La formation d'agrégats d'anticorps monoclonaux en solution est difficile à empêcher. Même si la présence de gros agrégats est assez rare, leur existence peut avoir des effets dramatiques dans les systèmes d'injection, en menant à des situations de colmatage partiel ou total de la restriction dans ce dernier. Cela entraîne une injection mal contrôlée ou même une obstruction totale du système d'injection. Très peu est connu sur le rôle de la taille des agrégats et de la pression appliquée sur de tels évènements de colmatage. Dans cette thèse, nous présentons un système microfluidique modèle, imitant les systèmes médicaux d'injection afin de comprendre fondamentalement le colmatage de restrictions d'une taille donnée. Des solutions très concentrées en anticorps monoclonaux nous permettent de créer des agrégats de protéines (plus grands que 50 micromètres) en utilisant un stress mécanique ou thermique. Nous montrerons que le colmatage a lieu quand les agrégats atteignent la taille de la restriction et peut dans certains cas être défait en augmentant la pression appliquée. La possibilité observée d'éjecter des agrégats de la restriction via une augmentation en pression indique le rôle important de la déformabilité des agrégats de protéines, à ce jour complètement inexplorée. Nous réalisons des expériences systématiques pour différentes tailles relatives d'agrégats et de pressions appliquées, et nous mesurons le débit en sortie. Malgré leurs formes et densités différentes, nous pouvons prédire le nombre d'évènements de colmatage pour une taille donnée de restriction par des mesures utilisant le Flow Imaging Microscopy (MFI). De plus, notre système peut détecter l'occurrence de très gros agrégats (très rares) souvent non détectés par d'autres techniques. Avec un modèle mécanique simple, nous pouvons estimer pour la première fois un ordre de grandeur du module d'Young et un diamètre effectif de pores pour des agrégats d'anticorps monoclonaux. Nous avons également développé une autre expérience modèle dans un canal hyperbolique couplé avec un flow focusing afin d'observer la déformation d'agrégats sous écoulement élongationnel. Nous décrirons leur comportement en analysant leurs trajectoires qui sont pour la plupart d'entre eux du tumble et de l'alignement avec l'écoulement. De plus, nous développerons un modèle mécanique qui tient compte de la force de friction dans une expérience modèle contrôlée avec une solution polymérique de PEGDA. Nous étudierons ainsi le rôle d'une différence de pression minimale à appliquer pour remettre la particule en mouvement dans la restriction, et ainsi relier cela aux agrégats de protéines. / The formation of aggregates in solutions of monoclonal antibodies is difficult to prevent. Even if the occurrence of large aggregates is rather rare, their existence can have dramatic effects in injection devices, as they can lead to partial or total clogging of constrictions in the latter. This leads to badly controlled injection or even total obstruction of the device. Little is know on the role of aggregate size and applied pressure on such clogging events. In this thesis, we present a microfluidic model system, mimicking medical injection devices to gain fundamental understanding of the clogging of constrictions of given size. Highly concentrated solutions of monoclonal antibodies allow us to create protein aggregates (bigger than 50 micrometers) using mechanical or heat stress. We show that clogging occurs when aggregates reach the size of the constriction and can in some cases be undone by increasing the applied pressure. The observed possibility to eject aggregates from constrictions via an increase in pressure indicates the important role of protein aggregate deformability, so far completely unexplored. We perform systematic experiments for different relative aggregate size and the applied pressure, and measure the flow-rate. Despite their different shapes and density, we can predict the number of clogging events for a given constriction size by Flow Imaging Microscopy (MFI) measurements. In addition our device can detect the occurrence of very rare big aggregates often overlooked by other detection techniques. With a simple mechanical model where we neglected the friction, we could estimate for the first time an order of magnitude for the Young modulus and a porous diameter for monoclonal antibodies aggregates. We also develop another model experiment with an hyperbolic channel coupled with a flow focusing to observe deformation of the aggregates under extensional flow. We describe their behavior by analyzing their trajectories which are for most of them tumbling and alignment with the flow. Moreover, we develop a mechanical model which took into account the friction force in a controlled model experiment with polymeric solution. We thus investigate the role of a minimal applied pressure to generate the particle movement into the constriction, and then link it with protein aggregates.

The investigation of the physical properties of deoxyribonucleic acid under confinement is an essential step for the all-embracing understanding of the replication and transcription in living cells as well as for the development of the biomimetic nanotechnology. The following report addresses the measure-ment and interpretation of the intramolecular diffusion along stretched λ-DNA-molecules. This work comprises the fabrication of submicroscopic channels via softlithography, the integration of the chan-nels in an experimental setup with a fluorescence microscope and a source-measurement unit, and the experiments with the DNA-stretching in electrical field. The important results are the development of a measuring assembly with stable, softlithographic structures in Ormostamp, the direct imaging and measurement of the intermolecular diffusion along stretched DNA-molecules in channels with cross sections down to 100 x 300 nm2, and the qualitative and quantitative analysis on the basis of models of polymer physics.

The mechanical parameters of biological cells are relevant indicators of their function or of disease. For example, certain cancerous cells are more deformable than healthy cells. The challenge consists in developing methods that can measure these parameters while not affecting the cell. The Optical Stretcher is a microfluidic system that deforms single suspended cells without contact using lasers and determines the cells’ viscoelastic properties. The advantage compared to standard methods of molecular biology is that cells do not need to be treated with additional markers. Basic versions of the Optical Stretcher have existed for some years. These allow the measurement of homogeneous cell populations. Up until now, it was only possible to calculate average population values of compliance. To characterize inhomogeneous populations however, it is necessary to consider each single cell and measure additional mechanical or optical parameters such as the refractive index. This work highlights various extensions of the Optical Stretcher. A novel procedure, including an improved image processing algorithm, is presented to analyze mechanical data in real time. In combination with measurements of the optical refractive index, single cells can now be characterized in more detail. Moreover, it is now possible to extract interesting subpopulations that can be further examined with molecular biology techniques. Depending on the intended purpose, novel devices for cell measurements, based on microfluidic and optical considerations, are presented. The fundamental concept involves microstructured chips that can be integrated into a commercial microscope. These chips offer the possibility of separating measured cell populations according to their mechanical properties. This separation, including mathematical classification, is demonstrated. These methods are tested with cell types of differing mechanical properties to prove their applicability in practice. Single cells are sorted into their respective population of origin. These novel methods offer the possibility of a versatile device to be applied in biophysical research.

Le sol constitue le milieu naturel contenant la plus grande diversité de micro-organismes (typiquement 109 cellules de 104 espèces différentes par gramme de terre). Pourtant il n’est possible d’obtenir la croissance que d’une fraction en laboratoire (moins de 5 %). Réaliser des cultures de cette diversité, inaccessible pour le moment, aurait des applications considérables en agriculture (création d’engrais ou pesticide biologique et respectueux de l’environnement) et en pharmacologie (découverte d’antibiotiques ou anticancéreux). Ce travail de thèse est principalement axé sur l’étude de la croissance de micro-organismes difficiles à cultiver issus d’échantillons naturels tels que les sols. Des gouttes de tailles micrométriques, créées par microfluidique digitale, sont utilisés comme microréacteurs afin d’obtenir la croissance en laboratoire des espèces microbiennes encapsulées à l’intérieur. Une première étape consiste à obtenir une solution ne contenant que les microbes provenant de notre échantillon naturel de sol pour pouvoir réaliser l’encapsulation sans matières minérales et obtenir une diversité la plus proche possible de celle du sol. Une deuxième étape consiste à encapsuler les cellules contenues dans notre solution en faisant varier certaines conditions comme : la concentration initiale de microbes, le milieu de culture ou le temps d’incubation. Par l’observation des gouttes après croissance et séquençage des gènes ARNr 16S des cellules contenues à l’intérieur nous démontrons qu’il est possible d’obtenir la croissance de jusqu’à 40 % des espèces. Cette méthode microfluidique ouvre la voie du criblage à haut débit des interactions entre une espèce donnée (pathogène humain ou de plante, phage/virus) avec le microbiote qu’il est susceptible de contaminer (flore intestinal, sols, mers …) et ainsi déterminer quantitativement la réaction du milieu étudié, en plus de son utilisation pour la croissance d’espèces difficilement cultivables en laboratoire. / Soil is the natural medium containing the highest microbial diversity (109 cells from 104 different species per gram of soil). Yet we still can’t grow in laboratory more than 5 % of them. Having access to this diversity will lead to crucial applications for farming (production of organic fertilizers or environmentally friendly pesticides) and to pharmacology (discovery of new antibiotics or new anticancer molecules). This work focuses on the study of growth of non culturable micro-organisms from natural samples, like soil. This method uses microfluidics droplets as microreactors to obtain the growth of microbial species encapsulated inside. The first step is to achieve a solution with nothing but the microbes from our natural sample (no minerals) for a successful encapsulation and obtain diversity as close to the one found in the soil. The second step is to encapsulate the cells from this solution with different set of condition like : initial concentration, growth media and incubation time. By coupling observation of the droplets after growth and the rRNA 16S sequencing of their content we demonstrate that it is possible to obtain the growth of up to 40 % of the species. This microfluidic method, besides its use in growing unculturable species in laboratory, opens the way towards high-throughput screening of interactions between a given species (human or plant pathogens, phage/virus) and the microbiota it is likely to contaminate (gut flora, soil, sea …) and obtain the quantitative determination the reaction of microbiota.

The goal of this study is to design and manufacture a microfluidic device capable of measuring changes in impedance valuesof microfluidic cell cultures. Tocharacterize this, an interdigitated array of electrodes was patterned over glass, where it was then bonded to a series of fluidic networks created in PDMS via soft lithography. The device measured ethanol impedance initially to show that values remain consistent over time. Impedance values of water and 1% wt. saltwater were compared to show that the device is able to detect changes in impedance, with up to a 60% reduction in electrical impedance in saltwater. Cells were introduced into the device, where changes in impedance were seen across multiple frequencies, indicating that the device is capable of detecting the presence of biologic elements within a system. Cell measurements were performed using NIH-3T3 fibroblasts.

Developing microdevices on flexible material attracts scientific community to explore applications in different aspects of health and point of care diagnostics. Flexible substrates offer unique characteristics such as flexibility, stretchability, portability, low-cost, and simple fabrication. Fabrication of cost-effective paper-based analytical devices by wax printing has recently become popular using cellulose filter papers. Paper-based devices need higher temperature to form hydrophobic barrier across paper substrate, rely on large working channels (≥ 500 μm) for liquid handling, and exhibit lower efficiency (~50%) of sample mobility. Such limitations confine applications of wax-based fabrication. In this dissertation, we report printability, fidelity, and applications of wax micropatterns on polyethylene terephthalate-based substrate (PET), which is a a non-cellulosic, non-fibrous, and non-porous material. Resolution, sustainability against heat and biocompatibility was tested on wax micro-features. The patterned devices were explored for variety of applications.First, wax microwells on PET showed mouse embryonic stem cell (mESC) self-renewal or direct differentiation. Second, microfluidic flow was demonstrated on wax printed microchannels on PET which was used to develop distance-based assay. Third, fluidic properties of trinucleotide repeat sequences were investigated on wax microchannels. Fourth, multilayer wax-on-plastic device was fabricated using wax printing with hand painting of conductive materials for electrochemical immunosensing.

Rapidly diagnosing the precise drug resistance present in sepsis-inducing bacteria is a continuing need to maintain the efficacy of our medical systems. Diagnostics currently being developed for such scenarios are either sensitive or rapid, but not both. Sequence-specific single DNA molecule analysis could fill this gap if it could be adapted to work on smaller targets, similar to those produced by classical biological methods. In this work, I demonstrate that immobilized ssDNA in the appropriate hybridization buffer can rapidly pull its complementary sequence out of solution. I also demonstrate that such systems in a microfluidic chip can be used to capture bacterial plasmids as a step toward subsequent multiplexed analysis. Finally, I demonstrate that a 120 bp double stranded polynucleotide with an overhanging single stranded 25 bp probe sequence can be modified with multiple fluorophores and used to label captured targets in a sequence-specific manner. This system shows that it is possible to label bacterial plasmids in a manner that can bridge the technological gap between single molecule counting and small oligonucleotide targets. Such a system can achieve lower limits of detection for clinically relevant samples while maintaining rapid processing times.

This thesis presents a novel method of modulating silicon photonic circuits using 3D printed microfluidic devices. The fluids that pass through the microfluidic device interact directly with the silicon waveguides. This method changes the refractive index of the waveguide cladding, thus changing the effective index of the system. Through using this technique we demonstrate the shift in resonant wavelength by a full free spectral range (FSR) by increasing the concentration of the salt water in the microfluidic device from 0% to 10%. On a 60 μm microring resonator, this equals a resonant wavelength shift of 1.514 nm when the index of the cladding changes by 0.017 refractive index units (RIU), or at a rate of 89.05 nm/RIU. These results are confirmed by simulations that use both analytical and numerical methods. This thesis also outlines the development of a process that uses two-photon absorption(TPA) in silicon to produce a photoelectrochemical (PEC) etching effect. TPA induces free carriers in silicon that then interact with the Hydroflouric Acid (HF) solution that the wafer is submerged in. This interaction removes silicon away from the wafer, which is the etching observed in our experiments. Non-line-of-sight PEC etching is demonstrated. The optical assemblies used in these experiments are presented, as are several of the results of the etching experiments.

In this study a biosensor based on real time quartz crystal microbalance (QCM) monitoring is optimized and characterized for the application in the Norosensor. This biosensor is aimed to recognise, capture and amplify Norovirus (NoV). In an initial step a simplified bioassay was developed that focuses on the latter parts of the assay which consists of DNA-guided probing and amplification of the captured virus and includes the development of an amplification model assay directly to the functionalised crystal surface. A padlock probe with matching sequence to the conjugated oligonucleotide on the quartz crystal surface is used as target in the model assay. Although a number of studies have been carried out based on padlock probe ligation and rolling circle amplification (RCA) based QCM sensing, these studies utilize the entire crystal surface to capture and amplify the biomolecule. In this research work the QCM monitoring is explored on a centrally functionalised electrode surface through conjugation only at the centre of the electrode for increased mass sensitivity. Thus, allowing capture and amplification of the padlock probe only at the centre of the quartz crystal. A 14mm diameter, thermoncompensated AT-cut, nonpolished quartz crystal with a 10mm diameter gold surface coating acting as electrode was utilized for QCM measurements. The detection system is based on mass binding and amplification on the QCM to produce a negative frequency shift in the fundamental frequency of the vibrating quartz crystal. The amplification products were additionally fluorescently labelled and fluorescent microscopy images were also obtained at the end of every experiment to verify the presence or absence of DNA capture and amplification. Experimental findings show that the current flow chamber with a 15ul capacity is able to detect a specific padlock probe concentration of 1nM on a conjugated region of ~2.5mm diameter. RCA amplified the mass with an average frequency shift of -80Hz in 60mins RCA incubation time. Further, the specificity and sensitivity of the QCM system was explored. However, the system has limitations where sensor binding of reaction proteins, such as DNA ligase and BSA, to some extent is observed. The storage stability of the functionalized self-assembled monolayer (SAM) on the QCM is also observed to deteriorate and thus, is of concern. Nevertheless the combination of RCA based amplification with QCM real-time monitoring has the potential for rapid and simple, low cost detection of the Norovirus. / I det här arbetet har vi optimerat och karateriserat en biosensor för detektion av Norovirus som orsakar häftiga utbrott av kräksjuka under vinterhalvåret vilket leder till både försämrad vård samt stora ekonomiska förluster för samhället. Målet inom EU projektet “Norosensor” är att utveckla ett snabbtest som kan tillämpas efter ett utbrott på till exempel en vårdavdelning och som ska mäta mängden virus i luften vilket kan fungera som riktlinje för om en avdelning är säker att användas eller ej. Tekniskt är målet med testet att fånga in viruspartiklar från luften som specifikt binds till sensorytan. Därefter ökar vi känsligheten från bundna partiklar genom en DNA-baserad amplifiering. Detta genererar specifik, viruskorrelerad massa som mäts med en kvartskristall mikrovågs sensor. När massan ökar minskar frekvenser vid vilken kristallen vibrerar och detta mäts i realtid. Det här arbetet har inte behandlat infångande eller inbindning av virus utan har fokuserat på den senare delen av protokollet som omfattar amplifieringen på sensorytan. En modell-assay har därför utvecklats där viruspartikeln istället representeras av en så kallad “padlock probe” (hänglås probe). Då sensorn är mycket känslig har först olika protokoll testats för effektiv rengöring av ytan med hjälp av ultraljud. I nästa steg har ytan funktionaliserats med thiol-modifierade syntetiska DNA molekyler som används för infångningen av målmolekylen på sensorytan (virus eller i detta fall padlock proben). Det har tidigare uppskattats att för att få maximal känslighet i massmätningen så är det fördelaktigt att binda viruset endast i mitten på en mycket liten yta av kristallen. Den här avhandlingen har därför fokuserat på att utveckla protokoll för detta där ytan först funtionaliserats i mitten innan resten av ytan blockats för att undvika ospecific inbindning. Resultaten visar att vi kan generera en centrerad funtionalisering och att vi får låg ospecifik binding. Protokollet består av flera biokemiska reakionssteg såsom (i) inbindning och lingering av padlock probe och (ii) amplifiering av den ligerade proben genom “rolling circle amplification”. För att kunna verifiera att vi fått amplifieringsprodukter på ytan har vi dels mätt frekvensändringen på grund av ökad massa men också märkt in dem med fluorescerande molekyler och detekterat dem i microskop. Under arbetets gång har ett flertal olika typer av kristaller testats. Det visade sig att om en polerad yta används (1μm grovhet) så migrerade molekylerna iväg från mitten när vi oscillerade kristallen medan vi fick bättre resultat om något grövre (3μm) ytor användes. Vi testade även ett flertal olika flödesceller av olika material och med olika reaktionsvolymer. Eftersom kristallen är mycket känslig så påverkar faktorer som flödeshastigheter och eventuella luftbubblor frekvensen. Vi optimerade därför detta och körde mätningarna vi6konstant flöde men med alternerande, låga hastigheter när vi tillsatte nya reagens eller inkuberade reaktionerna. Vi förvärmde även reaktionsmixarna för att minska ospeficika effekter och konstaterade att den funktionaliserade ytan påverkades av lagring över tid. I våra försök såg vi att protein såsom ligeringsenzymet och albumin, vilka har förhållandevis stor massa, hade effekter på frekvensen redan i sig genom att binda till ytan. Ytterligare optimeringar måste därför göras framöver för att minska denna inbinding bland annat genom bättre tvättsteg. Vi kunde dock påvisa linjär massökning med ökad amplifieringstid och har bevisad hög specificitet. Slutligen utvecklades ett litet mjukvaruprogram för att automatisera analysen och minska bruset. Sammanfattingsvis har vi lyckats utveckla ett enkelt och snabbt system för specifik massamplifering av Norovirus.

Anthropogenic pollution of water resources and the environment by various hazardous compounds and classes of substances raises concerns about public health impacts and environmental damage. Commercially available, portable and easy-to-use devices to detect and quantify these compounds are rather sparse, but would contribute to comprehensive monitoring and reliable risk assessment. The Soft Colloidal Probe (SCP) assay is a promising platform for the development of portable analytical devices and thus has a great potential for a transfer to industry. This assay is based on the differential deformation of an elastic particle, i.e., the SCP, as a function of analyte concentration, which affects the extent of interfacial interactions between the SCP and a biochip surface. The objective of this work was to adapt this assay for the detection of anthropogenic pollutants. Biomimetic molecular recognition approaches were used based on naturally occurring target proteins that specifically bind the anthropogenic pollutants of interest. This adaptation included the elaboration of strategies for site-specific immobilization of the respective proteins and functionalization of SCPs. In this work, it is demonstrated that the SCP method can be employed for the highly specific and sensitive detection of the critically discussed pesticide glyphosate by using the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Furthermore, a specific detection scheme for estrogens and compounds with estrogenic and antiestrogenic activity was developed by harnessing estrogen sulfotransferase as the biomimetic recognition element. In the second part of the thesis, improvements of the SCP sensing methodology are described. These improvements were achieved by accelerating data analysis and developing a novel synthesis method for SCPs that ensures monodisperse particles with superior reproducibility. Rapid extraction of interaction energies is achieved by using a pattern matching algorithm that reduces the time required for data analysis to a fraction. The microfluidics-assisted synthesis of SCPs enables the production of highly monodisperse SCPs with adjustable size and mechanical properties. Various functionalization approaches have been developed that allow easy and modular introduction of functional groups and biomolecules for SCP-based sensing approaches.