This thesis presents the development of microfluidic devices designed to facilitate research into mouse embryonic stem cells (ESCs). ESCs are a well-studied cell, largely due to their pluripotent nature, meaning they are able to differentiate into all cell types of the body and may self-renew indefinitely in appropriate culture conditions. ESCs, along with many other lines of biological enquiry, are increasingly studied with the use of micro uidic technology which enables fine tuning of physical and chemical environments unachievable on the macro scale. Two varieties of microfluidic technology are presented in this thesis, one for high- resolution mechanical phenotyping of ESCs and the second as a novel in-chip culturing platform to study cellular transitions. Chapter 1 presents a broad introduction to ESCs and biological enquiry with microfluidics, aimed to underpin the following Chapters. Chapters 2 and 3 present self-contained projects, thus each include a motivation and introduction section more specific than that presented in Chapter 1. These Chapters also contain their own methods, results and conclusion sections. Finally, Chapter 4 presents a summary of the work performed along with an outlook of upcoming investigations. In Chapter 2, I present a microfluidic device developed and utilised in collaboration with Christophe Verstreken (Department of Physics, University of Cambridge), which has been used to apply a mechanical stress to live cells enabling measurement of their nuclear deformability. The device facilitates detection of both nucleus and cytoplasm which can then be analysed with a custom-written MATLAB code. Quantitative measurements of nuclear sizes and strains of ESCs indicated a negative Poisson ratio for nuclei of cells cultured in specific medium conditions. Furthermore, we demonstrate that the device can be used to physically phenotype at high-throughput by detecting changes in the nuclear response after treatment with actin depolymerising and chromatin decondensing agents. Finally, we show the device can be used for biologically relevant high-resolution confocal imaging of cells under compression. The work from this chapter is presented in Hodgson et al. [1]. In Chapter 3, I present a novel microfluidic platform developed in collaboration with Prof. Austin Smith and Dr Carla Mulas (Centre for Stem Cell Research, Cambridge). The developed platform enables individual ESCs to be cultured under continued observation as they exit their pluripotent stem cell state. Each cell within the device may be extracted from the chip at any time for further investigation without disturbing other cells. Assessing the transition from the stem cell state in individual cells is paramount if we are to understand the mechanisms of pluripotency.

Droplet-based microfluidics can be used to enhance stem cell-based therapy by creating cell-laden hydrogel encapsulations to increase engraftment and retention while providing protection from immune responses caused by the host environment. Current research involves gaining better control over therapeutic mechanisms and one focus is to understand the mechanisms behind cell egress. Control over egress is vital to determining how long cells remain in proximity to the therapeutic target. We propose a microfluidic platform capable of encapsulating cells in two subsequent steps in order to create a double emulsion structure around the cell. In this project, hydrogel-in-hydrogel microdroplets are successfully manufactured without the presence of an intermediate oil layer and are used to observe model NIH 3T3 cell egress. In studying cell egress from singly or doubly encapsulated microcapsules, we are able to better understand the mechanisms that drive egress. Specifically, we hypothesize that cells egress when close to the edge of the microcapsule. In a double emulsion, cells are naturally located away from the edge and closer to the center. Results show that double emulsion microdroplets significantly reduce cell egress but do not eliminate it.

The aim of this thesis is to gain new insights into the physics underlying the long-term stability and instability of liquid foams and emulsions in the absence and presence of particles. By using Finite element based and mesoscopic Lattice Boltzmann techniques along with the microfluidic tools at our disposal, we tackled this question using two very different, yet complementary, approaches. In the first part, we went down to the smallest scale of foam, by observing a single bubble where the particle would straddle at interfaces of thin films. This brought a novel understanding to the observation that the torque on the particle is independent of film thickness and was mainly contributed by contact line stresses. We then precisely measured the hydrodynamic and dielectrophoretic interactions of a particle armored bubble treating the bubble as a flat surface and showed that its resistance to the motion was much less for hydrophobic particles compared to other wetting particles while the dielectrophoretic forces were more for hydrophobic particles as the latter protruded more in the oil phase. These findings are of utmost importance when designing particle-stabilized foams and dielectrophoresis-based particle separation techniques because they guide the choice of the particles to use for a particular application. In the second part, we studied the foam at a larger scale, by analyzing the evolution of a large population of identical bubbles produced in microfluidic geometries. This monodisperse foam destabilizes through Ostwald ripening or Coarsening toward a well-known self-similar state. However, we have shown that the transient regime leading to that state is not homogeneous in space. The microfluidic model that we develop predicts how the disorder grows in the foam, which is a valuable asset in applications where an ordered organization of the bubbles is required resisting foam coarsening. Furthermore, multiscale Lattice Boltzmann simulations of emulsion drainage based on frustrated long-range interactions are developed using the images from the microfluidic experiment as the initial phase thus providing a global understanding of emulsion stabilization and drainage dynamics. The key parameters investigated for particle-induced emulsion stabilization were solid particle concentration, particle size, wettability, heterogeneity and particle shape. The resulting emulsion droplets adopted pronounced non-spherical polyhedral shapes with time, indicating a high elasticity of the interface. The stability and the remarkable non-spherical shape of the emulsion droplets stabilized by the particles were features which bear resemblance with foam stabilization of bubbles using hydrophobic particles in flotation processes.

This thesis describes a novel approach for cell encapsulation in alginate gel microbeads. The main aim of the thesis was to optimise a microfluidic setup and chip to encapsulate cells in monodisperse alginate gel microbeads. A number of cytotoxicity tests were therefore carried out to determine the effect of formulations used for the production, degradation and gelation of calcium alginate gel beads. Results from these tests revealed that the formulations used had little or no significant effect on cell growth, and therefore, alginate was deemed to be a suitable cell encapsulating material for further investigations. Alginate gel microbeads were produced using hydrodynamic focusing techniques. For this purpose two different microfluidic setups were constructed. Fluids (oil, acidified oil and samples) were driven through the microfluidic setup by gravity. However, a number of drawbacks using this setup arose, such as polydispersity and reproducibility. Syringe pumps were introduced into the design of the second microfluidic setup as a means of driving fluids through the setup. In addition three different microfluidic chips were fabricated with the aim of producing the ideal alginate gel microbead. The first microfluidic chip (PMMA MC1) was fabricated from PMMA and involved producing alginate gel microbeads that were internally gelified. This chip suffered from a number of drawbacks such as continuous blockages within the microfluidic channels, which led to the development of the second microfluidic chip. This chip was also fabricated from PMMA (PMMA MC2) but in contrast to PMMA MC1, gelification occurred externally, i.e. gelation took place off chip, and in this case the alginate microdroplets were dropped into a well containing 1 mL of acidified oil. This encapsulating procedure caused immediate cell death, which indicated that the internal gelation of alginate gel microbeads was favoured. These results also indicated that the design of the microfluidic chip needed developing in order to produce the ideal microbeads that can be used for cell encapsulation. This led to the fabrication of a novel microfluidic chip (PC MC3) which was fabricated from polycarbonate (PC) and involved internal gelation of the calcium alginate gel microbeads. The combination of using the optimised microfluidic setup and PC MC3, in addition to alternations in some of the solutions used to make the alginate microbeads, resulted in the production of the desired ideal gel microbeads containing cells. Snap shots of the encapsulated cells obtained using fluorescence microscopy after 24 hours of encapsulation, revealed that the cells showed some characteristics of living cells, yet at the same time they also showed some characteristics of dead cells. These findings demonstrate the potential use of the optimised microfluidic setup and PC MC3 chip for many biological and medical applications.

Bacteria can quickly adapt to changing environmental conditions by activating alternative sigma factors. It has been shown previously that single cell approaches can reveal hidden dynamics in sigma factor activation. Here, we investigate the single cell response dynamics of the B. subtilis extracytoplasmic function sigma factors, which are an important part of the cell envelope stress response, under their specific stresses. To do this we use transcriptional reporters of sigma factors, quantitative single cell snapshots, time-lapse microscopy, and microfluidics. By developing an improved microfluidics setup for single cell time-lapse microscopy, as well as improved single cell analysis code, we are able to observe new sigma factor dynamics. First, we observe heterogeneous entry into a higher $\sigma^{V}$ activity state in response to lysozyme, which displays a memory, as the heterogeneity is lost on removal and reapplication of the stress. Next, we observe a pulse amplitude and duration modulated sigma factor response of $\sigma^{M}$ to bacitracin. Finally, for $\sigma^{M}$ under ethanol and acidic stress, and for $\sigma^{Y}$ under ethanol stress, we observe a noisy increase in activity to a new steady state level, where the degree of variability between cells depends on the stress condition. This thesis also discusses efforts on building a single cell microfluidic device based on the ”mother machine” design, for the rod-shaped cyanobacterium, S. elongatus, which forces the cells to grow in a straight line. Growing this organism in a traditional mother machine device has, so far, proved challenging. To adapt the mother machine for cyanobacteria we modify the channel geometry using electron beam lithography, and improve the loading protocol. The research presented here reveals the range of regulatory dynamics possible for ECF sigma factors in B. subtilis, and provides improved microfluidics and analysis code that will enable easier quantification of bacterial gene circuits at the single cell level in the future.

La cytométrie en flux en association avec la coloration et le marquage d'anticorps présente l'un des outils les plus précieux en biotechnologie actuelle fournissant des informations sur l'hétérogénéité des populations cellulaires, la taille et le volume des cellules, ainsi que l'expression de certaines molécules de surface et intracellulaires. L'augmentation du coût et la difficulté fondamentale de ces méthodes, cependant, sont attribués à l'exigence des molécules de marquage de surface. Diélectrophorèse (DEP) a été identifiée comme alternative sans marquage prometteuse. Cette thèse porte sur l'amélioration des technologies basée sur les DEP actuelles, et le développement d'une nouvelle méthode pour aborder les questions de cytometrie diélectrophorétique (DEP) permettant la mesure probabiliste des signatures diélectriques (DE) de cellules au niveau d’une population, ainsi que de permettre l'identification de biomarqueurs fiables pour les changements cellulaires.Tout d'abord, les améliorations de la cytométrie DEP sur la translation de cellules latérales induites par DEP sont explorées par fabrication. Un système de tri cellulaire benchmark microfluidique est présenté, et l'effet des désalignements des microcanaux sur les topologies des électrodes des cellules DEP vivantes est discuté. Un modèle de S. cerevisiae est présenté et validé expérimentalement dans des dispositifs microfluidiques fabriqués. Un nouveau procédé de fabrication permettant le prototypage rapide de dispositifs microfluidiques avec des électrodes intégrées bien alignées est présenté. Des dispositifs identiques ont été fabriqués avec des procédés standards de lithographie douce PDMS. Selon l'étude benchmark, la procédure standard PDMS est tombée bien en dessous de la gamme nécessaire pour le tri des cellules par DEP. Le temps de fabrication et les coûts de la méthode proposée se sont révélés être à peu près les mêmes.Deuxièmement, une nouvelle méthode appelée cytométrie DEP distribuée (2DEP cytométrie) a été développée. Elle utilise une translation verticale de cellules induite par effet de DEP en liaison avec la vélocimétrie par image de particules (PIV) afin de mesurer la répartition probabiliste de forces DEP sur une population cellulaire entière. La méthode a été intégrée dans un dispositif microfluidique avec des électrodes intégrées. Les cellules passant à travers le micro-canal sont sollicitées par des forces de sédimentation, tandis que les forces DEP soit s’opposent à la sédimentation, prennent en charge la sédimentation, ou aucun des deux, en fonction des signatures DE des cellules. Les hauteurs à laquelle les cellules se stabilisent correspondent à leur signature DE et sont mesurées indirectement en utilisant PIV.Les données expérimentales quantifient la signature DE d'une population de S. cerevisiae et la lignée cellulaire human immortalise leucemie myeloide K562. Tout d'abord, l'effet de la surexpression de certaines protéines membranaires a été étudié dans des cellules S. cerevisiae. La répartition mesurée des forces DEP a été comparée à la population de cellules exprimant une protéine cytoplasmique au même taux. Deuxièmement, 2DEP cytométrie a été appliquée à la lignée cellulaire K562. Les effets de la réponse à un stress provoqué par divers inducteurs sur la signature DE de la population cellulaire ont été analysées.Enfin, l'analyse statistique des données définies estimation par noyau ajustées pour surmonter la nature finie des données mesurées. En combinaison avec des spectres en distance de Wasserstein, notés signatures Wasserstein, ont été quantifiés et liée à certains changements cellulaires. Ces signatures peuvent être utilisées comme marqueurs biologiques fiables pour certains changements cellulaires.En conclusion, 2DEP cytométrie a montré être suffisamment sensible pour identifier certains changements d’états cellulaires. Le nouveau dispositif 2DEP cytométrie est donc une alternative prometteuse à la cytométrie en flux classique / Flow cytometry in combination with staining and antibody labelling presents one of the most valuable tools in current biotechnology providing information about cell population heterogeneity, cell size and volume, as well as expression of certain surface and intracellular molecules. The increased cost and the fundamental difficulty of these methods, however, are attributed to the requirement of the surface marker molecules. Attractive alternatives to flow cytometry are label-free methods, such as micro-filtration, dielectric spectroscopy, and electro-kinetic methods. Out of these methods, dielectrophoresis (DEP) was selected as the most promising approach. This thesis focuses on improvements of current DEP-based technologies, and development and establishment of a new method to address the issues of dielectrophoretic (DEP) cytometry enabling label-free non-invasive probabilistic measurement of cell dielectric (DE) signatures on population level, as well as enabling identification of reliable biomarkers for cell changes.First, improvements of DEP cytometry based on DEP-induced lateral cell translation through fabrication are explored. A benchmark microfluidic live cell sorting system is presented, and the effect of microchannel misalignment above electrode topologies on live cell DEP is discussed in detail. Simplified model of budding S. cerevisiae cell is presented and validated experimentally in fabricated microfluidic devices. A novel fabrication process enabling rapid prototyping of microfluidic devices with well-aligned integrated electrodes is presented and the process flow is described. Identical devices were produced with standard PDMS soft lithography processes. The presented fabrication process significantly improved the alignment of the microstructures. According to the benchmark study, the standard PDMS procedure fell well outside the range required for reasonable cell sorting efficiency. The fabrication time and costs of the proposed methodology were found to be roughly the same.Second, a method called distributed dielectrophoretic cytometry (2DEP cytometry) was developed. It uses a DEP-induced vertical translation of live cells in conjunction with PIV in order to measure probabilistic distribution of live cell DE signatures on an entire cell population. The method was integrated in a microfluidic device with integrated electrodes. Cells passing through the microchannel are acted on by sedimentation forces, while DEP forces either oppose sedimentation, support sedimentation, or neither, depending on the DE signatures of the cells. The heights at which cells stabilize correspond to their DE signature and are measured indirectly using particle image velocimetry (PIV).Experimental data quantify the DE signature of a S. cerevisiae population and Human immortalised myelogenous leukaemia cell line K562. First, the effect of over-expression of certain membrane protein was studied in S. cerevisiae cells. Measured distribution of DEP forces was compared to cell population expressing a cytoplasmic protein at the same rate. Second, 2DEP cytometry was applied to K562 cell line. Effects of stress response triggered by various inducers on the DE signature of the cell population were analysed.Finally, statistical data analysis defined adjusted kernel density estimation to overcome the finite nature of the measured data. In combination with Wasserstein pseudometrics from sampled data, the Wasserstein distance spectra, denoted as Wasserstein signatures, were quantified and linked to certain cell changes. These signatures may be used as reliable biomarkers for cell changes.In conclusion, 2DEP cytometry showed it is sensitive enough to identify certain changes in cell states. The novel 2DEP cytometry device is therefore a promising alternative to conventional flow cytometry

The use of biosensors and microfluidics devices is often limited by constraints in terms of volumetric throughput due to the small dimensions of devices in microfluidics and of expensive and complicated sample preparation steps necessary to ensure the operation of biosensing platforms. This can be due to high initial sample volume with low concentration analytes or complex media matrices from which analytes are extracted. While working to analyse Cryptosporidium presence in drinking water a novel technique was developed. The huge advantages from using a label-free, buffer-free hydrodynamic mechanism in terms of cost, coupled with the ease of simply scaling a single design to match any target size and the ability manufacture these quickly and easily using cheap and readily available robust materials (i.e. acrylic sheet) may allow a revolution in the scope of microfluidics applications. Using a cascaded array of hydrodynamic focusing devices uniquely designed for parallelised operation from a single pump or pressure source, the array can be tailored to meet the specific requirements of many applications, in particular high volume and low concentration target analyte enrichment from complex media.

abstract: Disease prevention and personalized treatment will be impacted by the continued integration of protein biomarkers into medical practice. While there are already numerous biomarkers used clinically, the detection of protein biomarkers among complex matrices remains a challenging problem. One very important strategy for improvements in clinical application of biomarkers is separation/preconcentration, impacting the reliability, efficiency and early detection. Electrophoretic exclusion can be used to separate, purify, and concentrate biomarkers. This counterflow gradient technique exploits hydrodynamic flow and electrophoretic forces to exclude, enrich, and separate analytes. The development of this technique has evolved onto an array-based microfluidic platform which offers a greater range of geometries/configurations for optimization and expanded capabilities and applications. Toward this end of expanded capabilities, fundamental studies of subtle changes to the entrance flow and electric field configurations are investigated. Three closely related microfluidic interfaces are modeled, fabricated and tested. A charged fluorescent dye is used as a sensitive and accurate probe to test the concentration variation at these interfaces. Models and experiments focus on visualizing the concentration profile in areas of high temporal dynamics, and show strong qualitative agreement, which suggests the theoretical assessment capabilities can be used to faithfully design novel and more efficient interfaces. Microfluidic electrophoretic separation technique can be combined with electron microscopy as a protein concentration/purification step aiding in sample preparation. The integrated system with grids embedded into the microdevice reduces the amount of time required for sample preparation to less than five minutes. Spatially separated and preconcentrated proteins are transferred directly from an upstream reservoir onto grids. Dilute concentration as low as 0.005 mg/mL can be manipulated to achieve meaningful results. Selective concentration of one protein from a mixture of two proteins is also demonstrated. Electrophoretic exclusion is also used for biomarker applications. Experiments using a single biomarker are conducted to assess the ability of the microdevice for enrichment in central reservoirs. A mixture of two protein biomarkers are performed to evaluate the proficiency of the device for separations capability. Moreover, a battery is able to power the microdevice, which facilitates the future application as a portable device. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019

Dans cette thèse, des systèmes nano-opto-mécaniques (NOMS) sont explorés et deux composants nano-opto-mécaniques sont conçus, simulés, fabriqués et analysés. Premièrement, un générateur de moment angulaire composé d'un résonateur en anneau, un guide d'onde et un ensemble de nano-plots est conçu, analysé théoriquement et simulé. L'analyse théorique et les résultats numériques montrent qu'une série de champs tournants optiques (ROFs) sont générés lorsque différentes longueurs d'onde de résonance sont couplées dans le générateur. Par la suite, la force optique, le potentiel optique et le couple optique du ROF généré sont analysés théoriquement, simulés numériquement et discutés. Les distributions de forces optiques sont affectées par le ROF en fonction des ordres angulaires et des différents nano objets considérés. Les couples optiques sont analysés et discutés pour des objets différents, àsavoir les nano-particules sphériques, des nano-fils et un nano-rotor. Enfin, un système accordable de transparence induite par résonateurs couplés (Coupled-Resonator-Induced Transparency –CRIT), qui est entraîné par la force optique exercée entre le résonateur en anneau et le substrat, est conçu, analysé théoriquement, simulé,fabriqué et mesuré expérimentalement. Le système CRIT accordable est constitué d'un guide d'onde et de deux résonateurs en anneau couplés, dans lequel un anneau est fixe et l’autre libre de se mouvoir. Différentes puissances d'entrée produisent différentes forces optiques sur l'anneau libéré, qui produisent différentes déformations et changements de l'accumulation du champ optique, et ainsi différents décalages du spectre de transmission optique et une variation du retard de groupe / In this PhD thesis, the nano-opto-mechanical system (NOMS) is explored and two nano-opto-mechanical devices are designed, analyzed, simulated and fabricated. Firstly, an angular momentum generator consisting of a ring resonator, a wave guide and a group of nano-rods is designed, theoretically analyzed and simulated. The theoretic alanalysis and numerical results show that a series of rotating optical field (ROF) are generated when different resonant wave lengths are coupled into the generator. Subsequently, the optical force, the optical potential and the optical torque of the generated ROF are theoretically analyzed, numerically simulated and discussed. The optical force distributions are affected by the ROF with different angular orders and different objects. The optical torques are analyzed and discussed for different objects, i.e. spherical nano-particle, nano-wire and nano-rotor. Finally, a tunable coupled-resonator-induced transparency (CRIT) system, which is driven by the optical force between the ring resonator and the substrate, is designed, theoretically analyzed, simulated, fabricated and experimented. The tunable CRIT system consists of a bus wave guide and two coupled ring resonators, in which one is the released ring and the other is the fixed ring. Different input powers produce different optical forces on the released ring, which produce different final deformations, change the optical field buildup, shift the transmission spectrum and vary the group delay

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on December 13, 2007) Vita. Includes bibliographical references.