INTEGRATED MICROSYSTEM-BASED APPROACH FOR DETECTION AND TREATMENT OF BACTERIAL BIOFILMS ON URINARY CATHETERS

Biofilms are a ubiquitous mode of growth for bacteria and present a significant challenge in healthcare due to their resistant nature towards traditional antibiotic therapy. Particularly, biofilms can form on indwelling urinary catheters, leading to catheter-associated urinary tract infections, which are one of the most prevalent healthcare-acquired infections. In recent years, microsystems-based approaches have been developed to measure and study bacterial biofilms. In this dissertation, microsystems are adapted for the catheterized urinary tract environment to address biofilm infections in situ. Specifically, a proof-of-concept device comprised of gold interdigitated electrodes on a flexible polyimide substrate is fabricated and characterized in vitro. This substrate allows the device to conform seamlessly with the cylindrical surface of a catheter. Real-time impedance sensing is demonstrated, showing an average decrease in impedance of 30.3% following 24 hours of biofilm growth. The device also applies the bioelectric effect, which yields an increase in impedance of 12% and the lowest biomass relative to control treatments. Furthermore, 3D-printed molds and commercial modeling software show that the cylindrical conformation does not have an appreciable impact on performance. This device is integrated with a commercially available Foley catheter using two disparate approaches: (1) integration of the flexible proof-of-concept device using a 3D-printed catheter insert and (2) electroless plating directly onto the catheter lumen. In addition to electrode integration, miniaturized electronic systems are developed to control sensing and treatment wirelessly with a minimal form factor. A smartphone mobile application is developed in conjunction with this effort, to provide a user-friendly interface for the system. Several functions are verified with the integrated system, including biofilm sensing, wireless signal transmission, bladder drainage, and balloon inflation. To decrease the risk associated with this system for future research in vivo and in a clinical setting, sensing and treatment are evaluated under realistic conditions. The biochemical aspect of the catheterized environment is recreated using artificial urine, and the physical aspect is recreated using a silicone model of a human bladder and a programmable pump. A 13.0% decrease in impedance is associated with bacterial growth; this decreased magnitude relative to the proof-of-concept device is due to the reduced degree of growth in artificial urine. The bioelectric effect is demonstrated as well, showing a reduction in planktonic bacteria of 1.50×107 CFU/ml and adhered biomass equivalent to OD590nm = 0.072 relative to untreated samples. This work provides a framework for developing microsystem-based tools for biofilm infection management and research from proof-of-concept to integrated system, particularly for CAUTI. The results demonstrate that the cylindrical conformation does not interfere with device sensing or treatment performance and that the system maintains functionality under realistic conditions, laying the groundwork for future in vivo and clinical testing. The system will provide in situ and real-time data regarding catheter biofilm colonization in a way that is not possible with existing techniques. Finally, the system can serve to reduce reliance on antibiotics and reduce the spread of antibiotic resistance in CAUTI and other vulnerable areas

Disposable Lab-on-Chip Systems for Biotechnological Screening

The main goal of this work was to develop different disposable Lab-on-Chip (LoC) systems for the application of biotechnological screening e.g. for bioprocess development through microorganisms or drug testing with human cell lines. Nowadays, microfluidics represents a highly promising field for the fabrication of microtools, as the increasing demand for screening data are difficult to meet with current platforms. This is mainly due to time and cost aspects as well as a limited amount of newly developed drugs. The ideal microfluidic platform for biotechnological screening should include three different groups of elements: (i) microbioreactors (MBR) in which cultivation takes place; (ii) auxiliary microfluidic systems (for transportation, filtration or mixing), and (iii) enzymatic biosensors for onchip analysis of substrate concentrations which are difficult to measure offline due to small available sample volumes. Within the scope of this work, various horizontally and vertically positioned MBR designs (resembling plug flow reactors, micro stir tanks or bubble columns) were developed, fabricated and successfully applied to the screening of different biological expression systems, such as yeast cells (S. cerevisiae), fungal spores (A. ochraceus) and primary human endothelial cells. Different integrated functional structures based on geometrical, optical or electrical elements allowed for online monitoring of various physical, chemical and biological process parameters during cultivation. In terms of the second group, passive and active microvalves, PZTand pneumatically actuated micropumps, passive filtration and mixing elements were produced. The third group comprised different types of enzymatic biosensors based on a hybrid detection principle (electrochemical-optical) and on different types of enzymatic responses. In general, the unique LoC setup (patterned element made of poly(dimethylsiloxane) and bonded to a glass substrate) allows an easy integration of systems into one monolithic LoC platform which are usually better suited for technically mature systems. Modular systems are advantageous for prototyping of new microfluidic applications. Therfore, an LoC construction kit was developed that offers a user friendly, standardized modular platform.Im Rahmen der Dissertation wurden verschiedene Einweg-Lab-on-Chip Systeme entwickelt, die beispielsweise bei biotechnologischen Parameterstudien von Mikroorganismen zur Bioprozesssteigerung oder von humanen Zelllinien zum Wirkstoffscreening Anwendung finden. Die Mikrofluidik ist ein vielversprechendes Forschungsgebiet für die Herstellung von kostengünstigen Mikrochips, womit der steigende Bedarf für Screening-Daten aufgrund von Vorteilen wie Zeit- und Kostenreduzierung erfüllt werden kann. Eine ideale mikrofluidische Plattform zum biotechnologischen Screenen sollte aus folgenden Gruppen bestehen: (i) dem Mikrobioreaktor zur Kultivierung, (ii) mikrofluidische Komponenten zum Transportieren, Filtrieren und Mischen von Suspensionen, und (iii) einem enzymatischen Biosensor für die on-Chip Analyse von Substratkonzentrationen. Innerhalb der Arbeit wurden diverse horizontal und vertikal positionierte Mikrobioreaktoren entwickelt, hergestellt und erfolgreich zum Screenen von unterschiedlichen biologischen Expressionssystemen (wie S. cerevisiae, A. ochraceus und humane Endothelzellen) angewendet. Die Integration von geometrischen, optischen und elektrischen Funktionselementen erlaubte eine online Überwachung von verschiedenen physikalischen, chemischen und biologischen Prozessparametern während der Kultivierung. Im Bereich der Gruppe (ii) wurden passive und aktive Mikoventile, PZT- und pneumatisch aktuierte Mikropumpen, Filtrations- und Mischkomponenten hergestellt und charakterisiert. Gruppe (iii) umfasste die Entwicklung eines enzymatischen Biosensors mit hybridem (elektrochemisch-optisch) Messumformer. Der einheitliche Chipaufbau aller Lab-on-Chip Systeme – bestehend aus einer Kombination von strukturiertem Polydimethylsiloxan und Glas – erlaubt das monolithische und modulare Zusammenschalten der Einzelsysteme zu der gewünschten Plattform. Da für erste Prototypen eine modulare Verschaltung zu bevorzugen ist, wurde ein Baukastensystem entwickelt, welches eine standardisierte und benutzerfreundliche Plattform für flexible Versuchsaufbauten bietet

Micro Surface Discharge for Plasma-Assisted Catalysis in Portable Fuel Cell Reforming Applications

The production of non-equilibrium plasma at atmospheric pressure has and continues to prove advantageous for low-temperature applications where use of vacuum pumps is not feasible. Microplasmas, plasma generated in sub-millimeter dimensions, have proven capable of producing such characteristics for homogeneous chemical reactions and, as of late, have been designed to allow a portable operation with low power requirements. In this work, the novel design and application of a barrier type electrical discharge reactor, a so called micro surface discharge (MSD) reactor, is presented for application in plasma-assisted catalytic fuel reformation for a portable High-Temperature (HT) Polymer Electrolyte Membrane Fuel Cell (PEMFC). The reactor is optimized for temperature durability and leak tightness based on material choice and geometry. The generation of the high electric fields needed for electrical breakdown were simulated and analytically contemplated. Using micro-technological processes, the combination of electrical and fluidic components was realized monolithically. The practical feasibility of the device was strongly taken into consideration, with focus on the device-fuel cell integration and minimal reactor volume, which was dependent on the adjustment of the reactor design with respect to the smallest industrially available generator components. The reactor was optically, electrically and thermally analyzed in order to characterize the breakdown mode and plasma properties. The use of the MSD reactor for the reformation of methane into hydrogen for application in fuel cells was investigated, using a purpose built enclosure to connect the micro-structured reactor to various macroscopic interfaces, such as standard pneumatic and electrical connections. The conversion rate, product selectivity and reaction efficiency were examined with varying flow rates, inlet gas compositions, heterogeneous catalyst presence and plasma power loads. Significant methane conversion, as well as thermal, mechanical and chemical durability of the reactor was observed. Specifically, a synergistic effect was discovered where the production of hydrogen was significantly higher with a combination of plasma and catalyst than the addition of their productions separately. Future experiments with other fuels and at elevated temperatures could continue to develop and prove the feasibility of this reactor in portable reforming applications. This case study has proven significant potential for portable power generation applications and is the first truly miniaturized portable reactor optimized for such applications. The results from this work should serve as a basis for further research of even more compact systems with even higher efficiency.Die Erzeugung eines Nichtgleichgewichtsplasmas bei Atmosphärendruck zeigt immer wieder Vorteile bei Niedertemperatur-Anwendungen und erlaubt den Betrieb, wo Vakuumanlagen nicht realisierbar sind. Mikroplasmen, Nichtgleichgewichtsplasmen in Geometrien von weniger als einem Millimeter, haben insbesondere gute Eigenschaften hinsichtlich der homogenen chemischen Reaktion und des niedrigen Leistungsbedarfes, so dass sie für portable Anwendungen geeignet sind. In dieser Arbeit wird das neuartige Design eines Barrierenentladungsreaktors, genauer eines sogenannten Mikrooberflächenentladungsreaktors (MSD), für die Anwendung in plasmaunterstützten katalytischen Brennstoffreformern präsentiert, um in einer Hochtemperatur (HT) Polymer-Elektrolyt-Membran-Brennstoffzelle eingesetzt zu werden. Die Erzeugung von hohen elektrischen Feldern, die für die Entladung benötigt werden, wurde simuliert und die einzelnen Komponenten analytisch betrachtet. Durch Anwendung von mikrotechnologischen Prozessen wurde die monolithische Kombination elektrischer und fluidischer Komponenten realisiert. Die praktische Machbarkeit des Reaktors wurde während der Auslegung berücksichtigt, mit einem Fokus auf der Integration in eine Brennstoffzelle und minimalem Volumen. Dieses war abhängig von der Anpassung des Reaktors an die kleinsten industriell verfügbaren Generatorkomponenten. Um die Entladungs- und Plasmaeigenschaften zu charakterisieren, wurde der Reaktor optisch, elektrisch und thermisch analysiert. Die Verwendung des MSD Reaktors wurde für die Reformierung von Methan in Wasserstoff für Brennstoffzellenanwendungen untersucht. Dazu wurde ein spezielles Gehäuse angefertigt, um den mikrostrukturierten Reaktor mit pneumatischen und elektrischen Makroverbindungen zu verschließen. Die Umsatzrate, die Produktselektivität und der Wirkungsgrad der Reaktion wurde für unterschiedliche Durchflussraten des Edukts, dessen Zusammensetzung, sowie bei Anwesenheit eines heterogenen Katalysators, bei verschiedenen Plasmaleistungen durchgeführt. Der wesentliche Umsatz des Methans, sowie thermische, mechanische und chemische Beständigkeit des Reaktors wurden demonstriert. Besonders wurde ein synergistischer Effekt entdeckt, wobei sich die Bildung von Wasserstoff signifikant steigerte, bei der Verwendung einer Kombination von Plasma und einem Katalysator gegenüber dem Betrieb ohne. Zukünftige Versuche mit anderen Brennstoffen und bei erhöhter Temperatur könnten zu einer Weiterentwicklung führen und einen Beweis für die Umsetzbarkeit des Reaktors liefern. Diese Fallstudie zeigt, dass derartige Reaktoren ein großes Potential für portable Leistungsquellen haben. Es handelt sich bei den untersuchten Reaktoren um die den ersten miniaturisierten, portablen Reaktoren. Die Ergebnisse dieser Arbeit sollen als Basis für weitere Forschung dienen

Development of a cell encapsulation technology for the production of functional, micro-encapsulated pancreatic islets for transplantation

Previously held under moratorium from 8 August 2019 until 9 December 2021Diabetes type 1 is an autoimmune disease in which the patient’s own immune system destroys the insulin producing β-cells, located in the pancreatic islets. Without enough insulin production, the blood glucose levels of the patient rise, which can lead to damages of blood vessels and nerves, blindness or even seizures and comas. For some patients that have trouble maintaining normoglycaemia allogeneic islet transplantation has become an alternative treatment option. Patients with these transplanted islets are no longer prone to hypoglycaemic episodes and can sometimes become completely insulin independent. However, this success is not long-lived. The life span of the transplanted islets is limited due to the host’s immune responses and the toxicity of modern immunosuppressive agents. In this thesis, islet encapsulation for clinical transplantation is investigated and further developed. Islet encapsulation can protect the islets from the immune system, without the aid of the immunosuppressants. The construction and optimization of a micro-encapsulator that can be used to create encapsulations is described, as well as the multiple parameters to create small, uniform encapsulations. To further enhance the biocompatibility and immunoprotective properties of alginate hydrogel, alginate was purified to eliminate most of the impurities and tested for its permeability. Encapsulating pancreatic islets in this purified alginate showed encouraging results, with the islets remaining viable and functional longer than their control counterparts. Larger islets can develop necrotic cores within encapsulations, due to the lack of vascularization. To create smaller islets out of dissociated larger islets, a single-step encapsulation and aggregation method was developed, that unfortunately was not suitable for islet cells, but was capable of developing functional hepatic organoids out of HepaRG cells, that could be used for drug testing. Finally, a proof of principle was given for the creation of pancreatic islet patches using 3D bioprinting methods.Diabetes type 1 is an autoimmune disease in which the patient’s own immune system destroys the insulin producing β-cells, located in the pancreatic islets. Without enough insulin production, the blood glucose levels of the patient rise, which can lead to damages of blood vessels and nerves, blindness or even seizures and comas. For some patients that have trouble maintaining normoglycaemia allogeneic islet transplantation has become an alternative treatment option. Patients with these transplanted islets are no longer prone to hypoglycaemic episodes and can sometimes become completely insulin independent. However, this success is not long-lived. The life span of the transplanted islets is limited due to the host’s immune responses and the toxicity of modern immunosuppressive agents. In this thesis, islet encapsulation for clinical transplantation is investigated and further developed. Islet encapsulation can protect the islets from the immune system, without the aid of the immunosuppressants. The construction and optimization of a micro-encapsulator that can be used to create encapsulations is described, as well as the multiple parameters to create small, uniform encapsulations. To further enhance the biocompatibility and immunoprotective properties of alginate hydrogel, alginate was purified to eliminate most of the impurities and tested for its permeability. Encapsulating pancreatic islets in this purified alginate showed encouraging results, with the islets remaining viable and functional longer than their control counterparts. Larger islets can develop necrotic cores within encapsulations, due to the lack of vascularization. To create smaller islets out of dissociated larger islets, a single-step encapsulation and aggregation method was developed, that unfortunately was not suitable for islet cells, but was capable of developing functional hepatic organoids out of HepaRG cells, that could be used for drug testing. Finally, a proof of principle was given for the creation of pancreatic islet patches using 3D bioprinting methods

Wafer scale integration of coulomb blockade-based nanobiosensors with microfluidic channels for label-free detection of cancer biomarkers

Dans cette thèse, nous proposons et démontrons un nouveau type de nanobiocapteur pour la détection de biomolécules à haute sensibilité et leur intégration à grande échelle (plaquette de 4 pouces). Le principe du nouveau nanobiocapteur électrique est basé sur la variation de conductivité électrique à travers des nano-îlots grâce au phénomène quantique appelé « blocage de coulomb ». Les nano-îlots de nickel (5nm de diamètre) sont placés entre les nano-électrodes interdigitées (IND) (~45nm de largeur). La conductivité de ces dispositifs à jonctions tunnel multiples (MTJ) est modifiée par l’adsorption de biomarqueurs impliqués dans la tumorogènese. Les oncologues ont récemment isolé et caractérisé un nouveau fragment d’anticorps à chaine simple (scFv) qui reconnaît sélectivement la forme active de RhoA. Ce biomarqueur potentiel a été trouvé surexprimé dans diverses tumeurs. Les fragments d’anticorps ont été adsorbés, par des liaisons de coordination, sur les nano-îlots de nickel. Ces fragments sont capables de reconnaître spécifiquement la forme active de RhoA. Nous avons étudié ce biomarqueur et validé la chimie de surface à base d’îlots de nickel pour la détection sans marquage, en utilisant une microbalance à quartz (QCM). Puis, nous avons mis au point et adapté à notre dispositif une méthodologie innovatrice pour réaliser, à l’échelle d’une plaquette, des microcanaux basés sur du photoPDMS. La caractérisation électrique finale des dispositifs intégrés a été testée en temps réel et à flux biologique continu. La forme active de RhoA a été détectée en discriminant la forme inactive. En annexe, je présente mon opinion épistémologique et éthique sur la nanotechnologie ___ In this thesis we propose and implement the fabrication on 4 inch wafer of a novel type of nanobiosensor capable of high sensitivity detection. The principle of the nanobiosensor is based on the variation of electrical tunnelling conductivity through metal nanoislands due to the quantum phenomenon called coulomb blockade. Nickel nanoislands(~5nm diameter), are placed between interdigitated nanoelectrodes devices (IND) (width~45nm). Hence, the conductivity of these Multiple-Tunnel-Junction (MTJ) devices is modified by the absorption of biomarkers involved in tumourigenesis. Oncologists have recently isolated and characterised a new conformational single chain variable fragment (scFv) which selectively recognises the active form of RhoA. This potential biomarker has been found overexpressed in various tumours. Antibodies fragments (scFv) are absorbed through coordinative bonds onto nickel nanoislands. Hence the scFv are capable of recognising specifically the active RhoA conformation. We have investigated this biomarker and validated the nickel nanoilands based chemical construction for label-free biodetection using quartz crystal microbalance (QCM) before implementing the methodology to our devices. An innovative methodology to realise photoPDMS-based microchannels was also developed. Encapsulation with an etched PDMS-nanocomposite finalised the integration of the devices. The final electrical characterisation of the integrated device was tested in real time and continuous biological flow. The active form of RhoA was discriminated against its inactive conformation. In annexe, I present my epistemological and ethical opinions in nanotechnolog