Das Ziel dieser Arbeit war die Entwicklung eine Gerätes, welches die Überwachung von mehreren hundert Mikrobioreaktoren ermöglicht. Dabei sollte es, neben der Reduzierung von Kosten und Arbeitskraft, die konventionellen Pipettiermethoden in der zeitliche Auflösung übertreffen. Weiterhin soll es über eine Gradientenerzeugung verfügen, um sehr feine Variationen der Zusammensetzung des verwendeten Mediums zu ermöglichen. Dafür wurde ein Analysator für fluoreszierenden Tropfen (Fluorescent Droplet Analyser) entwickelt, mit dem ein segmentierter Fluss von mehreren hundert Tropfen erzeugt werden kann und für viele Tage gemessen werden kann. Für die Messung wurde der Analysator mit einer flexiblen Fluoreszenzoptik ausgestattet um unterschiedliche Fluoreszenzfarbstoffe oder Molekühle detektieren zu können. Mehrere Experimente wurden durchgeführt, welche das Potentials des Gerätes zeigen. Einzelne Zellen des Pantoffeltierchen (Paramecium tetraurelia) konnten in einzelnen Tropfen eingeschlossen werden und mit einem metabolischen Farbstoffe ihre Stoffwechselaktivität gemessen werden. Ebenfalls wurden viele Experimente mit Pseudomonas fluorescens und E.coli YFP durchgeführt. Durch die flexible Fluoreszenzoptik konnte das Wachstum beider Arten in eine Experiment gemessen werden.:1 Introduction 1 1.1 Motivation 1 1.2 Scope of this thesis 2 1.3 State of the art 3 2 Fundamentals 2.1 Millifluidics vs. microfluidics 5 2.1.1 Droplet based microfluidics 6 2.1.2 Surfactant in droplet-based millifluidics 7 2.2 The model of microorganism growth 8 2.3 The fluorescence based detection 9 3 Materials & Methods 3.1 The culture of the microorganisms and preparation 11 3.1.1 The Paramecium tetraurelia 11 3.1.2 Pseudomonas fluorescens 12 3.1.3 Escherichia coli 12 3.1.4 The bacteria culture 13 3.2 The Poisson distribution . 15 3.3 The composition of the emulsion 16 3.4 The fluidic components 17 3.5 Modules of the Fluorescent Droplet Analyser 18 3.5.1 The optocoupled relay card 18 3.5.2 The used fluidic pumps 19 3.5.3 The photonic elements of the FDA 19 3.5.4 The light sources 21 3.5.5 The optical fibres 23 3.5.6 The used detectors 23 3.6 The operating Software 24 4 The Fluorescent Droplet Analyser 4.1 The fluidic network for droplet generation and shuttling 26 4.1.1 Generation of a droplet sequence 28 4.1.2 Measuring a droplet sequence 30 4.2 The electric schematics of the FDA 31 4.3 The light source, guidance, and detection 32 4.3.1 Preparation of the fibres 33 4.3.2 The detection setup 35 4.4 The developed LabView program for operating the FDA 37 4.4.1 The automated measurement process 40 4.4.2 The peak mode 41 4.4.3 The time mode 42 4.4.4 The combined mode 42 4.4.5 The dynamic threshold 42 4.5 The developed VI program for analysing the data of the FDA 44 4.6 Flow characteristics of the FDA 46 4.6.1 Basic flow characteristics 46 4.6.2 Characteristics for shuttling of a droplet sequence 47 4.6.3 Influence of the hydrodynamic resistance 48 5 Monitoring of Paramecium tetraurelia 5.1 Introduction 51 5.2 Generation of a droplet sequence for Paramecium tetraurelia 53 5.3 Metabolic dynamics in droplets 54 5.4 Ecotoxicity assays 58 6 Monitoring of bacteria 6.1 Introduction 63 6.2 Generation of a droplet sequence for the bacteria experiments 64 6.3 Cell number calibration of the FDA 65 6.4 Monitoring of the bacteria growth 68 6.5 Investigation of the of the fluorescence signal 70 6.6 One bacteria cell in a droplet 71 6.7 The determination of the minimum inhibitory concentration 74 6.8 Long-term monitoring 80 6.9 Sectioning of the droplet sequence 80 6.10 Multicolor fluorescence detection 83

The field of organ-on-chip is a relatively new area of research and builds upon the principle of engineering microfluidic systems to mimic the body’s internal environment as precisely as possible. Eventually these models could hopefully simulate whole organ-systems and enable the examination of the cell’s or organ’s reaction to foreign substances like new pharmaceuticals in a better way than current models. Previously this has been done with in vitro models such as petri dishes that only offer static culturing conditions. These are not very realistic environments compared to the human body where the cells are exposed to both variations in pressure and flows among other things. The purpose of this bachelor’s thesis project has been to evaluate and improve the design of an organ-on-chip system developed by the EMBLA-group at Ångströmslaboratoriet, Uppsala university. This has been done by evaluating the manufacturing process to find areas of improvements of the current chip design, as well as conducting a literature study to understand key components of similar organ-on-chip systems and see if it is possible to implement relevant parts to the organ-on-chip of this project. One of these important parts is a so-called bubble trap. A bubble trap is a construction that enables the capturing and elimination of bubbles in the system since the bubbles can harm the chips components, kill the cells, and compromise measurements.  A first prototype of the bubble trap was developed in Polydimethylsioxane (PDMS) and integrated on the EMBLA-group’s chip design. The principle behind the bubble trap was to use the natural buoyancy of the bubbles to trap them. This was done by introducing an upwards going slope before the inlets to the chip. In this manner the bubbles would float up to the top of the slope and accumulate at the roof as the liquid moved on into the chip without bubbles. To make the bubbles leave the chip a low-pressure chamber was added on top of the bubble trap to help the process of the bubble’s diffusion through the roof and out of the chip. The development of an improved chip design turned out to be a time-consuming endeavor and the time left for evaluation the functionality of the chip became too short. One test was performed which showed that the bubbles did accumulate at the top of the slope as expected, but it rapidly became full and thus started to let bubbles through to the microfluidic chip. The bubbles did not diffuse as efficiently as required and the removal of the bubbles became inefficient. To understand and correct the problem areas of this bubble trap design further tests and experiments will have to be conducted. / Organ-på-chip (Organ-on-chip eller OoC) är ett relativt nytt forskningsområde som bygger på att mikrofluidiksystem utvecklas till att efterlikna människokroppen i så stor utsträckning som möjligt. Detta då det är attraktivt att kunna undersöka cellers/organs beteende vid tillförsel av vissa substanser, till exempel nya läkemedel. I tidigare in vitro modeller har det endast observerats och utförts tester på celler odlade i statiska förhållanden vilket inte är likt den omgivning cellerna har i människokroppen där de tex utsätts för olika vätskeflöden och tryckförändringar.    Syftet med detta examensarbete har varit att utvärdera och förbättra designen på ett OoC system utvecklat av EMBLA-gruppen på Ångströmlaboratoriet vid Uppsala universitet. Detta har gjorts genom att studera den nuvarande tillverkningsprocessen för att hitta relevanta förbättringsområden samt att genom en litteraturstudie undersöka viktiga delar som bör ingå i dessa typer av system. En av dessa delar är en bubbelfälla (bubble trap eller BT) vilket innebär att det i chippet bör finnas ett sätt att eliminera/fånga upp bubblor. Detta eftersom bubblorna kan orsaka stor skada på både chipet, cellerna och mätningarna som skall utföras. En första prototyp av en BT design i Polydimetylsiloxan (PDMS) utvecklades och integrerades på EMBLA-gruppens OoC design. Principen bakom BT-designen var att utnyttja bubblornas flytkraft vilket gjordes genom att introducera en uppåtgående backe innan ingångskanalen. Bubblorna kan därmed flyta upp till toppen av lutningen och vätskan kan fortsätta in i mikrochipset utan bubblor. För att bubblorna ska ta sig ut ur chippet integrerades en tryckkammare ovanpå BT-designen för att få bubblorna att diffundera ut genom taket i den uppåtgående kammaren och ut ur chippet. Utvecklingen av den förbättrade chip-designen visade sig var tidskrävande och tiden för att utvärdera designens funktionalitet blev för kort. Ett test gjordes på den nya chip-designen vilket visade att den utvecklade BT som väntat fångade upp bubblor men att den snabbt blev full i och med att bubblorna inte diffunderade ut genom taket i den takt som behövdes. Vidare undersökningar och experiment behövs för att evaluera vad som orsakade detta och rätta till eventuella felkällor i design och experimentuppställning.

This work is devoted to investigate light confinement in rolled-up microtubular cavities and their optofluidic applications. The microcavities are fabricated by a roll-up mechanism based on releasing pre-strained silicon-oxide nanomembranes. By defining the shape and thickness of the nanomembranes, the geometrical tube structure is well controlled. Micro-photoluminescence spectroscopy at room temperature is employed to study the optical modes and their dependence on the structural characteristics of the microtubes. Finite-difference-time-domain simulations are performed to elucidate the experimental results. In addition, a theoretical model (based on a wave description) is applied to describe the optical modes in the tubular microcavities, supporting quantitatively and qualitatively the experimental findings. Precise spectral tuning of the optical modes is achieved by two post-fabrication methods. One method employs conformal coating of the tube wall with Al2O3 monolayers by atomic-layer-deposition, which permits a mode tuning over a wide spectral range (larger than one free-spectral-range). An average mode tuning to longer wavelengths of 0.11nm/ Al2O3-monolayer is obtained. The other method consists in asymmetric material deposition onto the tube surface. Besides the possibility of mode tuning, this method permits to detect small shape deformations (at the nanometer scale) of an optical microcavity. Controlled confinement of resonant light is demonstrated by using an asymmetric cone-like microtube, which is fabricated by unevenly rolling-up circular-shaped nanomembranes. Localized three-dimensional optical modes are obtained due to an axial confinement mechanism that is defined by the variation of the tube radius and wall windings along the tube axis. Optofluidic functions of the rolled-up microtubes are explored by immersing the tubes or filling their core with a liquid medium. Refractive index sensing of liquids is demonstrated by correlating spectral shift of the optical modes when a liquid interacts with the resonant light of the microtube. In addition, a novel sensing methodology is proposed by monitoring axial mode spacing changes. Lab-on-a-chip methods are employed to fabricate an optofluidic chip device, allowing a high degree of liquid handling. A maximum sensitivity of 880 nm/refractive-index-unit is achieved. The developed optofluidic sensors show high potential for lab-on-a-chip applications, such as real-time bio/chemical analytic systems.

With this thesis, I present an experimental study focusing on the provision of thermodynamic data of fluids at elevated pressure and temperature conditions. Hereby a microcapillary setup that is equipped with an in situ Raman Spectroscopy unit as well as with a high-speed camera, was further improved within the scientific employment of the author. The setup consists in principle of a fused-silica microcapillary embedded in a heating block, which is furthermore connected to high pressure syringe pumps. Pure compounds and mixtures were studied with the microfluidic setup and different thermodynamic properties were determined. For instance, vapor pressures of Poly(oxymethylene) Dimethyl Ethers (OME3 and OME4), a potential class of renewable diesel fuels, were the first time measured for temperatures exceeding the atmospheric boiling temperature. Hereby the regarded compound is pressurized at constant temperature, from what the vapor pressure is determined optically by detecting bubble or film formation, indicating the transition from vapor to liquid state. The main results of this thesis were however the vapor-liquid equilibria (VLE) of fuel/air-systems that were determined by in situ Raman Spectroscopy, whereby the Stokes-scattered Raman signal can be successfully separated phase-dependently by light barrier technology. A further task was the determination of saturated mixture densities of the validation system ethanol/CO2. With this study, I intend to contribute to the scarce literature data for the studied systems and properties. Therewith I want to help to enhance the understanding of microprocesses such as the evaporation and mixing formation in diesel combustion engines.

Confronted with the global demographic changes and the increasing pressure on modern healthcare system, there has been a surge of developing new technology platforms in the past decades. Droplet microfluidics is a prominent example of such technology platforms, which offers an efficient format for massively parallelized screening of a large number of samples and holds great promise to boost the throughput and reduce the costs of modern biomedical activities. Despite recent achievements, the realization of a compact and generic screening system which is suited for resource-limited settings and point-of-care applications remains elusive. To address the above challenges, the dissertation focuses on the development of a compact multifunctional droplet micro-magnetofluidic system by exploring the advantages of magnetic in-flow detection principles. The methodologies behind a novel technique for biomedical applications, namely, magnetic in-flow cytometry have been put forth, which encompass magnetic indexing schemes, quantitative multiparametric analytics and magnetically-activated sorting. A magnetic indexing scheme is introduced and intrinsic to the magnetofluidic system. Two parameters characteristic of the magnetic signal when detecting magnetically functionalized objects, i.e. signal amplitude and peak width, providing information which is necessary to perform quantitative analysis in the spirit of optical cytometry has been proposed and realized. Magnetically-activated sorting is demonstrated to actively select individual droplets or to purify a population of droplets of interest. Together with the magnetic indexing scheme and multiparametric analytic technique, this functionality synergistically enables controlled synthesis, quality administration and screening of encoded magnetic microcarriers, which is crucial for the practical realization of magnetic suspension arrays technologies. Furthermore, to satisfy the needs of cost-efficient fabrication and high-volume delivery, an approach to fabricate magnetofluidic devices on flexible foils is demonstrated. The resultant device retains high performance of its rigid counterpart and exhibits excellent mechanical properties, which promises long-term stability in practical applications.

Die vorliegende Arbeit befasst sich mit der Entwicklung von Biosensoren mit dem Ziel, mit Hilfe der Kopplung molekular geprägter Polymere (MIPs) als neuartiges Rezeptormaterial und dem sensitiven Nachweisprinzip der Fluoreszenz eine neue Qualität des Analytnachweises zu erreichen. Es wurde eine neue Strategie zur Optimierung der Bindungseigenschaften von molekular geprägten Polymeren in wässrigen Lösungsmitteln entwickelt, die die Kopplung aus Design of Experiments und der Optimierung multipler Zielgrößen umfasst. Damit konnten die Polymerbindungseigenschaften für alle vier betrachteten Parameter wesentlich verbessert werden. Mit Hilfe stationärer und zeitaufgelöster Fluoreszenztechniken wurde die Aufklärung der Wechselwirkung zwischen MIP und Analyt auf molekularer Ebene sowie die Charakterisierung einer neuen Nachweisstrategie basierend auf einen Förster-Resonanzenergietransfer-Mechanismus realisiert. Es wurde ferner ein MIP-Sensor für biologische Proben mit mikrofluidischer Probenzuführung aufgebaut und mittels Fluoreszenzspektrometer als konventionelles Nachweisverfahren etabliert. Darauf aufbauend wurde der optische Nachweis miniaturisiert und somit miniaturisierte Lichtquellen und Detektoren sowie eine faser-optische Lichtleitung eingesetzt. Davon ausgehend erfolgte die Optimierung des Messaufbaus hinsichtlich der Sensitivität und Nachweisgrenze des fluoreszierenden Analyten. Schließlich wurden erstmalig fluoreszenzmarkierte MIP-Partikel zur Lokalisation und Quantifizierung auf Zelloberflächen eingesetzt, d.h. diese dienten als Antikörperersatz der Immunfärbung. / This thesis deals with the development of biosensors with the aim to couple molecularly imprinted polymers (MIPs) as new receptor material with the sensitive detection principle of fluorescence in order to improve analyte detection. A new strategy for optimization of binding parameters of molecularly imprinted polymers in aqueous media was developed which is based on the coupling of design of experiments and the optimization of multiple objective parameters. Due to that the polymer binding properties for all four considered parameters could be optimized considerably. With the help of steady state and time-resolved fluorescence techniques the interaction between MIP and analyte could be clarified on a molecular basis. Furthermore the characterization of a new detection strategy based on a Förster resonance energy transfer mechanism was realized. Moreover a MIP sensor with microfluidic sample handling for biological samples was built-up and established with fluorescence spectroscopy as conventional detection method. Based on that, the optical detection was miniaturized with respect to light sources, detectors as well as optical fibers for light guidance. This set-up was optimized concerning sensitivity and limit of detection of the fluorescent analyte. Finally, for the first time fluorescently marked MIP particles were applied for imaging on cell surfaces – meaning that they were used for immunostaining as antibody mimics.

Die vorliegende Arbeit befasst sich mit der Entwicklung von Biosensoren mit dem Ziel, mit Hilfe der Kopplung molekular geprägter Polymere (MIPs) als neuartiges Rezeptormaterial und dem sensitiven Nachweisprinzip der Fluoreszenz eine neue Qualität des Analytnachweises zu erreichen. Es wurde eine neue Strategie zur Optimierung der Bindungseigenschaften von molekular geprägten Polymeren in wässrigen Lösungsmitteln entwickelt, die die Kopplung aus Design of Experiments und der Optimierung multipler Zielgrößen umfasst. Damit konnten die Polymerbindungseigenschaften für alle vier betrachteten Parameter wesentlich verbessert werden. Mit Hilfe stationärer und zeitaufgelöster Fluoreszenztechniken wurde die Aufklärung der Wechselwirkung zwischen MIP und Analyt auf molekularer Ebene sowie die Charakterisierung einer neuen Nachweisstrategie basierend auf einen Förster-Resonanzenergietransfer-Mechanismus realisiert. Es wurde ferner ein MIP-Sensor für biologische Proben mit mikrofluidischer Probenzuführung aufgebaut und mittels Fluoreszenzspektrometer als konventionelles Nachweisverfahren etabliert. Darauf aufbauend wurde der optische Nachweis miniaturisiert und somit miniaturisierte Lichtquellen und Detektoren sowie eine faser-optische Lichtleitung eingesetzt. Davon ausgehend erfolgte die Optimierung des Messaufbaus hinsichtlich der Sensitivität und Nachweisgrenze des fluoreszierenden Analyten. Schließlich wurden erstmalig fluoreszenzmarkierte MIP-Partikel zur Lokalisation und Quantifizierung auf Zelloberflächen eingesetzt, d.h. diese dienten als Antikörperersatz der Immunfärbung. / This thesis deals with the development of biosensors with the aim to couple molecularly imprinted polymers (MIPs) as new receptor material with the sensitive detection principle of fluorescence in order to improve analyte detection. A new strategy for optimization of binding parameters of molecularly imprinted polymers in aqueous media was developed which is based on the coupling of design of experiments and the optimization of multiple objective parameters. Due to that the polymer binding properties for all four considered parameters could be optimized considerably. With the help of steady state and time-resolved fluorescence techniques the interaction between MIP and analyte could be clarified on a molecular basis. Furthermore the characterization of a new detection strategy based on a Förster resonance energy transfer mechanism was realized. Moreover a MIP sensor with microfluidic sample handling for biological samples was built-up and established with fluorescence spectroscopy as conventional detection method. Based on that, the optical detection was miniaturized with respect to light sources, detectors as well as optical fibers for light guidance. This set-up was optimized concerning sensitivity and limit of detection of the fluorescent analyte. Finally, for the first time fluorescently marked MIP particles were applied for imaging on cell surfaces – meaning that they were used for immunostaining as antibody mimics.

Die vorliegende Arbeit beschreibt die Übertragung der aus der Siliziumtechnologie bekannten Präzision der Strukturierung und die Zuverlässigkeit der Verbindungstechnologie auf andere Materialien wie Kupfer und PMMA. Diese Untersuchung ist auf die Entwicklung der Teiltechnologien Strukturierung und Integration fokussiert und konzentriert sich insbesondere auf die Kombination von Mikrostrukturierung und dreidimensionalen Aufbautechniken einschließlich vertikaler fluidischer Durchkontaktierungen bei den Materialien Silizium, Kupfer und Kunststoff (PMMA). Eine begleitende Charakterisierung und messtechnische Bewertung gestattet die Weiterentwicklung während der Experimentedurchführung und erweitert den Stand der Wissenschaft hinsichtlich der genannten Kombinationen. / The work describes the transfer of well known high precisive and reliable micro technologies for patterning and packaging of Silicon to new materials like Copper and PMMA. This investigation is focused on special patterning technologies and system integration aspects. Furthermore the development of material-dependent micro patterning technologies and multi layer packaging techniques including vertical fluidic interconnects using materials like Silicon, Copper, and PMMA (polymer) is shown. An accompanying characterization and measurement-based evaluation enables the ongoing development while performing experimental analysis. At least a higher state of the art for these complex combinations is reached.