - 34
KurzfassungWe present the automation of an aliquoting process in an unmodified commercially available real-time PCR thermocycler (Rotor-Gene, QIAGEN, Germany) by integration of a centrifugal microfluidic LabDisk. With the help of a new centrifugo-thermopneumatic actuation principle we demonstrate the splitting of a DNA sample into 8 times 20 μl aliquots. Each aliquot serves for separate bacteria detection by real-time PCR. For demonstration purposes, DNA of the bacteria Corynebacterium glutamicum and Escherichia coli are automatically detected using pre-stored PCR master mixes inside the reaction cavities. The presented work is an example of how so-called Microfluidic Apps can increase the degree of automation of standard laboratory instruments through the microfluidic integration of workflows. In contrast to conventional automation via pipetting robots Microfluidic Apps do not require fixed costs.
S. Wadle, S. Rubenwolf, M. Lehnert, B. Faltin, R. Zengerle, F. von Stetten
Cost-effective geometric multiplex mediator probe PCR
2013 7. Senftenberger Innovationsforum Multiparameteranalytik, 18. - 19.04.2013
KurzfassungProbe-based real-time PCR is used in molecular diagnostics due to its superior specificity and clinical sensitivity. High synthesis costs for sequence-specific dual-labelled detection probes are still one reason why researchers are reluctant when larger numbers of probes need to be ordered. In order to reduce costs we suggested a novel real time PCR method, the mediator probe PCR [1, 2]. It replaces fluorescently labeled hydrolysis probes by sequence-specific label free mediator probes (MP). Cleavage of the MP during amplification results in release of a mediator which is detected by a universal fluorogenic reporter (UR) oligonucleotide. The key to cost savings is that the same UR can be used for all assays and therefore can be ordered in large scale. This way oligonucleotide synthesis costs can be reduced to less than 40 % compared to the synthesis costs in hydrolysis probe based assays. In this work, performance characteristics of mediator probe PCR (MP PCR) were compared to hydrolysis probe PCR (HP PCR).
S. Wadle, S. Rubenwolf, M. Lehnert, B. Faltin, R. Zengerle, F. von Stetten
Cost-effective real-time analysis by mediator probe (RT-)PCR
2013 qPCR & NGS 2013 Symposium, 18th -20th March 2013, Technical University of Munich, Freising, Germany
A. Kloke, S. Niekrawietz, A.R. Fiebach, J. Bernhardt, R. Kneusel, K. Schemel, J. Ritzel, F. von Stetten, R. Zengerle, N. Paust
Disposable LabTube cartridges for automated protein purification in standard lab centrifuges
2013 MicroTAS 2013, Freiburg, 27.-31.10.2013 , Seiten: 1628 - 1630
KurzfassungThe protein purification process is often the bottleneck for the efficient production of a large number of different proteins. Automation of these procedures is often the crux of the matter and is frequently a trade-off between efficiency and cost. Using our novel disposable LabTube cartridges we demonstrate how the process of His-tagged protein purification can be automated in standard laboratory centrifuges. LabTube cartridges include prestored reagents which are sequentially applied to a Ni-NTA purification matrix by an integrated ballpen mechanism actuated by acceleration changes of the centrifuge. Fully automated runs demonstrated similar yield and purity compared to manual purifications with sample addition as the only manual handling step. Thus, the user is available for parallel tasks during 95 % of the overall process time (33 min). In contrast manual processing requires the user to be present for 18 minutes out of the 33 minute overall process time. Since LabTube automation requires no investment in a special lab automation device, this platform lowers the market entry barrier for lab automation.
D. Kosse, F. Schwemmer, D. Buselmeier, R. Zengerle, F. von Stetten
Druckluftunterstützes Thermodiffusionsbonden zur Herstellung dünnwandiger Lab-on-a-Chip Kartuschen mit zwei fluidischen Ebenen
2013 Microsystemtechnik (MST) Kongress 2013, Aachen, 14. - 16.10.2013 , Seiten: 697 - 700
KurzfassungMikrothermoformen von Folienmaterialien ist eine neue sowie attraktive Technologie zur Herstellung dünnwandiger Lab-on-a-Chip Testträger. Der hier vorgestellte Prozess erlaubt das thermische Verbinden mehrerer mikrothermogeformter Folien zur Herstellung dünnwandiger mikrofluidischer 3D-Strukturen. Unter Verwendung von Druckluft wird der Folienstapel bestehend aus zwei mikrothermogeformten Folien außen und einer unstrukturierten Mittelfolie zusammengepresst. Diese Vorgehensweise ermöglicht den Verzicht auf aufwendig strukturierte Siegelwerkzeuge und garantiert eine gleichmäßige und Geometrie-unabhängige Einleitung der Siegelkraft. Die Herstellbarkeit dünnwandiger Testträger mit zwei fluidischen Ebenen wird anhand einer LabDisk demonstriert.
O. Strohmeier, M. Rombach, D. Mark, R. Zengerle, G. Roth, F. von Stetten
Erzeugung von Verdünnungsreihen auf einer Laborzentrifuge
2013 Microsystemtechnik (MST) Kongress 2013, Aachen, 14. - 16.10.2013 , Seiten: 353 - 356
KurzfassungVorgestellt wird ein neuartiges Verfahren zur automatischen Erzeugung von Verdünnungsreihen auf einer mikrofluidischen Kartusche. Die fluidische Prozessierung erfolgt lediglich durch definierte Rotation der Kartusche in einer Standard-Laborzentrifuge. Integrierte, aktiv gesteuerte Ventile sind hierbei nicht erforderlich. Das Verdünnungs-verhältnis wird lediglich durch das Volumen der zu verdünnenden Flüssigkeit bestimmt. Als Anwendungsbeispiel wird die Erzeugung von 5 Verdünnungsstufen aus Fluorescein in PBS Puffer in den Verhältnissen 1:3 und 1:5 gezeigt und anschließend die Reproduzierbarkeit mittels Fluoreszenzmessung gezeigt.
K. Mitsakakis, S. Hin, O. Strohmeier, D. Mark, F. von Stetten, R. Zengerle
Fully-automated point-of-care detection of malaria and other infectious diseases with a disc-shaped diagnostic platform
2013 Second WHO Global Forum on Medical Devices Duration, Geneva, Switzerland, 22-24 November 2013
KurzfassungMalaria is one of the highest mortality rate infectious diseases globally, mainly prevalent in sub-saharan Africa. Other diseases like dengue, pneumonia, typhoid fever are also present in the same areas, and, although they emerge from different pathogenic agents, they exhibit the same clinical symptom (acute fever). This makes reliable diagnosis extremely challenging especially due to the low resource nature of the endemic regions. Under these circumstances, existing diagnostic methods are often not efficient enough and unable to provide a generic solution: (i) blood smear microscopy is only malaria-specific; (ii) lateral flow Rapid Diagnostic Tests (RDTs) are cheap but single-target specific; (iii) existing molecular methods (e.g., PCR, ELISA) and/or pathogen cultivation require expensive equipment, well-trained users and long time-to-result. The presently suggested technology aims to provide a true point-of-care diagnostic platform, by addressing key application-oriented needs: (i) Portability and autonomous use, based on a disc-shaped plastic disposable cartridge (LabDisk) capable of handling liquid sample (blood) via centrifugal forces operated by a CD-player-like device (LabDisk Player). (ii) Full automation from sample collection to result, via a simple blood transfer device (patient-to-system interface) and on-disc integration of all biochemical components needed for the blood-based pathogen identification (e.g., molecular probes, buffers, etc). (iii) Rapid analysis, by using time-saving analytical protocols based on immunoassays and isothermal nucleic acid amplification (LAMP, instead of PCR). (iv) Multiplexity, by combining a broad diagnostic panel on the same disc (parasites, viruses, bacteria). The panel is flexible and can be tailored to the geographic-specific diseases. (v) Low-cost fabrication technology based on microthermoforming of thin polymer foils, adaptable from pharmaceutical and food package production. This work is part of the EU FP7 project DiscoGnosis, financed by the European Commission which is acknowledged, as well as all the consortium members for their contribution.
G. Czilwik, J. Jin, G. Roth, S. K. Vashist, T. van Oordt, O. Strohmeier, F. von Stetten, R. Zengerle, D. Mark
Immunoassay auf der LabDisk Plattform auf Basis einer Grundoperation zum Transfer magnetischer Partikel
2013 Microsystemtechnik (MST) Kongress 2013, Aachen, 14. - 16.10.2013 , Seiten: 406 - 408
KurzfassungWe present a novel technology for automated processing of immunoassays on our centrifugal-microfluidic LabDisk platform. Here, magnetic beads acting as a mobile phase are subsequently transported through liquid buffers by inter-play of magnetic- and centrifugal forces. Compared to state of the art technologies, the main advantage are short pro-cessing times, assay automation in a portable point-of-care player (LabDisk Player prototype) and a modular microflu-idic design. For demonstration of the automated processing scheme, we chose a magnetic immunoassay for quantifica-tion of human c-reactive protein (h-CRP). Due to a 1-step kinetics reaction of all immunoassay reagents and by only applying two successive washing steps the process time was only 20 minutes. Therefore the test fulfills the require-ments for rapid point-of-care diagnostics.
M. Keller, G. Czilwik, T. van Oordt, O. Strohmeier, J. Drexler, D. Mark, D. Kosse, N. Paust, R. Zengerle, F. von Stetten
LabDisk – novel centrifugal microfluidic unit operations for sample-to-answer analysis and Microfluidic Apps
2013 9. Jahrestagung des Arbeitskreises Mikrosysteme für Biotechnologie und Lifesciences e.V, Jena, 18. - 20.06.2013
A.R. Fiebach, S. Zhang, L. Drechsel, A. Kloke, N. Fritzemeier, D. Wulff, J. Steigert, R. Zengerle, F. von Stetten, N. Paust
LabTube – an innovative platform for assay automation in laboratory centrifuges
2013 Microsystemtechnik (MST) Kongress 2013, Aachen, 14. - 16.10.2013 , Seiten: 27 - 30
KurzfassungIn this paper we present the innovative LabTube platform: A fully automated, easy to handle and cost-effective system that automatizes diverse assays on standard laboratory centrifuges and thus offers a reasonable alternative to existing lab-on-a-chip systems. The LabTube has the size of a 50 ml centrifuge tube and is composed of three stacked revolvers that are operated by a centrifuge-triggered ball-pen mechanism. The upper revolver provides the reagent pre-storage, the middle revolver comprises the assay-specific fluidic unit operations and the lower revolver functions as a unit for the collection and separation of the processed reagents. To demonstrate the functionality of the LabTube on the basis of a frequently used assay, a DNA extraction was performed. The fully automated DNA extraction from rapeseed lysate in the LabTube resulted in a DNA yield of 17,7 +/- 8,1 ng μl -1 in comparison to a manual reference with 14,8 +/- 1,5 ng μl -1. Thus, the hands-on time was reduced from 15 minutes to 1 minute by employing the LabTube, without the need for special training or special laboratory equipment. Currently, the LabTube is also under investigation for protein analysis and for point-of-care diagnostic.
M. Keller, J. Naue, P. Papireddy Vinayaka, O. Strohmeier, D. Mark, U. Schmidt, R. Zengerle, F. von Stetten
Microfluidic APP featuring nested PCR for forensic screening assay on off-the-shelf thermocycler
2013 MicroTAS 2013, Freiburg, 27.-31.10.2013 , Seiten: 320 - 322
J. Liebeskind, A. Kloke, A. R. Fiebach, F. von Stetten, R. Zengerle, N. Paust
Mixing by on-chip generated gas bubbles for assay automation in standard laboratory centrifuges
2013 MicroTAS 2013, Freiburg, 27.-31.10.2013 , Seiten: 967 - 969
O. Strohmeier, S. Laßmann, B. Riedel, D. Mark, G. Roth, M. Werner, R. Zengerle, F. von Stetten
Multiplex SNP Genotyping of Tumorcell DNA for KRAS mutations by allele specific real-time PCR on a centrifugal microfluidic disk segment “GeneSlice”
2013 7. Senftenberger Innovationsforum Multiparameteranalytik, 18. - 19.04.2013
KurzfassungPoint Mutations (SNP) on the KRAS oncogene have been identified as an important predictive biomarker for response to EGFR-targeted therapy of colorectal carcinomas. Since KRAS mutations are prevalent in up to 40% of all colorectal carcinomas, routinely conducted KRAS genotyping is becoming mandatory to predict therapy success and to reduce therapy costs by avoiding ineffective treatment. We demonstrate a low-cost, disposable centrifugal microfluidic cartridge “GeneSlice” for the parallel detection of the seven most relevant KRAS point mutations by allele-specific real-time PCR, a recently discussed cost effective alternative technique to dideoxy-sequencing. Microfluidic processing of the GeneSlices as well as allele-specific amplification and real-time detection are conducted in a slightly modified, commercially available PCR thermocycler, requiring only minor hands on time. Intra-chip standard deviation of Cq values on the GeneSlices was negligible. In 23/24 experiments, DNA from 6 cancer cell lines (n = 4 per cell line) was genotyped correctly and concordant with dd-sequencing. Additionally, DNA derived from microdissected formalin-fixed and paraffin embedded colorectal carcinomas of two cases was genotyped correctly and reproducibly (n = 3 per patient; 1 GeneSlice excluded from evaluation). The GeneSlice therefore clearly demonstrates the potential to become a valuable tool for routine diagnostics of KRAS mutations by reducing costs and hands-on time.
O. Strohmeier, S. Laßmann, B. Riedel, M. Werner, D. Mark, R. Zengerle, F. von Stetten
Multiplex detection of KRAS point mutations from tumor cell DNA on a centrifugal microfluidic cartridge (GeneSlice) for choice of personalized cancer therapy
2013 MicroTAS 2013, Freiburg, 27.-31.10.2013 , Seiten: 1815 - 1817
S.K. Vashist, G. Czilwik, F. von Stetten, R. Zengerle
Rapid Immunodiagnostic Kits based on proprietary 1-step chemistry for covalent and leach-proof antibody immobilization
2013 Microsystemtechnik (MST) Kongress 2013, Aachen, 14. - 16.10.2013 , Seiten: 384 - 387
KurzfassungWe have developed a novel chemistry for the 1-step immobilization of antibodies on solid-substrate bioanalytical platforms. It enables the development of rapid immunodiagnostic (ID) kits that are analytically superior to the commercial ID kits using conventional antibody immobilization procedure. The developed chemistry is low-cost as it simply involves the dilution of capture antibodies in a particular concentration of 3-aminopropyltriethoxysilane (APTES), which is in fact cheaper than the phosphate buffered saline. It enables the leach-proof covalent binding of capture antibodies in just 30 min, which is >20-fold and >4-fold more rapid than the conventional (used in commercial ID kits) and our previously developed immobilization procedures, respectively. The immobilization chemistry was employed for the developed of rapid ID kit, based on sandwich enzyme-linked immunosorbent immunoassay (ELISA), to detect human Creactive protein (hCRP) in the dynamic range of 15.6-4000 pg/mL. The developed ID was very highly-sensitive and can detect the entire pathophysiological range of CRP in human serum after appropriate sample dilution. The developed chemistry has very high commercial potential as it will enable the development of very highly-sensitive ID kits for clinical, industrial and bioanalytical applications.
A. Kloke, L. Drechsel, S. Zhang, A.R. Fiebach, N. Paust, R. Zengerle, F. von Stetten
The LabTube Platform – Disposable Cartridges For Automated Processing Of Biochemical Assays In Standard Laboratory Centrifuges
2013 21st annual international BIODETECTION TECHNOLOGIES, Washington, DC, USA, 18-19.6. 2013
KurzfassungA laboratory centrifuge can be applied to automate biochemical assays for point-of-care diagnostics or sample preparation such as DNA or protein extraction. Key innovation is integration of liquid handling into a 50 ml centrifuge tube. This "LabTube" harbors three revolvers which are stepwise rotated against each other by a g-force operated ball pen mechanics. The first revolver sequentially releases pre-stored reagents into the second revolver which is equipped with a mixing chamber and a solid phase column. Fractions of processed liquids are collected by the third revolver. Automated LabTube based DNA-extractions showed comparable yields to manual reference extractions.

2012

T. van Oordt, O. Strohmeier, D. Mark, R. Zengerle, M. Eberhard, J. Drexler, P. Patel, M. Weidmann, A. Zgaga-Griesz, W. Bessler, F. von Stetten
The LabDisk - A Fully Automated Centrifugal Lab-on-a-Chip System for the Detection of Biological Threats
2012 7th Security Research Conference, Future Security 2012, Bonn, Germany , Seiten: 220 - 223
D. Mark, T. van Oordt, O. Strohmeier, G. Roth, N. Paust, D. Kosse, J. Drexler, M. Eberhard, F. von Stetten, R. Zengerle
A Functional Blister-Pack LabDisk System for Point of Care Testing
2012 Forum Biotechnologie Baden-Württemberg 19. September 2012, Freiburg
T. van Oordt, O. Strohmeier, D. Mark, D. Kosse, G. Roth, K. Achazi, P. Patel, S. Linke, N. Paust, M. Weidmann, J. Drexler, F. Hufert, R. Zengerle, M. Eberhard, M. Niedrig, F. von Stetten
A Functional Blister-Pack LabDisk System for Point of Care Testing
2012 64th Annual Meeting of the German-Society-for-Hygiene-and-Microbiology (DGHM) Location: Hamburg, GERMANY Date: SEP 30-OCT 03, 2012 Int. J. of Med. Microbiol., Band: 302, Ergänzungsband: 1, Seite: 7-7
D. Mark, T. van Oordt, O. Strohmeier, G. Roth, J. Drexler, M. Eberhard, M. Niedrig, P. Patel, A. Zgaga-Griesz, W. Bessler, M. Weidmann, F. Hufert, R. Zengerle, F. von Stetten
Automated and miniaturized detection of biological threats with a centrifugal microfluidic system
2012 Smart Biomedical and Physiological Sensor Technology IX Baltimore, Maryland, USA | April 23, 2012 Proc. SPIE 8367
O. Strohmeier, B. Kanat, D. Bär, P. Patel, J. Drexler, T. van Oordt, G. Roth, D. Mark, R. Zengerle, F. von Stetten
DNA based sample-to-answer analysis on a centrifugal microfluidic foil cartridge
2012 MicroTAS 2012, Okinawa/Japan, 28.10. - 01.11.2012 , Seiten: 779 - 781
P. Patel, M. Weidmann, K. Achazi, S. Linke, O. Strohmeier, D. Mark, T. van Oordt, J. Drexler, M. Eberhard, F. von Stetten, M. Niedrig
Development of Rapid Diagnostic Platform for Detection of Category A Biothreat Pathogens in the Field
2012 ASM Biodefense & Emerging Diseases, Washington, MD, USA, February. 26-29, 2012
KurzfassungIncreased threat of infectious diseases and bioterrorist incidents pose danger to the human population. As a preparedness of such incidents, a rapid detection system for identification of biothreat agents is needed. The S.O.N.D.E. project intends to develop a suitcase size platform for the detection of category A biothreat pathogens by nucleic acid and antigen detection. One subproject is dealing with the development of molecular methods such as enrichment of pathogens and nucleic acid isolation. These assays are designed to be used in an integrated bio-analytical system being developed by our S.O.N.D.E. project partners for rapid detection of a number of pathogens and toxins. To concentrate enveloped and non-enveloped viruses from large volume biological samples, an enrichment method based on polymer was developed. Viruses captured by polymer were successfully analyzed for nucleic acid detection, virus infectivity assay and electron microscopy. Also the nucleic acid purification using paramagnetic beads was successfully developed and tested on different model pathogens (gram+ and gram- bacteria, RNA and DNA viruses). Purified nucleic acid was detected by real-time PCR and an isothermal amplification method for different pathogens in spiked samples and the limit of detections was 10-100 genome copies/ml. Centrifugal microfluidic platform and portable device have been developed by our project partners for the detection of biothreat pathogens in automated “sample to answer”-platform. Developed methods like nucleic acid isolation and isothermal amplification were integrated into this platform. Furthermore, this platform is going to be evaluated and compared with conventional laboratory methods as well as commercial available rapid diagnostic platforms.
Y. Abbas, J. Miwa, R. Zengerle, F. von Stetten
Development of an active micromixer using an external mechanical actuator array
2012 1st International Conference on Microfluidic Handling Systems, October 2012, Enschede, The Netherlands , Seiten: 34 - 37
KurzfassungWe present an active continuous-flow micromixer based on channel-wall deflection in a polydimethylsiloxane (PDMS) chip using Braille display pins. The chip design comprises a main microchannel connected to a series of side channels with dead ends aligned on the Braille pins. Computercontrolled deflection of the side-channel walls induces chaotic advection in the main-channel, which substantially accelerates mixing in low-Reynolds number flow. Several influencing parameters such as the number of cross-channels, actuation frequency, side-channel width, actuation sequence, and flow rate velocities have been investigated. Sufficient mixing of fluids could be achieved within seconds (~500 ms). Finally, continuous dilution of yeast cell sample by a ratio down to 1:10 is successfully demonstrated.
J. Hoffmann, S. Hin, F. von Stetten, R. Zengerle, G. Roth
Fabricating DNA Microarrays By Copying A Next Generation Sequencing Chip
2012 MicroTAS 2012, Okinawa/Japan, 28.10. - 01.11.2012 , Seiten: 1528 - 1530
F Stumpf, D Mark, G Roth, R Zengerle, F von Stetten
Lab-on-a-chip disposable cartridges for DNA purification and genotyping processed in standard laboratory instruments
2012 Biosensors 2012; Cancun, Mexico , Seite: P2.62
S. Wadle, O. Strohmeier, M. Rombach, D. Mark, R. Zengerle, F. von Stetten
LabDisk integrated DNA Extraction from Whole Blood using Magnetic Particles
2012 MicroTAS 2012, Okinawa/Japan, 28.10. - 01.11.2012 , Seiten: 1381 - 1383
M. Trotter, F. von Stetten, G. Roth, R. Zengerle, J. Hoffmann
Massively-parallel digital solid-phase PCR for the in-situ generation of a sequencing-ready picowell array circumventing emPCR
2012 Digital PCR Conference, San Diego, USA, 15.10. – 16.10.2012
R. Zengerle, J. Hoffmann, G. Roth, O. Strohmeier, A. R. Fiebach, L. Drechsel, S. Zhang, A. Kloke, N. Paust, D. Mark, F. von Stetten
Microfluidic Apps on standard Lab-instruments
2012 MicroTAS, Okinawa, Japan, 28. Okt. - 01. Nov. 2012 , Seiten: 239 - 241
KurzfassungHere we promote a new way of thinking: Designing microfluidic chips that can be processed on standard instruments which are already present in typical laboratory setups. Those instruments could be standard lab centrifuges, real-time PCR cyclers or even second generation sequencers. We call this approach the Microfluidic App approach
S. Zehnle, M. Rombach, F. von Stetten, R. Zengerle, N. Paust
Microfluidic centrifugo-pneumatic siphon enables fast blood plasma extraction with high yield and purity
2012 MicroTAS 2012, Okinawa/Japan, 28.10. - 01.11.2012 , Seiten: 869 - 871
J. Burger, O. Stoevesandt, J. Hoffmann, F. von Stetten, R. Zengerle, G. Roth, F Stumpf
Microfluidic hand-helds for DNA to protein microarray replication by cell-free systems
2012 Biosensors 2012; Cancun, Mexico , Seite: P3.141
F. Schwemmer, S. Zehnle, N. Paust, C. Blanchet, D. Svergun, F. von Stetten, R. Zengerle, D. Mark, M. Rössle
SAXS-LabDisk: A centrifugal microfluidic screening platform for protein structure analysis
2012 EMBL Conference Microfluidics 2012, Heidelberg, Germany, July 25-27 , Seite: 186
KurzfassungA small angle x-ray scattering (SAXS) screening platform for protein structure analysis based on centrifugal microfluidics is presented. SAXS can be used to reconstruct low-resolution structures of macromolecules directly from solution scattering. This enables screening for conformational changes of proteins due to different environmental conditions. However, for state-of-the art techniques, consumption of the protein samples is still at least 6 µl per condition and the time per measurement is at best 3 min. In most cases this still renders multi-parameter screening impractical. We present a centrifugal microfluidic platform capable of decreasing both, time per measurement and consumed protein volumes in small angle scattering screenings by more than one order of magnitude. During an automated rotational protocol the presented compact disk prepares 20 different screening conditions for each protein using 2 µl protein solution, 3 µl buffer and 3 µl screening solution. The three input solutions are split into 40 nl aliquots, the aliquots are then combined at predefined ratios, mixed and can be measured on chip. Up to seven independent protein screenings can be performed on one chip. Protein solution (2 µl), screening reagent (3 µl) and buffer solution (3 µl) are pipetted in the disk. During an automated rotational protocol the liquids are split into 120 aliquots of 40 nl each. Then 6 of these aliquots are combined, respectively. This results in 20 mixtures of different predefined ratios. One disk has enough space for seven dilution matrices or 140 experiments, which can be performed on chip within a SAXS beamline. Including positioning within the beam, the expected time per measurement is less than 5 s. The SAXS-LabDisk will, for the first time, enable routine SAXS screening of minute protein volumes. The performance of the SAXS-LabDisk for protein structure determination will be evaluated at the beamline PETRA-III at EMBL Hamburg, Germany later this year.
M. Rombach, S. Lutz, C. Dumschat, A. Witt, S. Hensel, S. Frenzel, F. Aßmann, F. Gehring, T. Reiner, H. Drechsel, P. Szallies, D. Mark, G. Roth, R. Zengerle, F. von Stetten
Sample-to-Answer LabDisk for Point-of-care Analysis of Total Cholesterol from Whole Blood
2012 MicroTAS 2012, Okinawa/Japan, 28.10. - 01.11.2012 , Seiten: 782 - 784
T. van Oordt, O. Strohmeier, D. Mark, R. Zengerle, M. Eberhard, J. Drexler, F. von Stetten
The LabDisk – a fully automated point-of-care system for the detection of biological threats
2012 7. Lab-on-a-Chip World Congress in San Diego, USA, 25.09. – 26.09.2012
D. Mark, O. Strohmeier, T. van Oordt, G. Roth, D. Kosse, R. Zengerle, Felix von Stetten
The centrifugal microfluidic LabDisk platform for the automation of nucleic acid analysis and immunoassays
2012 EMBL Conference Microfluidics 2012, Heidelberg, Germany, July 25-27 , Seite: 66

2011

Jürgen Burger, David Lämmle, Felix von Stetten, Oda Stoevesandt, Michael J Taussig, Roland Zengerle, Günter Roth
A hand-held DNA to protein microarray translation system
2011 Book of Abstracts of Functional Genomics, Frankfurt am Main, 03.-04.02.2011 , Seiten: 14 - 16
Jürgen Burger, David Lämmle, Felix von Stetten, Oda Stoevesandt, Michael J Taussig, Roland Zengerle, Günter Roth
A hand-held device to copy DNA to protein microarrays
2011 Proceedings of 2011 International Conference on Microtechnologies in Medicine and Biology Lucerne, Switzerland, 4-6 May , Seiten: 127 - 128
KurzfassungThe DNA Array to Protein Array (DAPA) system presented in 2008 by He et al. [1,2] allows to replicate protein microarrays from one master DNA microarray by cell-free expression and diffusion. We improved the basic idea by designing a hand-held microfluidic device replacing the porous membrane used in [1,2] by a microfluidic gap of ~50 μm in height. Hence any mechanical damage inflicted by the membrane is prevented as only the cell free system is in physical contact with the microarrays. The capillary priming of the microfluidic gap leads to a defined protein expression start. Protein translation and microarray formation only needs 30 minutes at 25 °C, the amount of cell free system needed is reduced significantly. The DNA master array can be re-used for multiple protein expressions.
S. Lutz, D. Mark, G. Roth, R. Zengerle, F. von Stetten
Centrifugal microfluidic platforms for molecular diagnostics
2011 EuroMedLab Ber lin 2011 – Berlin, 15-19 May 2011 Clinical Chemistry and Laboratory Medicine, Band: 49, Ergänzungsband: 1, Seiten: 608 - 608
M. Focke, O. Strohmeier, P. Reith, G. Roth, D. Mark, R. Zengerle, F. von Stetten
Centrifugo-thermopneumatic liquid actuation for microfluidic genotyping of nucleic acids
2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS), October 2-6, 2011, Seattle, Washington, USA , Seiten: 659 - 661
KurzfassungWe report a novel principle to actuate liquids based on centrifugo-thermopneumatic effects. This approach enables us to reliably realize temperature-controlled valves and aliquoting structures on the centrifugal microfluidic platform. We present special geometries that force liquids to generate gas entrapments when exposed to a centrifugal field. The gas entrapments expand (or contract) when heated (or cooled) thus displacing the liquid volumes. Successful implementation of this principle is demonstrated by a microfluidic chip for automated real-time PCR based genotyping of nucleic acids.
M. Karle, G. Czilwik, J. Miwa, N. Paust, G. Roth, R. Zengerle, F. von Stetten
Continuous microfluidic DNA purification for online monitoring and process control
2011 Proceedings of 2011 International Conference on Microtechnologies in Medicine and Biology Lucerne, Switzerland, 4-6 May , Seiten: 197 - 198
KurzfassungWe demonstrate the first continuous microfluidic platform for flow-through DNA purification from cell lysate. Using superparamagnetic beads, DNA is continuously transported across interfaces between co-flowing laminar streams of extraction reagents. In on-chip experiments DNA was continuously purified over a time period of 110 min. After the on-chip purification, DNA content and purity of the eluate sampled at different stages of the continuous extraction experiment was analyzed off-chip via qPCR. Results show successful flow-through purification with constant output over almost the complete duration of the experiment. Possible applications are seen in biological safety and environmental monitoring or bioprocess control.
T. van Oordt, D. Mark, R. Zengerle, M. Eberhard, M. Niedrig, F. von Stetten
Development of a Fully Automated Centrifugal Lab-on-a-Chip System for Rapid Field Testing of Biological Threats
2011 Proc. of Future Security 2011, Berlin, Germany, September 5-7
KurzfassungThe world’s growing mobility, mass tourism and the threat of terrorism increase the risk of the fast spread of infectious microorganisms and toxins. Today’s procedures for pathogen detection involve complex, stationary devices. Their use is often too time consuming for a rapid and effective response. Therefore a robust and mobile diagnostic system is required. We are presenting a microstructured Lab-on-a-Chip test carrier which performs complex biochemical analysis and a mobile centrifugal microfluidic device to process this test carrier. This hand-held system will allow rapid automated detection of biological threats at the point of need.
Fabian Stumpf, Junichi Miwa, Nils Paust, Felix von Stetten, Roland Zengerle, Günter Roth
Development of segmented-flow microfluidic operations for single-cell nucleic acid analysis
2011 45. Deutschen Gesellschaft für Biomedizinische Technik (DGBMT), September 27-30, Freiburg
KurzfassungThe importance of single cell analysis is rapidly growing especially in the fields of biological and medical research. A cost- and time-efficient technology for handling and analyzing individual cells instead of averaging whole culture popu-lations is extremely attractive for applications like drug development, pathological screening, cancer development and many others. Segmented-flow microfluidics is thought to be highly suitable for this purpose, considering the ability to handle a series of minute reaction compartments at moderate-to-high throughputs. Our on-going development of segmented-flow microfluidic devices aims for the realization of an integrated, automated single-cell nucleic acid analysis system with complete sample preparation capabilities. Although some of the required unit operations such as droplet mixing and thermocycling are already fairly established in this field, key procedures such as accurate single-cell encapsulation and nucleic acid purification are still missing.
Dominique Kosse, Dirk Buselmeier, Claas Müller, Roland Zengerle, Felix von Stetten
Druckluft-unterstütztes thermisches Deckeln folienbasierter Lab-on-a-Chip Kartuschen
2011 Mikrosystemtechnik-Kongress 2011, Darmstadt, Deutschland, October 10-12, 2011 , Seiten: 579 - 582
KurzfassungPräsentiert wird ein druckluft-unterstütztes thermisches Bondverfahren zum Deckeln dünnwandiger folienbasierter Lab-on-a-Chip Kartuschen (Lab-on-a-Foil) [1-3]. Das Verfahren verzichtet auf eine aufwendige Siegelaufnahme zur Siegelkrafteinleitung und ermöglicht eine auch unter thermischer Belastung stabile Deckelung von Lab-on-a-Foil Kartuschen. Besonders variotherme mikrofluidische Anwendungen mit schnellen Temperaturwechseln (z.B. Polymerase-Kettenreaktion) [4] profitieren von den dünnen Wandstärken und der temperaturstabilen Deckelung. Die Herstellung der Lab-on-a-Foil Kartuschen erfolgt durch Mikrothermoformen von Foliensubstraten, die anschließend gedeckelt werden. Die vorgestellte Deckelungsmethode nutzt Druckluft zur Einleitung der benötigten Siegelkraft. Als Siegelfolie wird eine co-extrudierte COC-Folie eingesetzt. Diese besteht aus einer Temperatur stabilen COC 6013-Trägerschicht und einer nieder-schmelzenden COC 8007 Siegelschicht. Experimentell konnte eine Druckstabilität der Siegelverbindung bis 500 kPa bei Raumtemperatur und bis zu 150 kPa unter Temperaturbelastung von 95°C gezeigt werden.
J. Hoffmann, S. Hin, F. von Stetten, R. Zengerle, G. Roth
Enabling DNA-microarrays in polymeric lab-on-a-chip substrates for multiplexed target analysis via solid-phase PCR
2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS), October 2-6, 2011, Seattle, Washington, USA , Seiten: 1840 - 1842
KurzfassungA novel universal protocol is demonstrated for covalent grafting of solid-phase PCR primers oriented onto glass and various polymers (COP, PP, COC, PDMS) relevant for Lab-on-a-chip (LoaC) applications. Primers immobilized in a DNAmicroarray format feature spots with high homogeneity and integrity. PCR compatibility of the novel immobilization protocol was confirmed by applying such arrays to solid-phase PCR (SP-PCR), improving previously reported “enhanced SPPCR” [1] in terms of factorial signal increase from 9.9 to 86.8 and specificity from 11.7 to 45.9. Our method also enables to directly integrate DNA-microarrays amenable for SP-PCR into microfluidic LoaC cartridges of various materials circumventing the need of hybrid assemblies.
O. Strohmeier, M. Rombach, D. Mark, R. Zengerle, G. Roth, F. von Stetten
Fully integrated dilution series generation on a laboratory centrifuge
2011 16th International Solid-State Sensors, Actuators and Microsystems Conference (TRANSDUCERS), 5-9 June 2011, Beijing China , Seiten: 2952 - 2955
KurzfassungWe present a novel approach for the fully automated generation of dilution series using a disposable microfluidic cartridge. Fluids are metered, mixed and routed without active valving by defined rotational speed only. As a processing device, a standard lab centrifuge can be used. The dilution ratio can be freely choosen by loading the corresponding volume of sample into the cartridge without the need for changes in the microfluidic layout. Dilution series of Fluorescein in PBS buffer with ratios of 1:3 and 1:5 are shown with each dilution series comprising 5 single dilution steps. Reproducibility was determined by fluorescence measurement.
J. Burger, André Gross, Daniel Mark, Felix von Stetten, Roland Zengerle, Günter Roth
IR thermocycler for centrifugal microfluidic platform wit direkt on-disk wireless temperature measurement system
2011 Mikrosystemtechnik-Kongress 2011, Darmstadt, Deutschland, October 10-12, 2011 , Seiten: 916 - 918
KurzfassungWe present a novel infrared (IR) thermocycler for centrifugal microfluidic platforms [1]. It allows to directly heat liquids in reaction containers within microfluidic polymer film disks [2] for polymerase chain reactions (PCR) with average heating gradients of up to 4 K/s. This thermocycler mainly consists of an IR ring-heater, a direct on-disk wireless temperature measurement system with a resolution of 0.1 K and an embedded adaptive PID controller. That enables to precisely control the temperature of the liquid even at variant conditions e.g. manufacturing tolerances, different cavity locations, ambient temperature changes, whereas state-of-the-art thermocyclers with in-direct air temperature measurement are inflexible due to static thermal energy flow models. Hence our system has the potential for a higher efficiency, accuracy and robustness, thus for a better PCR reproducibility.
J. Burger, A. Gross, D. Mark, F. von Stetten, R. Zengerle, G. Roth
IR thermocycler for centrifugal microfluidic platform with direct on-disk wireless temperature measurement system
2011 16th International Solid-State Sensors, Actuators and Microsystems Conference (TRANSDUCERS), 5-9 June 2011, Beijing China , Seiten: 2867 - 2870
KurzfassungWe present an infrared (IR) thermocycler with closed loop temperature control for performing fast polymerase chain reactions (PCR) in centrifugal microfluidics [1]. It consists of an IR ring heater and an on-disk wireless temperature sensor module with a resolution of 0.1 K [2]. The closed loop system enables to precisely control the temperature of the reagents even at varying conditions e.g. manufacturing tolerances of the polymer film disks [3], different locations of the cavities, ambient temperature changes. Due to the direct heating of the reagents by IR absorption we achieve fast average heating gradients of up to 4 K/s. Average cooling gradients so far are limited to 1.3 K/s. Our system is superior in terms of energy efficiency, temperature accuracy and overall reproducibility and robustness.
J. Burger, A. Gross, D. Mark, G. Roth, F. von Stetten, R. Zengerle
IR thermocycler for centrifugal microfluidic platform with direct ondisk wireless temperature measurement system
2011 Smart Sensors, Actuators, and MEMS V, 18th April 2011, Prague, Czech Republic , Seite: p. 80661X
KurzfassungThe direct on-disk wireless temperature measurement system [1,2] presented at μTAS 2010 was further improved in its robustness. We apply it to an IR thermocycler as part of a centrifugal microfluidic analyzer for polymerase chain reactions (PCR). This IR thermocycler allows the very efficient direct heating of aqueous liquids in microfluidic cavities by an IR radiation source. The efficiency factor of this IR heating system depends on several parameters. First there is the efficiency of the IR radiator considering the transformation of electrical energy into radiation energy. This radiation energy needs to be focused by a reflector to the center of the cavity. Both, the reflectors shape and the quality of the reflecting layer affect the efficiency. On the way to the center of the cavity the radiation energy will be diminished by absorption in the surrounding air/humidity and especially in the cavity lid of the microfluidic disk. The transmission spectrum of the lid material and its thickness is of significant impact. We chose a COC polymer film with a thickness of 150 μm. At a peak frequency of the IR radiator of ~2 μm approximately 85 % of the incoming radiation energy passes the lid and is absorbed within the first 1.5 mm depth of liquid in the cavity. As we perform the thermocycling for a PCR, after heating to the denaturation temperature of ~ 92 °C we need to cool down rapidly to the primer annealing temperature of ~ 55 °C. Cooling is realized by 3 ventilators venting air of room temperature into the disk chamber. Due to the air flow itself and an additional rotation of the centrifugal microfluidic disk the PCR reagents in the cavities are cooled by forced air convection. Simulation studies based upon analogous electrical models enable to optimize the disk geometry and the optical path. Both the IR heater and the ventilators are controlled by the digital PID controller HAPRO 0135 [3]. The sampling frequency is set to 2 Hz. It could be further increased up to a maximum value being permitted by the wireless temperature data transmission system. As we are controlling a significantly non-linear process the controller parameters need to be optimized for all temperatures relevant for the PCR thermocycling process. Such we get a dynamic system for both, the heating and the cooling process. Heating rates up to 5 K/s with our IR heater (100 W electrical power) could be achieved. Cooling rates of instantly 1.3 K/s at 20 Hz rotation frequency could be even further increased by higher rotation frequencies, faster air circulation, optimization of the controller parameters or an active air cooling unit. Keywords: lab-on-a-chip, centrifugal microfluidics, PCR, Immunoassay, thermocycling, IR radiator, thermistor, wireless temperature measurement system
Strohmeier O., Mark D., Focke S., Lutz S., Burger J., Riegger L., Hoffmann J., Zengerle R., von Stetten F.
Lab-on-a-Chip solutions designed for being operated on standard laboratory instruments
2011 45. Deutschen Gesellschaft für Biomedizinische Technik (DGBMT), September 27-30, Freiburg
KurzfassungWe report lab-on-a-chip solutions that can be operated on standard laboratory instruments. Although disposable labchips can be produced at low cost, market penetration can be hindered by initial invests for instrumentation. Therefore, we upgrade standard laboratory instruments by microfluidic disposables for process automation. This could significantly increase the acceptance of lab-chips. We present: 1. Microfluidic disks for DNA pre-amplification, aliquoting and real-time PCR, operated on a Qiagen thermocycler with < 10 copy sensitivity. 2. Fully automated hematocrit measurement in a DVD ROM drive from <10 μL of whole blood.
Maximilian Focke, Daniel Mark, Fabian Stumpf, Martina Müller, Günter Roth, Roland Zengerle, Felix von Stetten
Microfluidic cartridges for DNA purification and genotyping processed in standard laboratory instruments
2011 Smart Systems Integration 2011, Dresden, 22 – 23 March 2011 , Seite: P 40 (6pp)
KurzfassungWe present two disposable microfluidic cartridges intended for upgrading standard laboratory instruments with automated liquid handling capability by use of centrifugal forces. Both cartridges integrate and automate laboratory protocols for analysis of nucleic acids. The first microfluidic cartridge enables purification of DNA from human whole blood and is operated in a standard laboratory centrifuge. The second microfluidic cartridge enables genotyping of pathogens by geometrically multiplexed real-time PCR. It is operated in a slightly modified off-the-shelf thermal cycler. Both solutions aim at smart and cost-efficient ways to automate work flows in laboratories.
M. Focke, D. Mark, F. Stumpf, M. Müller, G. Roth, R. Zengerle, F. von Stetten
Microfluidic cartridges for DNA purification and genotyping processed in standard laboratory instruments
2011 Smart Sensors, Actuators, and MEMS V, 18th April 2011, Prague, Czech Republic , Band: 8066, Seite: Art80660G
KurzfassungTwo microfluidic cartridges intended for upgrading standard laboratory instruments with automated liquid handling capability by use of centrifugal forces are presented. The first microfluidic cartridge enables purification of DNA from human whole blood and is operated in a standard laboratory centrifuge. The second microfluidic catridge enables genotyping of pathogens by geometrically multiplexed real-time PCR. It is operated in a slightly modified off-the-shelf thermal cycler. Both solutions aim at smart and cost-efficient ways to automate work flows in laboratories. The DNA purification cartridge automates all liquid handling steps starting from a lysed blood sample to PCR ready DNA. The cartridge contains two manually crushable glass ampoules with liquid reagents. The DNA yield extracted from a 32 μl blood sample is 192 ± 30 ng which corresponds to 53 ± 8% of a reference extraction. The genotyping cartridge is applied to analyse isolates of the multi-resistant Staphyloccus aureus (MRSA) by real-time PCR. The wells contain pre-stored dry reagents such as primers and probes. Evaluation of the system with 44 genotyping assays showed a 100% specificity and agreement with the reference assays in standard tubes. The lower limit of detection was well below 10 copies of DNA per reaction. Keywords: lab-on-a-chip, lab-on-a-foil, centrifugal microfluidics, DNA purification, DNA genotyping
M. Karle, G. Czilwik, J. Miwa, N. Paust, G. Roth, R. Zengerle, F. von Stetten
Microfluidic flow-through DNA purification for continuous monitoring applications
2011 EuroMedLab Berlin 2011 – Berlin, 15-19 May 2011 Clin Chem Lab Med, Band: 49, Ergänzungsband: 1, Seiten: S607 - S607
R. Zengerle, D. Mark, D. Kosse, G. Roth, F. von Stetten
Microfluidic solutions for miniaturization, integration, automation and parallelization of tests on commercially available instruments
2011 16th International Solid-State Sensors, Actuators and Microsystems Conference (TRANSDUCERS), 5-9 June 2011, Beijing China , Seiten: 12 - 15
KurzfassungWe demonstrate that Lab-on-a-Chip implementations can be designed for operation in common commercialized instruments like laboratory centrifuges, DVD drives, or real-time PCR cyclers. On the one hand this approach could render the expensive development of tailor-made instruments superfluous and could significantly reduce market entry barriers for developing Lab-on-a-Chip solutions. On the other hand this provides instrument companies with the opportunity to upgrade their devices e.g. by integrating complex sample preparation protocols prior to the detection for which the instruments are designed. We demonstrate this approach for the following applications: • automated DNA-purification operated on a standard laboratory centrifuges with 50% yield compared to gold standards, • DNA pre-amplification, aliquoting, and real-time PCR operated on a slightly modified real-time thermocycler (Rotor-Gene) with < 10 copy sensitivity, • isothermal DNA amplification by recombinase polymerase amplification with < 20 copy sensitivity and a time-to-result of less than 15 minutes, also operated on the Rotor-Gene platform, • hematocrit measurement of less than 10 μL of whole blood in a DVD drive.
D. Kosse, M. Focke, C. Müller, F. Von Stetten, R. Zengerle
Microthermoforming and Sealing of COP Films to Form Thin Walled Lab-On-A-Chip Cartridges
2011 8th International Conference on Multi Material Micro Manufacture, Stuttgart, Germany, November 08-10 , Seiten: 187 - 190
KurzfassungThis paper describes a process chain to form and seal thermoplastic films to obtain enclosed microfluidic cartridges with thin walls. Especially applications requiring fast thermocycling such as polymerase chain reaction (PCR) highly benefit from this lab-on-a-foil approach, due to an improved heat transfer. The cartridge fabrication is a two-step process comprising microthermoforming by soft lithography followed by a novel gas pressure assisted thermal sealing. As film material a 188 μm thick cyclic olefin polymer (COP) is used, which is microthermoformed over a male Polydimethylsiloxane (PDMS) mold to shape the intended thin walled microstructures. The process parameters have been optimized and feature sizes in the range of a few micrometers to millimeters with aspect ratios up to three are replicated. The forming of the thermoplastic film is caused by heating the film above glass transition temperature (Tg) and applying a gas pressure of 310 kPa. After cooling and demolding the COP structures are thermally sealed with a special coextruded COC film. The coextruded film consists of a temperature stable TOPAS COC 6013 (Tg ~ 135 °C) layer and a thin layer of TOPAS COC 8007 (Tg ~ 79 °C) acting like hot melt. An additional mold for bond support, which is commonly needed for positive thermoformed structures, has been eliminated by a gas pressure assisted bonding approach. The processes enable fast and flexible prototyping of thin walled microfluidic cartridges within 8 – 10 hours.
G. Roth, S. Lutz, G. Welte, D. Mark, R. Zengerle, F. von Stetten
Mikrofluidisches Lab-on-a-Chip System zur Prozessierung von Immunoassays mit integrierter Probenvorbereitung
2011 Mikrosystemtechnik-Kongress 2011, Darmstadt, Deutschland, October 10-12, 2011 , Seiten: 457 - 460
KurzfassungEs wird eine Plattform zur Prozessierung von kompetitiven und Sandwich-Immunoassays präsentiert. Die Plattform be-steht aus einer spritzgegossenen mikrofluidischen Kartusche und einem Prozessierungs-Gerät mit Detektionseinheit. Die Kontrolle der Fluide erfolgt unter gezielter Kombination von Zentrifugal- und Kapillarkräften durch rein passive Elemente wie z.B. Siphons, sowie hydrophile und hydrophobe Beschichtungen. Die Kartusche ermöglicht die Separati-on von 4 µL Blutplasma aus 10 – 15 µL Vollblut, sowie Mischprozesse, Inkubations- und Waschschritte und verfügt über einen integrierten Flüssigabfallbehälter zur Vermeidung von Kontaminationen. Beispielhaft werden ein Sandwich- Immunoassay zur Analyse von IL8 und ein kompetitiver Immunoassay zur Analyse von Estradiol demonstriert. Die Detektion unter Rotation erfolgt mittels Chemilumineszenz.
Thomas van Oordt, Yannick Barb, Roland Zengerle, Felix von Stetten
Miniature stick-packaging – an industrial technology for pre-storage and release of reagents in lab-on- a-chip systems
2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS), October 2-6, 2011, Seattle, Washington, USA , Seiten: 437 - 439
KurzfassungStick-packaging of goods in tubular shaped composite-foil pouches has become a popular technology for food packaging. We miniaturized stick-packaging for use in Lab-on-a-Chip (LOAC) systems to pre-store and release liquid and dry reagents in a volume range of 80 – 500 μl. A frangible seal integrated in the package allows for the pressure-controlled release of reagents, reducing the number of downstream valves required for liquid control. The frangible seal is fabricated by ultrasonic welding, enabling adjustment of burst pressures from 20 to 100 kPa, and allowing for packaging of temperature sensitive reagents. As an additional advantage, stick-packaging is also a scalable technology suitable for both rapid prototyping and low-cost mass production. KEYWORDS: reagent pre-storage, stick-packaging, centrifugal lab-on-a-chip
Oliver Strohmeier, Nico Marquart, Daniel Mark, Günter Roth, Roland Zengerle, Felix von Stetten
Real-time PCR based food pathogen detection on a centrifugal microfluidic foil disk including positive- and no-template-controls
2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS), October 2-6, 2011, Seattle, Washington, USA , Seiten: 506 - 508
KurzfassungWe designed and evaluated a novel microfluidic structure for the real-time PCR based detection of six common food pathogens on a centrifugal microfluidic foil disk, including onboard positive- and no-template-controls for each of the targets. The microfluidic design enables for geometric multiplexed PCR in a standard centrifugal real-time PCR thermocycler. The limit of detection was 0.1 pg target DNA what corresponds to 17-56 DNA copies per PCR reaction. KEYWORDS: Lab-on-a-Chip, Centrifugal microfluidics, PCR on a chip, Food pathogen testing
J. Hoffmann, M. Trotter, F. von Stetten, R. Zengerle, G. Roth
Single-Molecule PCR in a Picowell Array Simultaneously Immobilizing PCR Products to a PDMS Coverslide
2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS), October 2-6, 2011, Seattle, Washington, USA , Seiten: 900 - 902
KurzfassungFor the first time, we amplified single DNA-molecules (“Digital PCR”) randomly distributed in a picowell array and simultaneously immobilized the generated PCR-products to the surface of a PDMS coverslide (“solid-phase PCR”) which was used as sealing of the picowells during PCR. First, by this unprecedented technique PCR-products can be recovered for both, further reactions/analysis and counting the number of initial DNA-molecules from digital signals with a good signal to noise ratio of 10. Second, our experiments demonstrate the currently smallest low-volume on-chip PCR in an array of 18.5 pL wells. This may enable single-cell PCR experiments by filling PCR microreactors with truly single-cells due to geometrical constriction. KEYWORDS: Digital PCR, solid-phase PCR, parallelization
T. van Oordt, J. Smetana, R. Zengerle, F. von Stetten
Stickpacks zur langzeitstabilen Vorlagerung von Reagenzien in Lab-on-a-Chip-Systemen
2011 Mikrosystemtechnik-Kongress 2011, Darmstadt, Deutschland, October 10-12 2011 , Seiten: 555 - 558
KurzfassungStickpacks sind eine in der Lebensmittelindustrie etablierte Methode zur Verpackung. Wir zeigen, dass diese nach einer Miniaturisierung auch zur Speicherung kleinster Flüssigkeitsvolumina (80 – 500 µl) verwendet werden können und demonstrieren dies am Beispiel einer langzeitstabilen Vorlagerung von Flüssigreagenzien in Lab-on-a-Chip-Systemen. Durch einen einstellbaren Berstdruck lassen sich die Reagenzien in den Stickpacks automatisiert freisetzen und können somit kontaktierungsfrei in zentrifugale Lab-on-a-Chip-Systeme integriert werden. Die Öffnungsdrücke sind im Bereich von 200 - 1200 mbar variierbar und die Stickpacks entleeren sich zentrifugal nahezu vollständig (99 % ± 1 %, ab Δp = 400 mbar). Die Eignung für eine langzeitstabile Lagerung konnte in beschleunigten Lager-Tests nachgewiesen werden.
O. Strohmeier, G. Czilwik, A. Emperle, M. Focke, G. Roth, D. Mark, R. Zengerle, F. von Stetten
Verfahren zur DNA Aufreinigung mit magnetischen Partikeln auf einer zentrifugal-mikrofluidischen Einweg-Foliendisk
2011 Mikrosystemtechnik-Kongress 2011, Darmstadt, Deutschland, October 10-12, 2011 , Seiten: 66 - 69
KurzfassungVorgestellt wird ein neues Verfahren zur integrierten DNA Aufreinigung mit Hilfe magnetischer Partikel auf einer zentrifugal-mikrofluidischen Foliendisk. Wesentliche Vorteile gegenüber dem Stand der Technik [1-4] sind kurze Prozesszeiten und Ausbeuten von annähernd 100% (verglichen mit der manuell durchgeführten Referenzextraktion), automatischer Betrieb bei niedrigen Rotationsfrequenzen (< 8 Hz) und ein modulares Design der mikrofluidischen Kammern. Der Transport der magnetischen Partikel zwischen den einzelnen Kavitäten erfolgt durch ein Zusammenspiel magnetischer und zentrifugaler Kräfte. Mit dem vorgestellten System konnte vollautomatisch DNA in PCR Qualität aus 3 Verdünnungen (1,5 • 104 Kolonie bildende Einheiten (KbE); 1,5 • 106 KbE und 1,5 • 108 KbE pro Probe) eines Escherichia coli Lysats in ~ 6 min extrahiert werden.
M. Focke, M. Müller, G. Roth, R. Zengerle, F. von Stetten, D. Mark, F. Stumpf
Zwei Lab-on-a-Chip-Einweg-Kartuschen zur DNA-Aufreinigung bzw. Genotypisierung in leicht modifizierten Standard-Laborgeräten
2011 Mikrosystemtechnik-Kongress 2011, Darmstadt, Deutschland, October 10-12, 2011 , Seiten: 914 - 915
KurzfassungWir präsentieren zwei unabhängige Lab-on-a-Chip-Systeme, die zum „Upgrade“ von geringfügig modifizierten kommerziellen Standard-Laborgeräten dienen. Beide Systeme erlauben die automatisierte Prozessierung von gängigen Laborprotokollen zur Extraktion beziehungsweise Analyse von Nukleinsäuren (DNA) und erweitern dadurch das Nutzungsspektrum dieser Geräte. Das erste System besteht aus einer mikrofluidischen Kartusche, die in einer programmierbaren Laborzentrifuge (Sigma 1-15, Sigma GmbH) betrieben wird und die Aufreinigung von DNA aus humanem Vollblut ermöglicht (Abb. 1). Das zweite System ist eine mikrofluidische Kartusche, die in einem leicht modifizierten Thermocycler (Rotor-Gene 2000, Corbett Research Ltd., mittlerweile Qiagen GmbH, Abb. 2) betrieben wird und ein geometrisches Multiplexing sowie anschließende Genotypisierung mittels Real-Time PCR ermöglicht. Beide Systeme sind aus Kunststoff gefertigt und sollen häufige Arbeitsabläufe in Laboren kosten-effizient automatisieren.

2010

Jürgen Burger, Thomas Jäger, André Gross, Anton Lastochkin, Daniel Mark, Günter Roth, Felix von Stetten, Roland Zengerle, Leo Reindl
Direct on-disk wireless temperature measurement for centrifugal microfluidic platforms
2010 Proc. of the 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS), Groningen, The Netherlands, October 3 – 10 , Seiten: 1502 - 1504
KurzfassungFor the first time we present an on-disk wireless temperature measurement system (Fig.1) for centrifugal microfluidic platforms[1]. Tiny thermistors with a volume of 54nl allow the direct temperature measurement in almost any of cavity (Fig.2) of microfluidic chips with a resolution of 0.1 K. A rotating radio module transfers digitalized data to a stationary receiver with D/A converter being connected to a temperature controller. The total response time of the temperature measurement system is 9.6 ms. The rotating electronic elements are powered by inductive coupling with up to 2W. The radio transmission of sensor data can be parameterized by any commercial PC via USB port. This system enables to precisely measure the temperature of fluids in centrifugal microfluidic systems and hence allows closed-loop control of direct heating systems e.g. IR radiators for fast Polymerase Chain Reaction (PCR) thermocycling processes. KEYWORDS: Lab-on-a-Chip, Microfluidic, PCR, Thermocycling, wireless temperature measurement
M. Karle, G. Czilwik, J. Miwa, N. Paust, G. Roth, R. Zengerle, F. von Stetten, Felix von Stetten
High-performance flow-through DNA purification on a microfluidic chip
2010 Proc. of the 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS), Groningen, The Netherlands, October 3 – 10 , Seiten: 106 - 108
KurzfassungWe present a significant increase in performance for flow-through purification of nucleic acids by continuous microfluidic processing. After binding to superparamagnetic beads the nucleic acids are sequentially transported across the phase-interface of co-flowing laminar streams of purification reagents. The entire purification procedure is performed within only 2 minutes. Compared to classical batch-wise purification in test tubes, 150  50 % of total DNA have been recovered in on-chip purifications over a DNA concentration range of 7 orders of magnitude. With appropriate surface modification of the magnetic beads the chip is also suggested for the implementation of other continuous biomolecular purification tasks. KEYWORDS: Nucleic Acids Purification, Magnetophoresis, Continuous Processing, Flow-Through Assay
D. Mark, S. Lutz, M. Focke, M. Müller, G. Roth, R. Zengerle, F. von Stetten
Integrierte Analytik und Diagnostik auf der zentrifugal-mikrofluidischen Bio-Disk Plattform
2010 Tagungsband des 15. Heiligenstädter Kolloquiums, 27.-29.09 2010, Heiligenstadt , Seiten: 285 - 290
KurzfassungIn der modernen Medizin gewinnen hochspezifische und sensitive Verfahren wie Nukleinsäurediagnostik oder Immunoassays zunehmend an Bedeutung. Diese beinhalten eine Vielzahl von aufwendigen Prozessierungsschritten. Sie werden daher entweder durch hochqualifiziertes Personal in langwierigen Verfahren manuell durchgeführt oder in Großlabors mit großem apparativen Aufwand automatisiert. Entsprechend lange sind die Antwortzeiten nach der Probennahme, da die aufwendige Laborarbeit bzw. das Verschicken von Proben Stunden bis Tage in Anspruch nimmt. Für eine schnelle, patientennahe Diagnose sind daher kompakte, automatisierte Geräte wünschenswert, die vor Ort mit geringem Schulungsaufwand eingesetzt werden können. Mikrofluidische Systeme sind dabei vielversprechende Kanditaten [1]. Die zentrifugale Mikrofluidik ist eine solche Entwicklungsplattform zur Miniaturisierung, Integration und Automatisierung biochemischer Analyseprotokolle [2]. Sie beruht auf der kontrollierten Prozessierung kleinster Flüssigkeitsmengen in einem zentrifugalen Beschleunigungsfeld und kann so Analyseprotokolle automatisieren. Auf Grundlage der zentrifugalmikrofluidischen Bio-Disk Plattform [2] zeigen wir die Integration grundlegender Laborprotokolle der Analytik und Diagnostik: Nukleinsäure- Aufreinigung, Nachweise basierend auf Polymerase-Kettenreaktion (PCR), sowie isotherme Amplifikationsverfahren und Immunoassays.
D. Mark, M. Focke, S. Lutz, J. Burger, M. Müller, L. Riegger, M. Rombach, J. Hoffmann, G. Roth, O. Piepenburg, Y. Park, R. Zengerle, F. von Stetten
Lab-on-a-chip solutions designed for being operated on standard laboratory instruments
2010 Eurosensors XXIV, September 5-8, Linz, Austria , Seiten: 444 - 447
KurzfassungIn this paper, we propose the development of microfluidic disposables that can be processed with standard laboratory instruments. The use of prevalent processing devices could significantly reduce existing market entry barriers for lab-on-a-chip solutions and support the market uptake of microfluidic products. We demonstrate the concept with the following applications: - microfluidic chips for DNA-purification operated on a standard laboratory centrifuge with 42% yield compared to gold standards (QIAamp, Qiagen GmbH) - microfluidic foil disk for DNA pre-amplification, aliquoting, and real-time PCR operated on a slightly modified Corbett Life Science thermocycler (now Qiagen) with < 10 copy sensitivity - microfluidic disposable for isothermal DNA amplification by recombinase polymerase amplification also operated on a Corbett Life Science (now Qiagen) thermocycler with < 10 copy sensitivity and a time-to-result of < 15 minutes. - fully automated hematocrit measurement in a DVD ROM drive from < 10 μL of whole blood.
O. Strohmeier, A. Emperle, M. Focke, G. Roth, D. Mark, R. Zengerle, F. von Stetten
Magnetic bead based DNA purification on a disposable centrifugal microfluidic foil cartridge
2010 Proc. of the 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS), Groningen, The Netherlands, October 3 – 10 , Seiten: 402 - 404
Gregor Welte, Sascha Lutz, Berit Cleven, Hero Brahms, Claudia Gärtner, Günter Roth, Daniel Mark, Roland Zengerle, Felix von Stetten
Microfluidic lab-on-a-chip system with integrated sample preparation for processing immunoassays
2010 Proc. of the 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS), Groningen, The Netherlands, October 3 – 10 , Seiten: 818 - 820
KurzfassungWe present an advanced, injection molded microfluidic lab-on-a-chip cartridge to process immunoassays with unit operations for sample preparation, metering, mixing, incubation, washing, chemiluminescence detection and waste handling. A newly developed readout device is capable of recording data points under rotation. Latest assay results feature a lower average standard deviation (7 % of maximum signal) in contrast to previously achieved 27 % [1]. A competitive chemiluminescent Estradiol immunoassay is demonstrated on-chip with a series of different Estradiol concentrations. A lower limit of detection of 60 pg/mL is established with a time-to-result of 45 min compared to 60 min in a microtiterplate.
Martina Müller, Daniel Mark, Markus Rombach, Günter Roth, Jochen Hoffmann, Roland Zengerle, Felix von Stetten
On the way to a fully integrated DNA-purification system on a standard laboratory centrifuge
2010 Proc. of the 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS), Groningen, The Netherlands, October 3 – 10 , Seiten: 405 - 408
KurzfassungFor the first time, we combine liquid reagent storage in glass capillaries [1], time-controlled reagent release [2], and a novel solution for unidirectional centrifugal routing, paving the way to a fully integrated DNA extraction on a standard laboratory centrifuge. The average yield of the extraction is 192 ± 30 ng DNA out of 32 μl blood, corresponding to 53 ± 8 % of a reference extraction. The overall processing time of ~66 minutes can be reduced to ~8 minutes after optimization of the burst-valves. This novel approach demonstrates a convenient way for fully automated DNAextraction on a standard laboratory centrifuge. KEYWORDS: Microfluidics, centrifugal routing, DNA extraction, time-controlled release, liquid reagent storage
T. van Oordt, D. Mark, M. Focke, S. Lutz, F. von Stetten, R. Zengerle
Rapid Field Testing of Biological Threats with Lab-on-a-Chip Systems
2010 5th Security Research Conference, Berlin, 07-09 September 2010 , Seiten: 256 - 257
KurzfassungThe world’s increasing mobility, mass tourism but also possible terrorist activities increase the risk of a fast distribution of infections and toxins. Today’s procedures for pathogen detection involve complex, stationary devices and may be too time consuming for a rapid and effective response. A robust, mobile field diagnostic system is required. A microfluidic system that includes a mobile centrifugal platform and a disposable test carrier enabling complex biochemical analysis is currently being developed within the BMBF funded project SONDE.
Arne Kloke, Christian Köhler, Roland Zengerle, Sven Kerzenmacher, Felix von Stetten
Reaction-specific platinum electrodes for implantable glucose fuel cells:Versatile fabrication by cyclic electrodeposition
2010 Proc. of PowerMEMS, Leuven, Belgium, December 1–3 , Seiten: 73 - 76
KurzfassungWe present a new versatile method for the generation of ultra porous noble metal electrodes based on cyclic Pt-Cu electrodeposition and anodic Cu dissolution. Performing this method, electrodes with tailored roughness factors (RF exceeding up to 2031) and thus reaction-specificity can be fabricated. Applied in implantable glucose fuel cells, the novel process enables a by 14 % increased power density compared to state of the art, at significantly reduced platinum consumption and substantially facilitated fabrication.
Phillip Kuhn, Nils Paust, Roland Zengerle, Felix von Stetten, Günter Roth
Smaller Structures Taking the Lead - Analysis and Simulation of Structure Size Influences on Binding Kinetics Down to the Single Molecule Level
2010 Proc. of IEEE-MEMS, Hong-Kong, China, January 24-28 , Seiten: 919 - 922
KurzfassungThis paper describes a method for the quantitative detection of biochemical binding events onto mi-crostructured and functional surfaces on a truly single molecular level. The classic Streptavidin-biotin system is used to provide the last detection step and the binding is visualized via gold-nanoparticles in a SEM. We showed that this allows a spatial resolution down to the nanometer scale. It also allowed us to proof the spot size dependence of binding kinetics according to the theorem of Ekins in one single experiment. The method allows to analyze any binding event on a planar surface and is enabling to measure surface densities of func-tional groups like the amount of BSA molecules on a blocked glass surface.

2009

M. Karle, J. Miwa, G. Roth, R. Zengerle, F. von Stetten
A novel microfluidic platform for continuous DNA extraction and purification using laminar flow magnetophoresis
2009 Proc. IEEE-MEMS; 25 – 29 January 2009, Sorrento, Italy , Seiten: 276 - 279
KurzfassungFor the first time we present a novel microfluidic platform using laminar-flow magnetophoresis for combined continuous extraction and purification of DNA from cells. All essential unit operations (DNA binding, sample washing and DNA elution) are integrated on one single chip. The key function is the motion of magnetic beads given by the interplay of laminar flow and a time-varying magnetic field. The magnetic beads enable the transport of the DNA across the interfaces between co-flowing laminar streams in a circular channel arrangement around a central rotating permanent magnet inducing time-varying magnetic field, which prevents the beads from sticking to the channel walls and enables controlled transfer of beads between different extraction reagents. The system was fabricated by micromilling in polycarbonate. Pressure driven experiments were performed to analyze the magnetophoretic concept and the biotechnical functionality. Compared to a macroscopic reference system 25% of total DNA have been recovered. An inlet flow velocity of 12.5 mm∙s-1 lead to an average bead velocity of 1.6 mm∙s-1 The sample transition time is approximately 1 minute. The device is a central part of a complete biochemical sensing system for continuous monitoring of cell growth in bioreactors, but allows also continuous purification of DNA, RNA, proteins or cells, including their subsequent real-time analysis.
S. Lutz, P. Weber, M. Focke, B. Faltin, C. Mueller, G. Roth, D. Mark, N. Armes, O. Piepenburg, R. Zengerle, F. von Stetten
Automated analysis of genetic markers by isothermal recombinase polymerase amplification in a centrifugal microfluidic cartridge
2009 Proc. Eurosensors XXIII, Lausanne, Schweiz , Seite: 131
KurzfassungRecombinase polymerase amplification (RPA) is a new isothermal DNA amplification method that runs at 37 °C, amplifies single copies in less than 15 minutes, and allows real-time fluorescence detection. For the first time we automated this method by microfluidic integration into a centrifugal lab-on-a-chip system, comprising unit operations for reconstitution of reagents, mixing with the sample, and aliquoting to test cavities. As the cartridge contains all of the required liquid and dry reagents only the addition of sample DNA is required. The system was demonstrated by the qualitative detection of < 10 copies of the antibiotic resistance gene mecA in less than 15 minutes. The presented disk-shaped Lab-on-a-Chip cartridge consists of a COP-foil structured by blow-molding [1]. The surface is coated with BSA for blocking and hydrophilization. Glass ampoules with liquid reagents and lyophilized dry reagents are inserted into reservoirs and the disk is sealed by an adhesive foil (fig 1A). When operated the sample is added to the cartridge, the liquid reagent ampoule is manually disrupted through the foil to release the reaction buffer, and the cartridge is placed into a centrifugal analyzer with integrated fluorescence detection (modified RotorGene 2000, Corbett Research, Australia). At spinning frequency of 27 Hz 50 μl of reaction buffer is transferred from the disrupted ampoule to the lyophilized RPA reagents. After dissolving the lyophilisate the solution is divided into 5 x 10 μL aliquots and transferred into the test cavities via a centrifugopneumatic valve (fig 1B). Each of the test cavities may contain a different primer and probe system. The RPA reaction is based on a special recombinase-primer-complex that allows strand displacement and amplification at 37 °C [2].
Maximilian Focke, Bernd Faltin, Claas Müller, Roland Zengerle, Felix von Stetten
Blasformen von Mikrofluidikstrukturen für Lab-on-Foil Anwendungen
2009 Tagungsband Mikrosystemtechnik-Kongress 2009, Berlin, 12.-14. Oktober , Seite: P 22
KurzfassungPräsentiert wird ein neuartiger Prozeß zur schnellen Herstellung dünnwandiger Folienkartuschen aus thermoplastischen Kunststofffolien durch Blasformen. Der Prozeßdurchlauf benötigt vom CAD-Entwurf bis zur fertigen Folienkartusche 6-8 Stunden. Die Folienkartuschen eignen sich durch ihre dünnen Wände insbesondere für mikrofluidische Anwendungen die eine schnelle Temperierung erfordern. In der In-Vitro-Diagnostik ist zum Beispiel der Einsatz für Nukleinsäure-Analysen mit Hilfe der Polymerase Kettenreaktion (PCR) extrem vorteilhaft. Während in herkömmlichen Folienformprozessen Entformschrägen an den Formwerkzeugen von mindestens 3° erforderlich sind, sind bei der hier vorgestellte Technologie keine Entformschrägen erforderlich. Das vereinfacht sowohl den Konstruktions- als auch den Herstellungsprozess signifikant. Die hergestellten Kartuschen zeichnen sich durch eine sehr geringe Maßabweichung von lediglich 0,4% aus. Die Konturtreue einer typischen Kavität mit einem Durchmesser von 3 mm beträgt 18 µm (Halbwertsbreite). Ihre Eignung für diagnostische „Lab-on-a-Foil“-Anwendungen wurde erfolgreich mit einer komplexen mikrofluidischen Aliquotierstruktur sowie der Durchführung einer Real-Time PCR gezeigt.
D. Mark, M. Focke, G. Roth, R. Zengerle, F. von Stetten
Detection of Biological Threats with Fully Automated Lab-on-a-chip Systems in the project SONDE
2009 Proc. Future Security - 4th Security Research Conference Karlsruhe, Germany, September 29th – October 1st , Seiten: 487 - 490
KurzfassungIncidents and terrorist attacks with biological agents bear the risk of causing widespread damage and panic. Rapid detection of such threats is not possible with state of the art technologies, since laboratory methods are time-consuming and often not available in the vicinity. However, to enable a fast assessment of patient symptoms and allow an appropriate quarantine and decontamination response, a robust point-of-care diagnostic system is required. To address this problem, novel lab-ona- chip platforms can be used. One solution is the centrifugal microfluidic platform. It allows automated liquid handling on a rotating structured polymer disk enabling the integration and automation of complex biochemical analysis, such as nucleic acid tests and immunoassays for B-detection.
Kloke A, Kerzenmacher S, Zengerle R, von Stetten F
Electrodeposited thin-layer electrodes for the use in potentially implantable glucose fuel cells
2009 Proc. Transducers, Denver, USA Transducers '09 - Digest of Technical Papers, Seiten: 537 - 540
KurzfassungWe present a novel concept for the fabrication ofelectrodes to be used in thin-layer implantable glucose fuelcells employing platinum based materials as long-termstable abiotic catalysts. A highly porous anode for glucoseoxidation is fabricated by a multiple alternation ofelectrodeposition of platinum-copper alloy and subsequentelectrochemical extraction of the non-noble copper. Toform a permeable oxygen reduction cathode, platinumblack is deposited onto a gold-coated polycarbonate tracketchmembrane (PC-TEM). Power densities of up to3.1 ìW cm-2 are obtained for consequently assembled fuelcells. The novel electrode structures exhibit comparableperformance to state of the art at significantly reducedthickness and facilitated fabrication.
S. Rubenwolf, J. Kestel, S. Kerzenmacher, R. Zengerle, F. von Stetten
Enhancing the lifetime of laccase-based biofuel cell cathodes by sequential renewal of enzyme
2009 60th Annual Meeting of the International Society of Electrochemistry, Bejing , Seiten: S-01 - P-011
KurzfassungWe present a novel bio-inspired approach to enhance the lifetime of enzymatic biofuel cell electrodes. Due to limited enzyme stability the lifetime of such fuel cells is at present only in the order of weeks or months [1]. While current research approaches to overcome this problem focus on enzyme stabilization by immobilization [2], living organisms separate enzyme lifetime from system lifetime by the continuous regeneration of fresh enzyme. We transfer this principle to biofuel cells by sequentially exchanging the electrolyte to re-supply active enzyme to the electrode. Since this concepts demands reversible adsorption of the enzyme, we chose a mediator-less graphite felt cathode with adsorbed laccase as biocatalyst, operating in oxygen saturated 100 mM citrate buffer (pH = 5) [3].
A. Kloke, S. Kerzenmacher, R. Zengerle, F. von Stetten
Facile Fabrication of Ultra Porous Platinum Electrodes and Their Application for Energy Harvesting Glucose Fuel Cells, Abstract number #205
2009 Proc. of 215th ECS Meeting, San Francisco, USA, May 24-29 , Seite: 572
KurzfassungWe present a new method for the generation of porous noble metal surfaces utilizing cyclic repetition of alloy deposition and removal of a non-noble alloy partner in cyclic voltammetry. This method was applied in acidic solution of H2PtCl6 and CuSO4 to fabricate ultra porous platinum electrodes with a roughness factor of above 5000, higher than elsewhere reported. Such platinum surfaces were characterized and tested as electrodes for the use in implantable glucose fuel cells, showing a competitive performance to state of the art with several general technological advantages.
Marc Karle, Junichi Miwa, Günter Roth, Stefan Haeberle, Roland Zengerle, Felix von Stetten
Kontinuierlich arbeitende Mikrofluidik-Plattform zur Aufreinigung von Biomolekülen
2009 Tagungsband Mikrosystemtechnik-Kongress, Berlin, 12.-14. Oktober , Seite: P 31
KurzfassungWir präsentieren eine neue Mikrofluidik-Plattform, die eine kontinuierliche Extraktion und Aufreinigung von Biomolekülen z.B. aus einem Fermenter ermöglicht. Die Plattform beruht auf dem Einsatz der Magnetophorese in einer Laminarströmung. Alle erforderlichen Arbeitsschritte (Binden, Waschen und Eluieren der Biomoleküle) sind auf einem Chip integriert. Eine Schlüsselfunktion nehmen superparamagnetische Partikel in einer Laminarströmung in Verbindung mit einem zeitlich variierenden Magnetfeld ein. Die Partikel ermöglichen den Transport der Biomoleküle über die Phasengrenze der Laminarströmung in einer kreisförmig um einen zentralen, rotierenden Permanentmagneten angeordneten Kanalstruktur. Die Rotation des Permanentmagneten erzeugt ein periodisch variierendes Magnetfeld, das die kontrollierte Bewegung der magnetischen Partikel zwischen unterschiedlichen Extraktionsreagenzien ermöglicht, ohne die Partikel an der Kanalwand zu immobilisieren. Als erste Applikation wurde ein Chip zur DNA-Extraktion aus Polykarbonat mittels Mikrofräsen hergestellt. Um die biotechnische Funktionalität und das magnetophoretische Konzept zu untersuchen, wurde bakterielle DNA direkt aus dem Lysat extrahiert. Im Vergleich mit einem makroskopischen Referenzsystem wurden 80 % der DNA aufgereinigt. Eine Eingangsflussgeschwindigkeit von 12.5 mm∙s-1 führte zu einer durchschnittlichen Partikelgeschwindigkeit von 1.6 mm∙s-1 und zu einer DNA-Extraktion innerhalb ca. 1 Minute. Die Plattform ist ein zentrales Element eines Überwachungssystems zur kontinuierlichen Kontrolle des Zellwachstums in Bioreaktoren. Darüber hinaus kann sie, je nach Oberflächenmodifikation der magnetischen Partikel, auch für die Aufreinigung von RNA, Proteinen oder Zellen für nachfolgende Echtzeitanalysen verwendet werden.
S. Lutz, D. Mark, R. Zengerle, F. von Stetten
Lab-on-a-Chip Cartridge zur Durchführung von Immunoassays mit integrierter Probenaufbereitung
2009 Tagungsband Mikrosystemtechnik-Kongress 2009, Berlin, 12.-14. Oktober , Seite: P 7.3
KurzfassungWir stellen ein neuartiges Lab-on-a-Chip System vor, das unter Ausnutzung von Zentrifugalkräften eine integrierte Prozessierung von Immunoassays mit kompletter Probenaufbereitung ermöglicht. Unsere Lab-on-a-Chip Cartdige erlaubt die Separation von Blutzellen und Blutplasma, die Dosierung eines definierten Plasmavolumens, die Durchführung von Inkubations-, Misch- und Waschschritten sowie ein integriertes Abfallreservoir. Aus einem Blutvolumen von 10 µL extrahieren wir durch Sedimentation 4 µL Blutplasma mit einem cv von 6 %. Das Schalten der Fluide in der Cartridge wird mittels kapillarer und volumengesteuerter Siphonventile durchgeführt. Die Verlässlichkeit des fluidischen Systems wird erhöht durch eine Hydrophilisierung der Oberfläche mit BSA. Die Reaktionskammer ermöglicht effiziente Misch- und Waschprozesse und erlaubt die Vorlagerung von Trockenreagenzien. Um die Funktionalität unseres Lab-on-a-Chip Systems zu demonstrieren, zeigen wir eine quantitative Bestimmung des Hormons Estradiol in Konzentrationen von 25 pg/mL bis 1 ng/mL mittels eines Chemolumineszenz-basierten kompetitiven Immunoassays.
T Metz, D Mark, S Lutz, O Strohmeier, D Kosse, M Focke, B Faltin, J Burger, J Böning, J Miwa, M Karle, G Müller, C Müller, S Messner, H Reinecke, R Zengerle, F von Stetten
Lab-on-a-Chip Foundry Service – enabling the miniaturization of in vitro diagnostics
2009 Euromedlab, Innsbruck, Austria, 2009, 350-368 Clinical Chemistry and Laboratory Medicine, Seiten: 350 - 368
T. Metz, M. Karle, T. Preis, D. Mark, G. Roth, C. Müller, R. Zengerle, F. von Stetten
Lab-on-a-Chip Foundry Service: Schnelle kundenspezifische Implementierung miniaturisierter biochemischer Assays
2009 Tagungsband Mikrosystemtechnik-Kongress 2009, Berlin, 12.-14. Oktober , Seite: P 42
KurzfassungWir präsentieren eine konsistente Methodik zur effizienten Miniaturisierung, Integration und Automatisierung bio-chemischer Assays auf der Grundlage definierter mikrofluidischer Plattformen [1]. Eine mikrofluidische Plattform stellt einen Satz fluidischer Grundfunktionen (sogenannte Einheitsoperationen) zur Verfügung welche einfach kombiniert und durch eine günstige, wohl definierte Fertigungstechnologie hergestellt werden können. Anwendungsspezifische Assays lassen sich hierüber flexibel und mit geringem Entwicklungsrisiko durch die systematische Verknüpfung plattformspezifischer, validierter, Einheitsoperationen implementieren. Die mikrofluidischen Layouts der Einheitsoperationen werden in einem Design Handbuch, zusammen mit Validierungsergebnissen und den Fertigungsprozessen dokumentiert. Für die Validierung werden standardisierte Strukturen eingesetzt. Dies wird beispielhaft anhand einer Teststruktur für die kapillare Befüllung gezeigt.
Hoffmann J, Mark D, Zengerle R, von Stetten F
Liquid Reagent Storage and Release for Centrifugally Operated Lab-on-a-Chip Systems Based on a Burstable Seal
2009 Proc. Transducers, Denver, USA Proceedings of the 15th IEEE International Conference on Solid-State Sensors, Actuators and Microsystems, Band: 15, Seiten: 1991 - 1994
KurzfassungWe present a new approach for the pre-storage and release of liquid reagents in centrifugally operated Lab-ona- Chip (LoaC) cartridges. Liquids are stored in sealed cavities which are separated from the fluidic system by a weak-bonded interface. During centrifugal rotation, the liquid exerts an inertial force onto the predetermined area. This delaminates the sealing foil locally, resulting in a fluidic connection to the downstream channel. Time-torelease could be adjusted to a range between 31 s and 143 s by geometrical variations of the structure, enabling time controlled release. In sum, the burstable seal is a universal and robust valve: it is vapor tight, independent on wetting properties of liquids, and time controllable. Hence it excels siphon based valving concepts often used in centrifugal microfluidics and is most valuable for the design of LoaC cartridges for point of care applications.
T. Metz, D. Mark, S. Häberle, G. Roth, C. Müller, F. von Stetten, R. Zengerle
Microfluidic Platforms for Miniaturization, Integration and Automation of Biochemical Assays
2009 Proc. of International Conferences on Multi-Material Micro Manufacture (4M) / International Conferences on Micro Manufacturing (ICOMM), Karlsruhe , Seiten: 25 - 29
KurzfassungDuring the last decade many technological progresses were achieved in the field of Lab-on-a-Chip for the miniaturization, integration and automation of biochemical assays. Nowadays an unmanageable variety of alternative approaches exist that can perform these tasks. To cope with the complexity of those systems the paradigm of development changed from component oriented approaches towards the development of microfluidic platforms. A microfluidic platform provides a set of fluidic unit operations, which are designed for easy combination within a well defined (and low cost) fabrication technology. The platform allows implementation of different application specific systems (assays) in an easy and flexible way, based on the same fabrication technology. In this work we will discuss important issues for the successful implementation of a platform oriented development environment for Lab-on-a-Chip on the example of the newly set up Lab-on-a-Chip Foundry Service of the HSG-IMT. The systematic development of Lab-on-a-Chip will be shown on the example of a nucleidic acid assay on the centrifugal microfluidic platform. In the Lab-on-a-Chip Foundry Service, development is based on standardized microfluidics and standardized fabrication processes that are maintained in a knowledge management system, the Lab-on-a-Chip Design Handbook of the HSGIMIT. Facilities and experts are provided for microfabrication and microfluidics as well as for biochemistry and biology to enable fast development cylces in a one-stop shop approach. Prototyping is organized in rapid prototyping chains for polymer fabrication, sealing and assembly in a rapid prototyping workshop.
Daniel Mark, Markus Rombach, Sascha Lutz, Roland Zengerle, Felix von Stetten
Microfluidic unidirectional pneumatic switch for automated DNA-extraction on standard laboratory centrifuges
2009 Proc. of the Thirteenth International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS), Jeju, Korea, November 1-5 , Seiten: 110 - 112
KurzfassungFor the first time, we present a new, unidirectional liquid switching concept for the centrifugal microfluidic platform. The concept relies on a controlled liquid-air interface instability. It enables automated DNA-extraction and potentially other microfluidic protocols on standard laboratory centrifuges, superseding the need for expensive base instruments, which are common to all state of the art Lab-on-a-Chip platforms. As a proof of concept we fabricated a Lab-on-a-Chip cartridge for automated DNA extraction from 32 μL whole blood on a standard laboratory centrifuge. The obtained DNA yield was 88 ± 44 ng (42 % of the optimized reference extraction [1]).
J. Burger, D. Mark, G. Roth, C. Müller, R. Zengerle, F. von Stetten, Y. Park
Physiologische Assays in einem DVD Player am Beispiel der Messung des Hämatokrit
2009 Tagungsband Mikrosystemtechnik-Kongress 2009, Berlin, 12.-14. Oktober , Seite: P 78
KurzfassungDie Bestimmung des Hämatokrits (Erythrozytengehalt im Blut) mit zentrifugal mikrofluidischen Bio-Disks [1] in einem low-cost DVD Player wurde bereits in früheren Arbeiten untersucht [2,3]. Nun strukturieren wir konventionelle DVD Halbscheiben aus Massenproduktionsprozessen und Bonden diese zu mikrofluidischen Bio-DVD’s. Somit kann der Standard DVD-Player sowohl als Zentrifuge zur mikrofluidischen Prozessierung als auch zur Detektion der Ergebnisse mit dessen integrierter Opto-Elektronik verwendet werden. Der Hämatokrit gibt unter anderem Aufschluss über den Wasserhaushalt des Patienten oder den Erythrozytenanteil im Blut. Er wird häufig im Rahmen von point-of-care Tests neben anderen Parametern in der Notfallmedizin, in Dopingtests oder vor Blutspenden ermittelt. Somit stellt diese Arbeit einen weiteren Schritt hin zu kompletten DVD-basierten point-of-care Tests dar und bietet eine Grundlage für die Implementierung weiterer Assays.
Henning Hoefemann, Hans-Martin Trotter, Roland Gronmaier, Felix von Stetten, Roland Zengerle, Stefan Haeberle
Rheoplug – Segmented flow based viscometer
2009 Proc. of the Thirteenth International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS), Jeju, Korea, November 1-5 , Seiten: 76 - 78
KurzfassungWe present a novel method to measure the viscosity of liquid mixtures in μLvolume plugs based on a segmented flow platform [1]. Therefore, up to three different liquids of different viscosities are injected into a common plug which is afterwards transported through an air filled channel by a 15-20 mbar vacuum below ambient pressure at the chip outlet. Rapid mixing within the plugs is enabled by internal advection and the plug velocity directly depends on the viscosity of the plug. Following this approach, the viscosities of water/PEG mixtures in the range of 1-68 mPa s could be successfully measured on-chip.
M. Focke, R. Feuerstein, F. Stumpf, D. Mark, T. Metz, R. Zengerle, F. von Stetten
Self-blocking valve for a highly wetting fluids based on pinning of gas entrapments
2009 Proc. of the Thirteenth International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS), Jeju, Korea, November 1-5 , Seiten: 1397 - 1399
KurzfassungWe present a novel valving principle for a highly wetting fluid in centrifugal microfluidics by self-blocking pinning structures. While usual geometrical stopping valves prove instable for highly wetting liquids, the novel valve reproducibly generates gas entrapments in a fishbone-like structure. Their pinning pressures add up to a total burst pressure pburst that efficiently avoids uncontrolled capillary priming of microfluidic systems. In centrifugal experiments, burst pressures of 14-18 hPa (12 ± 2 Hz) were reproducibly achieved for isopropyl, a completely wetting alcohol (θ ~ 0°; σ = 22 mNm-1). The same structure also acts as a geometrical stopping valve for less wetting liquids (θ = 31°; σ = 30 mNm-1) [1].
J. Hoffmann, L. Riegger, D. Mark, F. von Stetten, R. Zengerle, J. Ducrée
TIR-based dynamic liquid-level and flow-rate sensing and ist application on centrifugal microfluidic platforms
2009 Proc. IEEE-MEMS; 25 – 29 January 2009, Sorrento, Italy , Seiten: 539 - 542
KurzfassungWe for the first time present a technique for the time-resolved localization of the liquid-gas interface on a centrifugal microfluidic platform based on total internal reflection (TIR) at the channel wall. The simple setup consists of a line laser and a linear image sensor array mounted in a stationary instrument. The linear detector allows a meniscus detection which can be realized at high speed rotation up to 30 Hz and features a spatial resolution of 50 µm. Apart from checking the presence of gas bubbles, the unique combination of centrifugation for compacting liquids, the dynamic meniscus detection allows to accurately measure liquid volumes with a high precision of 1.9 % as well as flow rates and viscosities (range: 1 – 12 mPas) with a precision of 4.7 % and 4.3 %, respectively.
Daniel Mark, Stefan Haeberle, Junichi Miwa, Patrick Weber, Guenter Roth, Marc Karle, Max Focke, Sascha Lutz, Jochen Hoffmann, Claas Müller, Felix von Stetten, Roland Zengerle
Two Microfluidic Platforms for Miniaturization, Integration and Automation of Assays
2009 Proc. of COMS, Copenhagen , Seite: -
KurzfassungTwo different platform concepts for microfluidic miniaturization, integration and automation of biochemical assays are presented. First, unit operations for batch-wise nucleic acid analysis on the centrifugal microfluidic platform are demonstrated, including unit operations for DNA extraction, aliquoting and real-time PCR. Second, the newly developed continuous phase transfer magnetophoresis platform is introduced. It enables continuous online process monitoring, demonstrated by implementation of unit operations for DNA extraction.
Christian Köhler, Arne Kloke, Sven Kerzenmacher, Roland Zengerle, Felix von Stetten
Two reaction-specific electrodes fabricated with the same process for the use in tissue implantable glucose fuelcells
2009 Bunsen Colloquium, Dez. 2009 , Seiten: 60 - 61
KurzfassungAbiotically catalyzed glucose fuel cells (GFCs) represent a promising approach for a self-sufficient power supply of low power implants from electrochemical conversion of the in tissue fluids simultaneously present glucose and oxygen. uch systems require two reaction-specific electrodes to achieve a reasonably high cell voltage. This is because both electrodes are operated in the same media and therefore a mixed electrode potential results from the two competing electrode reactions. Hence state of the art GFCs use two electrocatalysts differing in fabrication and material. Here we demonstrate the applicability of a novel catalyst deposition process to fabrication of both electro-des using the same material. Here, Pt-catalysts are fabricated from alternation of Pt-Cu co-deposition and Cu-dissolution
Mark D, Haeberle S, Lutz S, Zengerle R, von Stetten F
Vacuum supported liquid waste handling for DNA extraction on centrifugally operated Lab-on-a-chip systems
2009 Denver, USA Proc Transducers, Seiten: 1230 - 1233
KurzfassungWe present a reliable liquid waste containment for centrifugally operated Lab-on-a-Chip systems that works even for highly wetting reagents. It is based on the passive generation and enclosure of vacuum in a closed storage chamber that prevents all liquids from capillary reflux into the microfluidic channel network.The new waste handling presented here enabled the implementation of an integrated deoxyribonucleic acid (DNA) extraction chemistry without contamination risks based on purely passive structures. Using a 32 µL whole blood sample we achieved an extraction of 290 ng ± 80 ng DNA in 100 µL of eluate.

2008

J. Böning, D. Mark, S. Lutz, B. Faltin, M. Focke, M. Karle, J. Ducrée, S. Messner, R. Zengerle, F. von Stetten
"Lab-on-a-Chip Foundry Service": A Systematic Approach to the Development of Centrifugal Microfluidic Technologies
2008 Actuator 2008 / Bremen Messe Bremen, Seiten: 814 - 817
KurzfassungThis contribution provides a novel approach to a time and cost-efficient development of polymeric lab-on-a-chip applications. Rather than starting from scratch, new designs are derived from a library of validated laboratory unit operations (LUOs) and prototyped by standard operating procedures (SOPs). LUOs as well as SOPs are systematically documented and retrieved from a "BlueBook". The essential ingredients for the development of lab-on-a-chip applications are liquid handling technologies and fabrication technologies as well as test and development tools. This paper outlines a streamlined approach based on a microfluidic platform concept to reduce the cost, time and risks for the development of lab-on-a-chip technologies. As an example illustrating the platform concept, we describe our centrifugal microfluidic “Lab-ona- Disk” platform. The paradigm of the platform concept is to establish a library of LUOs such as metering, mixing and routing which are validated by experiments and accompanying simulations for a certain parameter range. The concatenation of these LUOs allows to realize complex applications. The layout is then transferred into hardware according to SOPs and afterwards measured at a designated test and development stand. Design rules for the LUOs, procedures (SOPs) and other relevant information such as materials or device components are documented and retrieved from a wiki-based software-based knowledge management portal called “BlueBook”. Keywords: Lab-on-a-Chip Foundry Service, centrifugal microfluidic platform, standard operating procedures, laboratory unit operations, knowledge management system
S. Kerzenmacher, U. Kräling, J. Ducrée, R. Zengerle, F. von Stetten
A Binder-less Glucose Fuel Cell with Improved Chemical Stability Intended as Power Supply for Medical Implants
2008 Proceedings of the 4th European Conference of the International Federation for Medical and Biological Engineering (eMBEC), Antwerpen, 23–27 November , Seiten: 2379 - 2384
KurzfassungWe present an improved abiotically catalyzed glucose fuel cell, intended as tissue implantable power supply for medical implants. A novelty is the application of binderless platinum electrodes for both, anode and cathode. This overcomes the limited chemical stability of glucose fuel cells fabricated from activated carbon particles dispersed in a hydrogel matrix. For the first time the diffusion resistance to be expected from tissue capsule formation has been taken into account during performance characterization under physiological concentrations of glucose and oxygen. Despite the resulting limited oxygen supply, the binder-less fuel cells exhibit a power density of (2.3 ± 0.2) μW cm-2, which is comparable to our previous prototypes. We show that this is due to the increased performance of the novel electrodes. Keywords — glucose, fuel cell, implantable, energy harvesting, platinum
Kloke A, Kerzenmacher S, Kräling U, Zengerle R, von Stetten F
A Permeable Foil-Based Thin Layer Oxygen Cathode for Biofuel Cell and Sensor Applications
2008 Tenth World Congress on Biosensors, Shanghai, China 2008 , Seite: P2.24
KurzfassungIn fuel cell and sensor research foil-based electrodes offering high surface area are of special interest. Here we demonstrate the development of an oxygen cathode established on a polyimide track etch membrane. Furthermore this cathode is permeable and therefore especially interesting for applications desiring mass transport through the cathode. We developed this electrode for abiotically catalyzed glucose fuel cells which require a permeable oxygen cathode on top of a glucose anode to enable separate electrode reactions [1], but this approach is also interesting for enzymatic fuel cells and sensor technology. Following a protocol similar to van der Wal et al. [2], we formed platinum alloys with aluminum (figure 1). Layers of 500 nm platinum and 500 nm aluminum were vapor-deposited on silicon. Samples were annealed at 300 °C for 120 minutes and subsequently activated in NaOH. We observed roughness factors comparable to van der Wal (about 150) using cyclic voltammetry (figure 2). Operated in aerated phosphate buffered solution these electrodes showed their applicability as oxygen reduction cathodes even after adding glucose to the solution and lowering the oxygen concentration (figures 3, 4). Using this process with 250 nm thick layers of platinum and aluminum on polyimide track etch membranes having straight pores of 2 μm in diameter we succeeded in developing thin (25 μm), permeable and flexible electrodes (figure 5). Their roughness factor is higher than 30. As cathode the foil-based electrode polarizes less under load than the ones fabricated on silicon but show a slightly lower open circuit voltage (figure 4). We showed the applicability of platinum-aluminum alloy formation for production of an oxygen cathode working under physiological concentrations of oxygen and glucose and succeeded in transferring this process onto a thin permeable foil substrate. Their electrochemical performance is comparable to carbon based oxygen cathodes which are state of the art in abiotically catalyzed glucose fuel cells (figure 3) [3].
A. Kloke, B. Biller, S. Kerzenmacher, U. Kräling, R. Zengerle, F. von Stetten
A single layer biofuel cell as potential coating for implantable low power devices
2008 Proc. Eurosensors XXII, Dresden, Germany, September 7-10 , Seiten: 1416 - 1419
KurzfassungAbiotically catalyzed glucose fuel cells were considered to only work with specific assemblies for electrode reaction separation. Using a highly porous oxygen tolerant catalyst for glucose oxidation we were able to realize a fuel cell with anode and cathode placed side by side. These results open the opportunity of having a sustainable power source implemented as single layer coating of medical implants. Further potential for power enhancement and cost reduction is identified within the variation of anode to cathode proportions.
Mark, Daniel, Häberle, Stefan, Metz, Tobias, Lutz, Sascha, Ducree, Jens, Zengerle, Roland, von Stetten, Felix
Aliquoting structure for centrifugal microfluidics based on a new pneumatic valve
2008 Proc. IEEE-MEMS 2008, Tucson, USA , Seiten: 611 - 614
KurzfassungWe present a new microvalve that can be monolithically integrated in centrifugally driven lab-on-achip systems. In contrast to existing operation principles that use hydrophobic patches [1], geometrically defined capillary stops [2] or siphons [3], here we present a pneumatic principle. It needs neither additional local coatings [1] nor expensive micro sized geometries [2]. The valve is controlled by the spinning frequency and can be switched to be open when the centrifugal pressure overcomes the pneumatic pressure inside an unvented reaction cavity. We designed and characterized valves ranging in centrifugal burst pressure from 6700 Pa to 2100 Pa. Based on this valving principle we present a new structure for aliquoting of liquids. We experimentally demonstrated this by splitting 105 μL volumes into 16 aliquots with a volume CV of 3 %.
S. Kerzenmacher, S. Zehnle, T. Volk, D. Jansen, F. von Stetten, R. Zengerle
Autarke Energieversorgung eines Herzschrittmachers mittels Glukosebrennstoffzelle und effizientem DC-DC-Wandler
2008 Tagungsband 14. Heiligenstädter Kolloquium - Technische Systeme für die Lebenswissenschaften; Heiligenstadt 22.-24.09.2008 , Seiten: 397 - 404
KurzfassungImplantierbare Glukosebrennstoffzellen auf Basis abiotischer Katalys-atoren sind ein vielversprechender Ansatz zur batterieunabhängigen Energieversorgung von aktiven Implantaten. Während sich unter physiologischen Bedingungen Leistungsdichten von 3 µW/cm² erreichen lassen, ist es in der praktischen Umsetzung des Konzepts erforderlich die mit 0,2 V recht geringe Zellspannung auf die für elektronische Schaltkreise typische Betriebsspannung von 3 V zu transformieren. Bei Ausgangs-leistungen im µW-Bereich zeigen kommerziell erhältliche DC-DC-Wandler jedoch nur sehr geringe Wirkungsgrade von etwa 4 %. Folglich wäre zum Betrieb eines 15 µW Herzschrittmachers eine Brennstoffzelle von mindestens 125 cm² erforderlich – zu groß um direkt auf dem Schrittmachergehäuse platziert werden zu können. Um die Wandlungsverluste zu minimieren, wurde daher eine optimierte Aufwärtswandler-Schaltung mit einem Wirkungsgrad von über 40 % aufgebaut, die den kontinuierlichen Betrieb eines 15 µW Herzschrittmachers mit einer nur 18 cm² großen Glukosebrennstoffzelle erlaubt. Der verringerte Flächenbedarf würde die zukünftige Ausführung der Brennstoffzelle als direkt auf der Oberfläche des Implantats integrierte Stromversorgung erlauben.
A. Kloke, S. Rubenwolf, C. Bücking, J. Gescher, S. Kerzenmacher, R. Zengerle, F. von Stetten
Bausatz für einen modularen Miniatur-Bioreaktor und seine Anwendung als elektrochemische Testzelle für Bio-Brennstoffzellen
2008 Tagungsband 14. Heiligenstädter Kolloquium - Technische Systeme für die Lebenswissenschaften; Heiligenstadt 22.-24.09.2008 , Seiten: 303 - 310
KurzfassungReproduzierbare Untersuchungen an Biomikrosystemen erfordern einen sehr flexiblen und vollständig charakterisierten experimentellen Aufbau, der in seinen Eigenschaften einem Bioreaktor entspricht. Wir stellen einen zu diesem Zweck neu entwickelten Miniatur-Bioreaktor vor, der aus Baukastenelementen zusammengesetzt wird. Der modulare Aufbau ermöglicht eine Anpassung in Volumen und verfügbaren Schnittstellen für Begasung, Medienwechsel, Sensorik und Probenahme. Ferner können mehrere einzelne Bioreaktoren zusammengeschlossen betrieben oder für elektrochemische Messungen über Membranen gekoppelt werden. Die Charakterisierung des Reaktors ergibt kLa-Werte und Mischzeiten, die im üblichen Bereich für nicht-modulare Blasensäulenreaktoren liegen. Die vielseitige Anwendbarkeit des neuen Systems wurde anhand der Wachstumskurve einer Escherichia coli Kultur sowie durch elektrochemi-sche Untersuchungen an einer enzymatischen Kathode und einer mikrobiellen Anode für Bio-Brennstoffzellen demonstriert.
M. Focke, B. Faltin, T. Hösel, C. Müller, J. Ducrée, R. Zengerle, F. von Stetten
Blow molding of polymer foils for rapid prototyping of microfluidic cartridges
2008 Proceedings of The 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2008) , Seiten: 988 - 990
KurzfassungWe present a novel process for small-lot manufacturing of microfluidic lab-on-achip cartridges made of thin polymer foils within 8 hours starting from a CAD layout. A master tool is micromilled and then casted with polydimethysiloxane (PDMS) to obtain an elastic replication mold. This is used for blow molding of thermoplastic foils in order to manufacture lab-on-a-chip cartridges with very thin sidewalls of 100 to 180 μm. These are suited for microfluidic applications requiring fast heat transfers, most prominently thermocycling for polymerase chain reactions (PCR) as demonstrated with our technology.
S. Lutz, P. Lang, L. Riegger, W. Streule, M. Daub, P. Koltay, F. von Stetten, R. Zengerle, J. Ducrée
Contact-Free Dispensing of Living Cells in Nanoliter Droplets
2008 Actuator 2008 / Bremen Messe Bremen
KurzfassungWe present a novel method for automated dispensing of living cells in nanoliter range droplets using a disposable pipette tip combined with an elastic polymer tube. After introduction of an unmetered suspension of cells into a reservoir connected to the pipette tip, a tuneable volume of 10 - 80 nL of cells suspension is issued in a non-contact procedure. Droplet ejection is enabled by a piezostack driven piston squeezing the tube at a defined position. We achieve a reproducibility of the printed cell culture medium volumes better than 5% and survival rate of the cells of 97% directly after dispensing. In addition we demonstrated good culturability and cell differentiation in order to consider potential long term effects of the dispensing process that could harness the cells. Keywords: nanoliter dispensing, cells, piezo actuation
Rubenwolf S, Kloke A, Kerzenmacher S, Zengerle R, von Stetten F
Direct Electron Transfer from Adsorption-Bound Laccase to Different Carbon Based Electrodes
2008 Tenth World Congress on Biosensors, Shanghai, China 2008 , Seite: P2.23
S. Rubenwolf, O. Strohmeier, R. Zengerle, F. von Stetten
Influence of Carbon Fiber Morphology on Direct Electron Transfer
2008 213th ECS Meeting, Phoenix, USA, 2008 , Seite: Abstr. 205
T Metz, D Mark, S Lutz, R Simon, M Focke, B Faltin, J Burger, J Böning, J Miwa, M Karle, G Müller, C Blattert, C Müller, S Messner, H Reinecke, R Zengerle, F von Stetten
Lab-on-a-Chip Foundry Service – speeding up the miniaturization of in vitro diagnostics
2008 Proc. 3rd Technology Forum Diagnostics & Bioanalytical Devices, Frankfurt, 2008, 3 , Band: 3
KurzfassungWe present the Lab-on-a-Chip Foundry Service of the HSG-IMIT that was founded to speed up the realization of in vitro diagnostic assays (IVD) as miniaturized microfluidic systems (Lab-on-a-Chip). The Lab-on-a-Chip Foundry Service opens the advantages of Lab-on-a-Chip - smaller consumption of reagents and faster turnaround times in compact, automated diagnostics - to a broad community. We offer everything from one hand: Layout, prototyping, fluidic and biochemical testing.
S. Lutz, P. Lang, I. Malki, D. Mark, J. Ducree, R. Zengerle, F. von Stetten
Lab-on-a-chip cartridge for processing of immunoassays with integrated sample preparation
2008 Proceedings of The 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2008) , Seiten: 1759 - 1761
KurzfassungWe present a novel centrifugal microfluidic lab-on-a-chip cartridge for the fully integrated processing of immunoassays including sample preparation and metering, incubation, washing and waste handling. From a sample of 10 μL human whole blood we extract 4 μL of blood plasma by sedimentation of blood cells driven by centrifugal forces with a CV of 6%. Routing of fluids is realized by capillary and volume triggered siphon valves. The reliability of the capillary-driven fluid channels is increased by surface coating with BSA for hydrophilization and blocking. The reaction chamber allows efficient mixing of fluids and dissolution of prestored reagents [1]. For demonstration purposes we quantified estradiol with a range of 25 pg/mL to 1 ng/mL using a chemiluminescent competitive immunoassay within 30 minutes [2].
D. Mark, M. Focke, B. Faltin, J. Ducrée, R. Zengerle, F. von Stetten
Nukleinsäurediagnostik auf der Bio-Disk Plattform
2008 Tagungsband 14. Heiligenstädter Kolloquium - Technische Systeme für die Lebenswissenschaften; Heiligenstadt 22.-24.09.2008 , Seiten: 321 - 328
KurzfassungDie zentrifugale Mikrofluidik gehört zu den vielversprechenden, mikrofluidischen Entwicklungsplattformen zur Miniaturisierung, Integration und Automatisierung biochemischer Analyseprotokolle [1]. Sie beruht auf der kontrollierten Prozessierung kleinster Flüssigkeitsmengen in einem zentrifugalen Beschleunigungsfeld und ermöglicht die Portierung von Analyse- und Diagnoseaufgaben von Großlabors in den Point-of-Care (POC) Bereich [2]. Auf Grundlage der zentrifugal-mikrofluidischen Bio-Disk Plattform [3] zeigen wir die Integration grundlegender Laborprotokolle für die Nukleinsäureanalytik: Nukleinsäure-Extraktion und Real-Time-PCR. Die Entwicklungen sollen eine vollständig integrierte und automatisierte Point-of-Care Plattform für die molekulare Diagnostik ermöglichen. Die Isolierung und Aufreinigung von DNA aus Probenmaterial wird durch einen Lyseschritt und eine Festphasenextraktion mit einer Silikamembran erreicht. Die Membran ist im Gegensatz zu Silika-Beads einfach zu handhaben und kann durch PCR-kompatible Klebstoffe oder mechanische Halterungen gut in mikrofluidische Strukturen integriert werden. Aus einer 20 µl Vollblutprobe konnten durchschnittlich 0,21 µg ± 0,09 µg DNA extrahiert werden. Die PCR Tauglichkeit der extrahierten DNA wurde in einem Real-Time PCR Thermocycler nachgewiesen und war nahezu identisch zu einer Referenzextraktion (QIAamp DNA Blood Mini Kit). Der Schwellwertzyklus (CT = Threshold Cycle) lag bei 2300 extrahierten Kopien eines vollständigen Genoms bei beiden Extraktionen bei 30. Um die extrahierte DNA zur parallelen Bestimmung mehrerer genetischer Parameter in kreuzkontaminationsfreie Kammern zu verteilen, wurde eine Aliquotierstruktur entwickelt, die auf einem passiven zentrifugo-pneumatischen Ventil basiert. Dieses robuste Ventil weist Schaltfrequenzen auf, die um mindestens das vierfache über denen von gleich dimensionierten kapillaren Ventilen liegen, und die Fertigung erfordert keine lokale Oberflächenmodifikation [4]. Die Aliquotierstruktur ermöglicht eine Aufteilung eines ca. 100 µl großen Probenvolumens in z. B. 16 Aliquote mit einem Volumen-Variationskoeffizenten von 3 %. Wir demonstrierten ferner die Durchführung einer Real-Time PCR in einer thermogeformten folienbasierten Bio-Disk mit Mikrofluidikstruktur. Die Foliendisk eignet sich aufgrund ihrer geringen thermischen Masse besonders für ein rasches Thermocycling. Standardkurven für die Amplifikation verschiedener Kopienzahlen des Exfoliatin-A-Gens (Staphylococcus aureus) wurden auf der Foliendisk sowie in standard Polypropylen (PP) PCR-Tubes durchgeführt. Die Schwellenwertzyklen für 100, 1000, und 10.000 Kopien waren 35,6, 31,5 und 27,5 für die die PP-Tubes und 33,6, 29,7 und 25,7 für die Foliendisk. Die Integration der vorgestellten Module für DNA-Extraktion und Real-Time PCR auf der Bio-Disk Plattform zeigt gutes Potential für die automatisierte und vollintegrierte Durchführung von kompletten Nukleinsäureanalysen.
Clemens Bücking, Stefanie Rubenwolf, Felix von Stetten, Johannes Gescher
Reductase activity of outer membrane c-type cytochromes in Shewanella oneidensis
2008 Jahrestagung der Vereinigung für Allgemeine und Angewandte Mikrobiologie und der Gesellschaft für Biochemie und Molekularbiologie, Frankfurt/Main, Germany , Seite: 91
KurzfassungDissimilatory iron reduction (DIR) is a respiratory process in which microorganism couple the reduction of metals to energy production. This process widely shaped the bio- and geosphere on our planet (Nealson, et al 2002). A better understanding of DIR is crucial for applications in biotechnology like remediation of contaminated soils or current production in microbial fuel cells. An important model organism to investigate DIR is the γ-proteobacterium Shewanella oneidensis which has to use an extended respiratory pathway to transport electrons through the periplasmic space to insoluble crystalline Fe(III)-oxides. C-type cytochromes are thought to mediate the electron transfer to the transition metal. Genome analysis revealed five genes for outer membrane c-type cytochromes, three of those have an unknown function so far. We wanted to investigate the specificity of all five putative outer membrane cytochromes towards metallic electron acceptors. Therefore we constructed a mutant with an in-frame-deletion of genes mtrD-omcB and inserted an inducible promotor in front of the known key players mtrA and mtrB. Genes for membrane cytochromes were cloned into pBad202-vector and transformed into the mutant. After characterizing protein expression, we measured reduction rates of cell suspensions with different metal-oxides. To further investigate the functionality of the proteins, we measured the ability to generate electricity in a microbial fuel cell. The results so far point towards different reduction abilities of the cytochromes for reducing soluble and insoluble iron forms.
S. Lutz, V. Reitenbach, D. Mark, J. Ducree, R. Zengerle, F. von Stetten
Unidirectional shake-mode for mixing highly wetting fluids on centrifugal platforms
2008 Proceedings of The 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2008) , Seiten: 748 - 750
KurzfassungWe present a novel approach for washing and mixing on centrifugal microfluidic platforms. Mixing and washing are essential unit operations for lab-on-a-chip platforms but often cause valving problems if they are performed with “real life fluids” like washing buffers containing high detergent or salt concentrations which are often used in diagnostic or biological assays. We therefore improved the shakemode mixing technique to find a method that combines reliable siphon-based valving with high mixing and washing efficiency. Therefore the so called unidirectional shakemode, is introduced and correlated to the already established technologies.

2007

Fleischhauer,B., Kloke,A., Rubenwolf,S., Schuh,K., Schlipf,C., Krüger,M., Müller,C., Prucker,O., Reinecke,H., Rühe,J, von Stetten,F., Zengerle,R., Woias,P.
"Micro Engery Harvesting" durch Mikro-Brennstoffzellen
2007 MikroSystemTechnik Kongress, Dresden, Germany, 15.-12. Oktober 2007 Proceedings, VDE, Seiten: 349 - 352
S. Kerzenmacher, R. Sumbharaju, J. Ducrée, R. Zengerle, F. von Stetten
A Surface Mountable Glucose Fuel Cell for Medical Implants
2007 14th Intern. Conf. on Solid-State Sensors, ..., Lyon/France, Jun 2007 Transducers '07 - Digest of Technical Papers, Band: 1+2, Seiten: U66 - U67
Kerzenmacher,S., Ducree,J., Zengerle,R., von Stetten,F.
A novel fabrication route yielding self-supporting porous platinum anodes for implantable glucose micro fuel cells
2007 Proceedings of PowerMEMS 2007, Freiburg, Germany, 2007 , Seiten: 31 - 34
Haeberle,S., Pausch,S., Burger,R., Lutz,S., von Stetten,F., Zengerle,R., Ducree,J.
Automation of nucleid acid extraction by a coriolis-force actuated droplet router
2007 MicroTAS 2007, Paris, France, 07.10. - 11.10.2007 , Seiten: 1231 - 1233
Kerzenmacher,S., von Stetten,F., Ducree,J., Zengerle,R.
Glukose-Brennstoffzellen als autarke Engergieversorgung für medizinische Mikro-Implantate: Stand der Technik und aktuelle Entwicklungen
2007 MikroSystemTechnik Kongress, Dresden, Germany, 15.-12. Oktober 2007 Proceedings, VDE, Seiten: 341 - 344
Haeberle,S., Pausch,S., Burger,R., Lutz,S., von Stetten,F., Zengerle,R., Ducree,J.
Integration, Automatisierung und Parallelisierung von Silika-basierten Nukleinsäure-Extraktionen mit dem virtuellen Coriolis-Schalter
2007 MikroSystemTechnik Kongress, Dresden, Germany, 15.-12. Oktober 2007 Proceedings, VDE, Seiten: 465 - 468
Haeberle,S., Ducree,J., von Stetten,F., Zengerle,R.
Microfluidic Platforms for Miniaturization, Integration, and Automation of Biochemical Assays
2007 MikroSystemTechnik-Kongress 2007, Dresden, Germany, 15.10. - 17.10.2007 Proceedings, Seiten: 449 - 455
Lutz,S., Lang,P., Faltin,B., Haeberle,S., von Stetten,F., Zengerle,R., Ducree,J.
Towards a comprehensive centrifugal process integration by rotationally induced lyophilisate dissolution and cell lysis
2007 Eleventh International Conference on Miniaturized Systems for Chemistry and Life Sciences 7 – 11 October 2007, Paris, FRANCE , Seiten: 1516 - 1518

2006

F. von Stetten, S. Kerzenmacher, A. Lorenz, V. Chokkalingam, N. Miyakawa, R. Zengerle, J. Ducree
A one compartment, direct Glucose fuel cell for powering long-term medical implants
2006 Proc of IEEE MEMS 2006, Istanbul, Turkey, Seiten: 934 - 937
F. von Stetten, J. Ducrée
Biofuel cells as power generators for implantable devices (invited key-note presentation)
2006 Proc. Eurosensors XX, Göteborg, Sweden, 2006, 1, 222-225

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