Development of microfluidic devices for cancer cell isolation

[eng] The emergence of liquid biopsies has been useful for the diagnosis of physiological conditions, inflammatory processes, and especially represents a good alternative tool for non-invasive analysis of tumor-derived materials. Currently, tissue biopsies are still the gold standard for tumor profiling. Nevertheless, this technique presents many limitations that include invasiveness, risk and depending on some anatomical locations is not easy (or even impossible) to obtain. Moreover, it provides a limited picture of the tumor profile, considering that tumors are heterogeneous entities composed of different subpopulations of cells, which display a variability of genetic and epigenetic changes. In this context, liquid biopsies are a cheaper, faster, non-invasive alternative to conventional biopsies, that can be used for personalized cancer therapy. In a broad sense, liquid biopsy is based on the isolation of biomarkers from the blood that can be used for cancer diagnosis and monitoring. This definition englobes Circulating Tumor Cells (CTCs), circulating tumor DNA (ctDNA) and nanovesicles. CTCs are cancer cells, which leave the primary tumor and enter the bloodstream initiating a process called metastasis. Nevertheless, one of the most relevant challenges in this field involves the processing and analysis of CTCs, due to their low amount in peripheral blood (1 to 100 CTCs per 109 blood cells) and high heterogeneity. Furthermore, the approaches for isolating CTCs from blood samples are limited due to high cell contamination rates or substantial loss of cancer cells, and high-cost methods. To overcome these limitations, microfluidic devices have been designed for isolating CTCs based on their intrinsic properties like density, size, deformability, and difference in membrane protein expression. This project was undertaken to develop microfluidic devices for isolating CTCs based on inertial focusing and affinity binding principle methods. We first developed a spiral microfluidic device that can efficiently separate the CTCs from most of the blood cells by their differences in size by applying a hydrodynamic sorting principle. The CTC output sample is contaminated by the largest leukocytes (~12 to 21 μm) which are in the same size range as the CTCs (~9 μm to 30 μm). The research has also explored the development of microfluidic spiral devices using a 3D printer, in which the geometry dimensions were adapted to remove Leukocytes binding to polystyrene particles functionalized with CD45 antibody, allowing a more CTCs sample purity. Alternatively, second type of microfluidic device known as a Herringbone chip was designed to capture the remaining leukocytes (negative enrichment of CTCs) from the spiral CTC output sample. This device uses an affinity-binding principle based on a mixed Self Assembled Monolayer (SAM) composed of a Silane-PEG-Biotin, Silane-PEG-OH and CD45- antibody (common antigen for leukocytes).Moreover, the microfluidic platform was optimized for highthroughput blood sample processing including a lysis pre-treatment, guaranteeing a high recovery of CTC and its viability for further analysis. On the other hand, an electronic circuit was successfully developed using piezoelectric micropumps MP6 controlled by Raspberry PI zero, which allowed to overcome some limitations of traditional syringe pumps, as well as facilitate the use of this platform in a clinical environment. Finally, the clinical proof of concept was initiated with samples from colon cancer patients in collaboration with the Vall d'Hebron hospital.[spa] Las biopsias líquidas representan una buena herramienta alternativa para el análisis no invasivo de materiales derivados de tumores. Esta definición engloba las células tumorales circulantes (CTC), el ADN tumoral circulante (ctDNA) y las nanovesículas. Las CTC son células cancerosas que abandonan el tumor primario e ingresan al torrente sanguíneo iniciando un proceso llamado metástasis. Sin embargo, uno de los desafíos más relevantes implica el procesamiento y análisis de las CTC, debido a su baja cantidad en sangre periférica (1 a 100 CTC por 109 células sanguíneas) y su alta heterogeneidad. Además, los enfoques para aislar CTC de muestras de sangre son limitados debido a las altas tasas de contaminación celular, la pérdida sustancial de células cancerosas y los elevados costos. Para superar estas limitaciones, se han diseñado dispositivos microfluídicos para aislar CTC en función de sus propiedades intrínsecas como densidad, tamaño, deformabilidad y diferencia en la expresión de proteínas de membrana. Durante esta tesis se desarrollaron dispositivos microfluídicos para aislar CTC basados en métodos de principio de unión por afinidad y enfoque inercial. Primero, desarrollamos un dispositivo microfluídicos en espiral que puede separar eficientemente las CTC de la mayoría de las células sanguíneas por sus diferencias de tamaño mediante la aplicación de un principio de clasificación hidrodinámica. La muestra de salida de CTC está contaminada por los leucocitos más grandes (~12 a 21 µm) que están en el mismo rango de tamaño que las CTC (~9 µm a 30 µm). La investigación también ha explorado el desarrollo de dispositivos en espiral utilizando una impresora 3D, en los que las dimensiones geométricas se adaptaron para eliminar la unión de leucocitos a partículas de poliestireno fucionalizadas con anticuerpo CD45, permitiendo una mayor pureza de la muestra de CTC. Alternativamente, se diseñó un segundo tipo de dispositivo conocido como chip en espiga para capturar los leucocitos restantes (enriquecimiento negativo de CTC) de la muestra de salida de CTC en espiral. Este dispositivo utiliza un principio de unión por afinidad basado en una monocapa autoensamblada (SAM) mixta compuesta de silano-PEG-biotina, silano-PEG-OH y anticuerpo CD45 (antígeno leucocitario común). Por otro lado, se desarrolló con éxito un circuito electrónico utilizando microbombas piezoeléctricas MP6 controladas por Raspberry PI zero, lo que permitió superar algunas limitaciones de las bombas de jeringa tradicionales, así como facilitar el uso de esta plataforma en un entorno clínico. Finalmente, se inició la prueba clínica de concepto con muestras de pacientes con cáncer de colon en colaboración con el hospital Vall d'Hebron

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

Micro/Nano-Chip Electrokinetics

Micro/nanofluidic chips have found increasing applications in the analysis of chemical and biological samples over the past two decades. Electrokinetics has become the method of choice in these micro/nano-chips for transporting, manipulating and sensing ions, (bio)molecules, fluids and (bio)particles, etc., due to the high maneuverability, scalability, sensitivity, and integrability. The involved phenomena, which cover electroosmosis, electrophoresis, dielectrophoresis, electrohydrodynamics, electrothermal flow, diffusioosmosis, diffusiophoresis, streaming potential, current, etc., arise from either the inherent or the induced surface charge on the solid-liquid interface under DC and/or AC electric fields. To review the state-of-the-art of micro/nanochip electrokinetics, we welcome, in this Special Issue of Micromachines, all original research or review articles on the fundamentals and applications of the variety of electrokinetic phenomena in both microfluidic and nanofluidic devices

Micro/nano devices for blood analysis

[Excerpt] The development of microdevices for blood analysis is an interdisciplinary subject that demandsan integration of several research fields such as biotechnology, medicine, chemistry, informatics, optics,electronics, mechanics, and micro/nanotechnologies.Over the last few decades, there has been a notably fast development in the miniaturization ofmechanical microdevices, later known as microelectromechanical systems (MEMS), which combineelectrical and mechanical components at a microscale level. The integration of microflow and opticalcomponents in MEMS microdevices, as well as the development of micropumps and microvalves,have promoted the interest of several research fields dealing with fluid flow and transport phenomenahappening at microscale devices. [...

Investigation and characterization of MP derived from media conditioned by various cancer cell lines and their effect on human umbilical vein endothelial cells (HUVECs) under static and flow conditions

Microparticles (MP) are procoagulant due to tissue factor and phospholipid exposure on the surface. MP are tumour-derived and can be a beneficial biomarker of cancer to recognize individuals who are susceptible to venous thrombosis. The aim of the presented work was to develop and validate an in vitro microfluidic system consisting of two distinct microfluidic biochips to enable the investigation of the relationship between tumour MP and endothelial cells in vitro. Firstly, a range of tumour cell lines were assessed for procoagulant activity (PCA) of the cells and also MP released into the media. Pancreatic AsPC-1, human glioma U87 ovarian ES-2 and SKOV-3, were found to have the highest PCA in both cell suspension and cell- free media, while pancreatic MIAPaCa-2 and ovarian A2780 had a lower PCA. Cell lines were then investigated as to whether or not they could form stable spheroids in 3D cell culture U87, AsPC-1 and ES-2 produced the most compact spheroids and had the fastest PCA. In contrast, PANC-1, MIAPaCa-2 and A2780 formed loose shaped spheroids and slower PCA. However, SKOV-3 showed small compact spheroid and slower PCA. Following the application of media flow, ES-2 and U87 were selected and transferred into the developed dual microfluidic biochips model. Labelled MP were quantified via flow cytometry and this showed MP concentration reduced over time suggesting attachment of tumour MP to HUVECs. This reduction in MP was further reflected with a loss of PCA associated with the media. The effect of Doxorubicin on tumour spheroids resulted in an increased PCA of an endothelial cell layer under flow condition. In conclusion, in this study a microfluidic two-chip dynamic model mimics the interstitial fluid flow showed that tumour MPs released from tumour spheroids attach to endothelial cells and potentially could be a mechanism of clot formation in cancer patients

Determination of epithelial growth factor receptor mutations in circulatory tumour cells from non-small cell lung cancer patients isolated using a novel microfluidic device

Patients with epidermal growth factor receptor (EGFR) sensitizing mutations in non small cell lung cancer (NSCLC) receive benefit from Tyrosine Kinase inhibitors. Accurate selection of patients before treatment is highly dependent on precise molecular diagnosis of EGFR mutations. Presently in the clinic, the diagnostic samples routinely used tumour biopsy and/or cell free DNA (cfDNA), are not sufficiently effective for precise diagnosis. Circulatory tumour cells (CTC) in blood have been explored successfully as alternative and complementary diagnostic markers to the current clinical tools. However, utility in the clinics has been hampered by the relatively low concentration of CTC in blood, and the lack of robust technologies that are adaptable for routine use. The present study describes the design and optimization of an immunomagnetic based microfluidic device (Lung card version II) that isolates CTC expressing the epithelial cell adhesion molecule (EpCAM) from blood with high capture efficiency and purity. The device is a 2-part system comprising a disposable chip that is simple in design and a reusable microfluidic unit that contains a mobile magnetic arm. The simple design and work-flow process of the device ensures cost efficiency for scalability and, ultimately, use in the clinic. The device was initially validated for its capability to isolate EpCAM positive cells. Results from spiking carboxylfluorescein succinimidyl ester stained EpCAM positive cells in media/blood showed a capture efficiency of ≥ 65% and a purity ≥ 97% from a 13ml sample in 50 minutes. The isolated CTC from NSCLC patients (n=38) were analysed for mRNA markers specific to malignant cells and were characterized for EGFR mutations following PCR and next generation sequencing. The mutational status of CTC was compared to that obtained from matched, tumour biopsy, samples. Significantly more mutations (P=0.0173) were detected in CTC enriched samples than the matched biopsy. Interestingly, mutations were detected in only 4 biopsy samples and the mutations detected in the biopsy were only concordant with results from CTC enriched samples for 1 patient. Exon 19 deletion was the most frequent mutation detected (86.7%) with rare mutations such as: L792P, C797S, H509R also been detected in CTC, and the present study reports the detection of K708R mutation in NSCLC for the first time. The clinical outcomes of patients who were positive for EGFR mutation from CTC, but had been placed on therapies based on mutation results from tissue biopsy were evaluated in this study. The results showed that no significant progression free survival (PFS) benefit was attained when comparing treatment response between patients whose CTC possessed an EGFR mutation and patients whose CTC possessed no EGFR mutation (10 months vs26 months p value-0.3420 HR- 0.76 95% CI- 0.2498-2.319). In summary the results from this study showed that the microfluidic device captured CTC with efficiency equal to other immuno-affinity based devices but had better purity rates and throughput and also that the device can be utilized for CTC processing for downstream analysis. Results from this current study further demonstrated the clinical potential of CTC+NGS matrix for the detection of EGFR mutations and the prospective impact it would have for precision oncology in NSCLC are discussed

Nanostructured biosensors with DNA-based receptors for real-time detection of small analytes

In zahlreichen lebenswichtigen Bereichen haben sich Biosensoren als unverzichtbare Messgeräte erwiesen. Der Nachweis von spezifischen Molekülen im Körper für eine frühzeitige Krankheitserkennung erfordert empfindliche und zugleich zuverlässige Messmethoden. Ein rasantes Fortschreiten im Bereich der Nanotechnologie führt dabei zur Entwicklung von Materialien mit neuen Eigenschaften, und damit verbunden, auch zu innovativen Anwendungsmöglichkeiten im Bereich der Biosensorik. Das Zusammenspiel von Nanotechnologie und Sensortechnik gewährleistet die Konstruktion von Sensoren mit empfindlicheren Nachweisgrenzen und kürzeren Reaktionszeiten. Die Option zur Integration und Miniaturisierung stellen daher einen erfolgreichen Einsatz in direkter Patientennähe in Aussicht, sodass Nanobiosensoren die Brücke zwischen Laborddiagnostik und Standardanwendungen schließen können. Die folgende Arbeit widmet sich der Anwendung von nanostrukturierten Biosensoren für einen empfindlichen und markierungsfreien Nachweis von Zielmolekülen. Ein Hauptaugenmerk liegt dabei auf der kontinuierlichen Messung von Biomarkern mit kompakten Auslesesystemen, die eine direkte Signalmeldung und somit eine Detektion in Echtzeit ermöglichen. Dies erfordert zunächst die sorgfältige Funktionalisierung von Sensoroberflächen mit geeigneten DNA-basierten Rezeptoren. Infolgedessen werden beispielhaft verschiedene Sensorsysteme, Analyten und Charakterisierungsmethoden vorgestellt sowie universelle Strategien für die erfolgreiche Konfiguration von Nanobiosensorplattformen präsentiert. Das erste Anwendungsbeispiel widmet sich einem plasmonischen Biosensor, bei dem vertikal ausgerichtete Gold-Nanoantennen Signale mittels sog. lokalisierter Oberflächenplasmonenresonanz (LSPR) erzeugen. Mit dem Sensor konnte erfolgreich die Immobilisierung, das nachträgliche Blocken sowie die anschließende Hybridisierung von DNA nachgewiesen werden. Mithilfe des LSPR-Sensors wurden gleichzeitig grundlegende Hybridisierungsmechanismen auf nanostrukturierten und planaren Oberflächen verglichen und damit verbunden die einzigartigen optischen Eigenschaften metallischer Nanostrukturen betont. In einem zweiten Anwendungsbeispiel misst ein elektrischer Biosensor kontinuierlich die Konzentration des Stressmarkers Cortisol im menschlichen Speichel. Der direkte, markierungsfreie Nachweis von Cortisol mit Silizium-Nanodraht basierten Feldeffekttransistoren (SiNW FET) wurde anhand zugrunde liegender Ladungsverteilungen innerhalb des entstandenen Rezeptor-Analyte-Komplexes bewertet, sodass ein Nachweis des Analyten innerhalb der sog. Debye-Länge ermöglicht wird. Die erfolgreiche Strategie zur Oberflächenfunktionalisierung im Zusammenspiel mit dem Einsatz von SiNW FETs auf einem tragbaren Messgerät wurde anhand des Cortisolnachweises im Speichel belegt. Ein übereinstimmender Vergleich der gemessenen Corisolkonzentrationen mit Werten, die mit einer kommerziellen Alternative ermittelt wurden, verdeutlichen das Potential der entwickelten Plattform. Zusammenfassend veranschaulichen beide vorgestellten Nanobiosensor-Plattformen die vielseitige und vorteilhafte Leistungsfähigkeit der Systeme für einen kontinuierlichen Nachweis von Biomarkern in Echtzeit und vorzugsweise in Patientennähe.:Kurzfassung I Abstract III Abbreviations and symbols V Content VII 1 Introduction 1 1.1 Scope of the thesis 4 1.2 References 6 2 Fundamentals 9 2.1 Biosensors 9 2.2 Influence of nanotechnology on sensor development 10 2.3 Biorecognition elements 12 2.3.1 Biorecognition element: DNA 13 2.3.2 Aptamers 14 2.3.3 Immobilization of receptors 15 2.4 Transducer systems 17 2.4.1 Optical biosensors - surface plasmon resonance 17 2.4.2 Electric Biosensors – Field-effect transistors (FETs) 21 2.5 Metal oxide semiconductor field-effect transistor - MOSFET 21 2.6 Summary 26 2.7 References 27 3 Materials and methods 33 3.1 Plasmonic biosensors based on vertically aligned gold nanoantennas 33 3.1.1 Materials 33 3.1.2 Manufacturing of nanoantenna arrays 34 3.1.3 Surface modification and characterization 35 3.1.4 Measurement setup for detection of analytes 38 3.2 SiNW FET-based real-time monitoring of cortisol 40 3.2.1 Materials 40 3.2.2 Manufacturing of silicon nanowire field effect transistors (SiNW FETs) 42 3.2.3 Integration of SiNW FETs into a portable platform 42 3.2.4 Biomodification and characterization of electronic biosensors SiNW FETs 42 3.2.5 Electric characterization of FETs 47 3.3 References 50 4 Plasmonic DNA biosensor based on vertical arrays of gold nanoantennas 51 4.1 Introduction - Optical biosensors operating by means of LSPR 53 4.2 Biosensing with vertically aligned gold nanoantennas 56 4.2.1 Sensor fabrication, characterization, and integration 56 4.2.2 Integration of microfluidics 58 4.2.3 Immobilization of probe DNA and backfilling 58 4.2.4 Hybridization of complementary DNA strands 62 4.2.5 Surface coverage and hybridization efficiency of DNA 69 4.2.6 Refractive index sensing 72 4.2.7 Backfilling and blocking 73 4.3 Summary 75 4.4 References 77 5 Label-free detection of salivary cortisol with SiNW FETs 83 5.1 Introduction 85 5.2 Design, integration, and performance of SiNW FETs into a portable platform 89 5.2.1 Structure and electrical characteristics of honeycomb SiNW FETs 89 5.2.2 Integration of SiNW FET into a portable measuring unit 91 5.2.3 Performance of SiNW FET arrays 93 5.3 Detection of biomolecules with SiNW FETs 102 5.3.1 General considerations for biodetection with FETs 102 5.3.2 Sensing aptamers with FETs 103 5.3.3 Biodetection of the analyte cortisol with SiNW FETs 104 5.3.4 Detection of cortisol with SiNW FETs 112 5.4 Summary 119 5.5 References 121 6 Summary and outlook 131 6.1 Summary 131 6.2 Perspectives – toward multiplexed biosensing applications 134 6.3 References 137 Appendix i A.1 Protocols i A.1.1 Functionalization of gold antennas with thiolated DNA i A.1.2 Functionalization of SiO2 with TESPSA and amino-modified receptors i A.1.3 Functionalization with APTES and carboxyl-modified receptors ii A.1.4 Preparation of microfluidic channels via soft lithography ii A.2 Predicted secondary structures iv A.2.1 Secondary structures of 100base pair target without probe-strands iv A.2.2 Secondary structures of 100base pair target with 25 base pair probe-strand x Versicherung xvii Acknowledgments xix List of publications xxi Peer-reviewed publications xxi Publications in preparation xxi Selected international conferences xxii Curriculum Vitae xxiiiBiosensors have proven to be indispensable in numerous vital areas. For example, detecting the presence and concentration of specific biomarkers requires sensitive and reliable measurement methods. Rapid developments in the field of nanotechnology lead to nanomaterials with new properties and associated innovative applications. Thus, nanotechnology has a far-reaching impact on biosensors' development, e.g., delivery of biosensing devices with greater sensitivity, shorter response times, and precise but cost-effective sensor platforms. In addition, nanobiosensors hold high potential for integration and miniaturization and can operate directly at the point of care - serving as a bridge between diagnostics and routine tests. This work focuses on applying nanostructured biosensors for the sensitive and label-free detection of analytes. A distinct aim is the continuous monitoring of biomarkers with compact read-out systems to provide direct, valuable feedback in real-time. The first step in achieving this goal is the adequate functionalization of nanostructured sensor surfaces with suitable receptors to detect analytes of interest. Due to their thermal and chemical stability with the possibility for customizable functionalization, DNA-based receptors are selected. Thereupon, universal strategies for confining nanobiosensor platforms are presented using different sensor systems, analytes, and characterization methods. As a first application, a plasmonic biosensor based on vertically aligned gold nanoantennas tracked the immobilization, blocking, and subsequent hybridization of DNA by means of localized surface plasmon resonance (LSPR). At the same time, the LSPR sensor was used to evaluate fundamental hybridization mechanisms on nanostructured and planar surfaces, emphasizing the unique optical properties of metallic nanostructures. In a second application, an electric sensor based on silicon nanowire field-effect transistors (SiNW FET) monitored the level of the stress marker cortisol in human saliva. Based on evaluating the underlying charge distributions within the resulting receptor-analyte complex of molecules, the detection of cortisol within the Debye length is facilitated. Thus, direct, label-free detection of cortisol in human saliva using SiNW FET was successfully applied to the developed platform and compared to cortisol levels obtained using a commercial alternative. In summary, both presented platforms indicate a highly versatile and beneficial performance of nanobiosensors for continuous detection of biomarkers in real-time and preferably point-of-care (POC).:Kurzfassung I Abstract III Abbreviations and symbols V Content VII 1 Introduction 1 1.1 Scope of the thesis 4 1.2 References 6 2 Fundamentals 9 2.1 Biosensors 9 2.2 Influence of nanotechnology on sensor development 10 2.3 Biorecognition elements 12 2.3.1 Biorecognition element: DNA 13 2.3.2 Aptamers 14 2.3.3 Immobilization of receptors 15 2.4 Transducer systems 17 2.4.1 Optical biosensors - surface plasmon resonance 17 2.4.2 Electric Biosensors – Field-effect transistors (FETs) 21 2.5 Metal oxide semiconductor field-effect transistor - MOSFET 21 2.6 Summary 26 2.7 References 27 3 Materials and methods 33 3.1 Plasmonic biosensors based on vertically aligned gold nanoantennas 33 3.1.1 Materials 33 3.1.2 Manufacturing of nanoantenna arrays 34 3.1.3 Surface modification and characterization 35 3.1.4 Measurement setup for detection of analytes 38 3.2 SiNW FET-based real-time monitoring of cortisol 40 3.2.1 Materials 40 3.2.2 Manufacturing of silicon nanowire field effect transistors (SiNW FETs) 42 3.2.3 Integration of SiNW FETs into a portable platform 42 3.2.4 Biomodification and characterization of electronic biosensors SiNW FETs 42 3.2.5 Electric characterization of FETs 47 3.3 References 50 4 Plasmonic DNA biosensor based on vertical arrays of gold nanoantennas 51 4.1 Introduction - Optical biosensors operating by means of LSPR 53 4.2 Biosensing with vertically aligned gold nanoantennas 56 4.2.1 Sensor fabrication, characterization, and integration 56 4.2.2 Integration of microfluidics 58 4.2.3 Immobilization of probe DNA and backfilling 58 4.2.4 Hybridization of complementary DNA strands 62 4.2.5 Surface coverage and hybridization efficiency of DNA 69 4.2.6 Refractive index sensing 72 4.2.7 Backfilling and blocking 73 4.3 Summary 75 4.4 References 77 5 Label-free detection of salivary cortisol with SiNW FETs 83 5.1 Introduction 85 5.2 Design, integration, and performance of SiNW FETs into a portable platform 89 5.2.1 Structure and electrical characteristics of honeycomb SiNW FETs 89 5.2.2 Integration of SiNW FET into a portable measuring unit 91 5.2.3 Performance of SiNW FET arrays 93 5.3 Detection of biomolecules with SiNW FETs 102 5.3.1 General considerations for biodetection with FETs 102 5.3.2 Sensing aptamers with FETs 103 5.3.3 Biodetection of the analyte cortisol with SiNW FETs 104 5.3.4 Detection of cortisol with SiNW FETs 112 5.4 Summary 119 5.5 References 121 6 Summary and outlook 131 6.1 Summary 131 6.2 Perspectives – toward multiplexed biosensing applications 134 6.3 References 137 Appendix i A.1 Protocols i A.1.1 Functionalization of gold antennas with thiolated DNA i A.1.2 Functionalization of SiO2 with TESPSA and amino-modified receptors i A.1.3 Functionalization with APTES and carboxyl-modified receptors ii A.1.4 Preparation of microfluidic channels via soft lithography ii A.2 Predicted secondary structures iv A.2.1 Secondary structures of 100base pair target without probe-strands iv A.2.2 Secondary structures of 100base pair target with 25 base pair probe-strand x Versicherung xvii Acknowledgments xix List of publications xxi Peer-reviewed publications xxi Publications in preparation xxi Selected international conferences xxii Curriculum Vitae xxii

Microfluidics for Biosensing

There are 12 papers published with 8 research articles, 3 review articles and 1 perspective. The topics cover: Biomedical microfluidics Lab-on-a-chip Miniaturized systems for chemistry and life science (MicroTAS) Biosensor development and characteristics Imaging and other detection technologies Imaging and signal processing Point-of-care testing microdevices Food and water quality testing and control We hope this collection could promote the development of microfluidics and point-of-care testing (POCT) devices for biosensing