The integration of droplet microfluidics devices with nanopore sensors offers a powerful and miniaturized sensing platform. Such devices can utilize the pre-processing capabilities of microfluidics in conjunction with single molecule sensing offered by nanopores. Microfluidics devices utilizing segmented flow (droplets) allow the compartmentalization of chemical and biological reagents in droplets reducing the processing time and associated cost, which is advantageous to biomolecular applications. Droplet microfluidics have been used in diagnostics and therapeutic applications such as cell and biomarker detection, gene amplification, and drug delivery. Nanopore sensors are currently used in investigating DNA and gene detection, protein-protein interactions, protein folding, and enzymatic kinetic reactions. This thesis proposes a design and outlines a methodology to integrate nanopore sensors within a droplet microfluidic device. The chapters are organized in highlighting three main objectives. The first objective is creating the segmented flow of oil-KCl droplets using a T-junction microfluidic design. The second objective is measuring the conductance of the segmented flow prior to the nanopore integration by using two side channel-AgCl electrodes. Subsequently, the third objective is integrating the droplet microfluidic device with a silicon nitride chip for nanopore fabrication. The nanopore is then created using controlled dielectric breakdown (CBD) method for DNA detection within droplets. The results show the feasibility of sensing individual DNA molecules within droplets using a nanopore sensor. The implemented approach expands upon nanopore applications to detect different samples simultaneously, fast food-borne pathogens and tumor discrimination in cancer biology. We anticipate that this integration is the future of nanopore sensors.

Fast mixing remains a major challenge in droplet-based microfluidics. The low Reynolds number operating regime typical of most microfluidic devices signify laminar and orderly flows that are devoid of any inertial characteristics. To increase mixing rates in droplet-based devices, a novel technique is presented that uses a high Reynolds number gaseous phase for droplet generation and transport and promotes mixing through binary droplet collisions at velocities near 1m/s. Control of multiple gas and liquid streams is provided by a newly constructed microfluidic test bed that affords the stringent flow stability required for generating liquid droplets in gaseous flows. The result is droplet production with size dispersion and generation frequencies not previously achievable. Limitations of existing mixing diagnostic methods have led to the development of a new measurement technique for measuring droplet collision mixing in confined microchannels. The technique employs single fluorophore laser-induced fluorescence, custom image processing, and meaningful statistical analysis for monitoring and quantifying mixing in high-speed droplet collisions. Mixing information is revealed through three governing statistics that that separate the roles of convective rearrangement and molecular diffusion during the mixing process. The end result is a viewing window into the rich dynamics of droplet collisions with spatial and temporal resolutions of 1μm and 25μs, respectively. Experimental results obtained across a decade vi of Reynolds and Peclet numbers reveal a direct link between droplet mixing time and the collision convective timescale. Increasing the collision velocity or reducing the collision length scale is the most direct method for increasing droplet mixing rates. These characteristics are complemented by detaching droplets under inertial conditions, where increasing the Reynolds number of the continuous gaseous phase generates and transports smaller droplets at faster rates. This work provides valuable insight into the emerging field of two-phase gas-liquid microfluidics and opens the door to fundamental research possibilities not offered by traditional oil-based architectures. / text

Acoustophoresis is a label free method where the acoustic radiation force is used to manipulate microparticles inside microfluidic channels. The magnitude of the force is dependent of several parameters, which include the density, speed of sound and size of the microparticles, as well as the amplitude of the pressure waves. Recently, acoustophoresis has been used in microfluidics to manipulate microparticles inside moving droplets. In this Master's thesis project, two microfluidic chip designs are used to enrich droplets with polystyrene beads (10 μm in diameter) using acoustophoresis. The microchips have been fabricated with two different fabrication methods; crystalline dependent wet etching and crystalline independent dry etching. In the microchips, water droplets in oil are generated with microparticles suspended in them. By using a channel width that is half a wavelength of the incoming acoustic waves, pressure nodal lines are created in the middle of the channel in which the microparticles align. The droplets then enters a droplet splitting feature, where they are divided into three daughter droplets. Since the majority of the incoming particles are recovered in the center daughter droplet while some of the droplet volume is removed, the center droplet is enriched with the microparticles. For the wet etched design stable droplet splitting was observed when the volumetric flow was 18 μL/min and the incoming droplets had a length-to-width ratio larger than 3. The maximum recovery for this design was 81.1% ± 13.8% with an applied voltage at 10 Vpp. Stable droplet splitting was observed for the dry etched chip at 10.5 μL/min and 18 μL/min at 10 and 20 Vpp, when the incoming droplet had a length-to-width ratio of 3. In this chip the maximum recovery was 93.2% ± 8.3% at the volumetric flow of 10.5 μL/min and an applied voltage of 20 Vpp.

Les anticorps sont des protéines en forme de Y, trouvées comme composant du sérum circulant, qui aident le système immunitaire à cibler et à répondre aux agents pathogènes et aux molécules étrangères, mais peuvent aussi contribuer à la maladie en réagissant aux protéines constitutives. Les anticorps sont produits par des Plasmocytes, qui les sécrètent dans la circulation. Parce qu'il n'y a pas de lien physique entre les plasmocytes et leurs anticorps sécrétés, la détection d'anticorps spécifiques d’un antigène est problématique. Dans cette thèse, j'explore l'utilisation de la microfluidique en gouttelettes pour générer et manipuler des compartiments aqueux homogènes dans lesquels des cellules sécrétant anticorps peuvent être isolées et analysées à haut débit a'échelle d'une seule cellule. Pour caractériser les cellules sécrétant des anticorps à l'intérieur des gouttelettes, j'utilise un nouveau test qui permet d'interroger les cellules en fonction de la spécificité de leur sécrétion. Ces compartiments de gouttelettes peuvent être triés pour le séquençage d'anticorps, ou analysés au cours du temps pour obtenir des informations cinétiques de l'interaction anticorps-antigène à l'intérieur de chaque gouttelette. En utilisant une nouvelle technologie, j'ai pu obtenir le répertoire d'anticorps de souris immunisées contre deux antigènes différents à partir de cellules sécrétant des anticorps spécifiques d’un antigène, avec des capacités égales ou supérieures aux technologies disponibles actuelles. Aussi, j'ai pu suivre le processus de maturation d'affinité des anticorps à l'échelle de la cellule unique, à la fois dans l'immunisation et la maladie auto-immune. Avec ces outils, je démontre comment la microfluidique peut être utilisée pour caractériser les réponses immunitaires et auto-immunes à travers l'évaluation de cellules sécrétant des anticorps. / Antibodies are Y shaped proteins, found as a component of circulating serum, that help the immune system target and respond to pathogens and foreign molecules, but can also contribute to disease when reacting to constitutive self-proteins. Antibodies are produced Plasma Cells, which secrete them into circulation. Because there’s no physical link between Plasma Cells and their secreted antibodies, the detection of antigen-specific antibodies is problematic. In this thesis I explore the use of droplet microfluidics to generate and manipulate homogeneous aqueous compartments in which single antibody secreting cells can be isolated and analyzed in a high-throughput manner. To characterize single antibody secreting cells inside the droplets I use a novel assay that allows to interrogate cells based on the specificity of their secretion. These droplet compartments can be sorted for single cell antibody sequencing, or analyzed over time to obtain kinetic information of the antibody-antigen interaction inside each droplet. Using new established technology I was able to obtain the antibody repertoire of mice immunized against two different antigens from single antigen-specific antibody secreting cells, with equal or better capacities than current available technologies. Also, I was able to follow the affinity maturation process of antibodies at the single-cell level, both in immunization and autoimmune disease. With these tools I demonstrate how microfluidics can be used to characterize the immune and the autoimmune responses through the evaluation of single antibody secreting cells.

At the cutting edge of the chemical and biological research, innovation takes place in a field referred to as Lab on Chip (LoC), a multi-disciplinary area that combines biology, chemistry, electronics, microfabrication, and fluid mechanics. Within this field, droplets have been used as microreactors to produce advanced materials like quantum dots, micro and nanoparticles, active pharmaceutical ingredients, etc. The size of these microreactors offers distinct advantages, which were not possible using batch technologies. For example, they allow for lower reagent waste, minimal energy consumption, increased safety, as well as better process control of reaction conditions like temperature regulation, residence times, and response times among others. One of the biggest drawbacks associated with this technology is its limited production volume that prevents it from reaching industrial applications. The standard production rates for a single droplet microfluidic device is in the range of 1-10mLh-1, whereas industrial applications usually demand production rates several orders of magnitude higher. Although substantial work has been recently undertaken in the development scaled-out solutions, which run in parallel several droplet generators. Complex fluid mechanics and limitations on the manufacturing capacity have constrained these works to explore only in-plane parallelization. This thesis investigates a three-dimensional parallelization by proposing a microfluidic system that is comprised of a stack of droplet generation layers working on the liquid-liquid ow regime. Its realization implied a study of the characteristics of conventional droplet generators and the development of a fabrication process for 3D networks of microchannels. Finally, the combination of these studies resulted in a functional 3D parallelization system with the highest production rate (i.e. 1 Lh-1) at the time of its publication. Additionally, this architecture can reach industrially relevant production rates as more devices can be integrated into the same chip and many chips can compose a manufacturing plant. The thesis also addresses the concerns about system reliability and quality control by proposing capacitive and radio frequency resonator sensors that can measure accurately increments as small as 2.4% in the water-in-oil volume fraction and identify errors during droplet production.

Droplet microfluidics is a promising field since it overcomes many of the limitations of single phase microfluidic systems. The improved mixing time scale, the increase of number of samples and the isolation of droplets are some of its virtues. The core of droplet microfluidics is a two-phase flow condition that is subjected to scaling of the confining geometry. With the scaling the complexities of the flow phenomena arise. For that reason both the processes of droplet generation and transport are not fully understood for various flow and fluid conditions. The work in this thesis aims to experimentally examine droplet generation and transport in microchannels for flow and fluid conditions that are experimentally challenging to perform. Examination of droplet generation in a T-junction microchannel design was performed with a quantitative velocity field approach known as micro particle image velocimetry (μPIV). The studies on droplet generation focused on very fast generation regimes, namely transition and dripping that have not been studied for a T-junction design. This achievement was accomplished because of the development of a fast optical detection and triggering system that allowed for acquiring images of different identical droplets at the same position. μPIV results indicate that the quantitative velocity field patterns of different regimes share some similarities. The filling stage in the transition and dripping regimes had some resemblance in their velocity patterns. The velocity patterns for the start of droplet pinch-off were alike for the squeezing and transition regimes. Furthermore, the presence of a surfactant in the droplet phase above the critical micelle concentration (CMC) did not have an effect on the general velocity patterns as long as the capillary number Ca was matched with the no-surfactant condition. The studies of hydrodynamic properties of droplet transport were performed in hard materials to avoid cumulative error sources, such as material pressure compliance and swelling effects. The project had several parts: designing a microchannel network that allowed studying the hydrodynamic properties of small droplets, surface treatments of the channel material for stable droplet generation and examining the hydrodynamics of small liquid droplets with sizes that have not been reported in the literature. The studies examined effects of changing the interfacial tension, viscosity, and flow conditions on the transport of droplets. The experimental results from the hydrodynamic transport studies indicated that for the droplet sizes that were examined the pressure drop of droplets was affected by the capillary number Ca and length of the droplet Ld. Also, the presence of surfactants altered the hydrodynamic properties of droplets. At a high concentration of surfactants the droplets pressure drop was reduced significantly. Moreover, the type of surfactant affected the magnitude of the pressure drop. Experimental results indicate that if the concentration of surfactants was very low (below CMC) it did not have an effect on the droplet excess pressure. These findings are important to consider in designing droplet microfluidic systems with complex channel networks that involve droplet sorting, splitting, and merging for droplets that contain surfactants.

Le cancer constitue un problème majeur de santé publique en France. La nécessité de disposer d’un test capable de détecter très précocement une tumeur, avant même qu’elle ne soit décelable par l’imagerie (et surtout avant les métastases) ne fait pas doute. Pour l’heure, la voie la plus prometteuse pour détecter la maladie demeure la mise au point des tests simples, fiables, rapides et hautement sensibles, reposant sur le dosage de marqueurs génétiques (des biomarqueurs). Nous avons développé une procédure non-invasive, innovante et au moins 1000 fois plus sensible que les méthodes actuelles (0,0005% de séquences mutées détectées parmi un excès de séquences non-mutées), pour le criblage des biomarqueurs spécifiques des cancers. Elle peut facilement être utilisée pour le diagnostic, le pronostique ou la prédiction des procédures de gestion clinique des patients souffrant du cancer colorectal et par après pour les patients souffrant d’autres types de cancer. Cette méthode est basée sur l’utilisation des millions de microgouttelettes en tant qu’autant de bioréacteurs indépendants. Apportant ainsi la possibilité d’analyse chaque molécule d’ADN indépendamment, elle permettra de détecter spécifiquement une minorité de séquences mutantes au sein d’une forte quantité de séquences non mutées et avec un débit important. Nous avons développé cette stratégie pour la détection des mutations de l’oncogène KRAS (responsables des non-réponses aux thérapies ciblées) et nous l’avons validé avec la détection de mutations KRAS dans les échantillons de plasma sanguin et de tumeur des patients atteints d’un cancer colorectal métastatique. Notre méthode trouvera son utilité non seulement dans le domaine du diagnostic précoce des cancers, mais aussi dans cas de la prédiction de la réponse aux traitements ciblés grâce à la détection de biomarqueurs spécifiques. / The aim of this work is to establish novel strategies for the highly sensitive screening of cancer biomarkers in biological samples.To achieve this goal, we developed droplet-based microfluidic dPCR technique. Using a limiting dilution, individual DNA molecules are encapsulated within monodisperse droplets of a water-in-oil emulsion created with a microfluidic device. Fluorescent TaqMan® probes targeting the screened cancer biomarkers allow the detection of mutations. We focused on the mutations in the human KRAS gene for the development of our test. This method is also transposable in a multiplexed format for the parallel detection of multiple mutations in clinical samples.The developed technique allowed the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of un-mutated KRAS genes and enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines. We validated our technique by screening for KRAS mutations in the blood plasma and tumor samples from patients with metastatic colorectal cancer.

Lab-on-Chip, the miniaturization of the chemical and analytical lab, is an endeavor that seems to come out of science fiction yet is slowly becoming a reality. It is a multidisciplinary field that combines different areas of science and engineering. Within these areas, microfluidics is a specialized field that deals with the behavior, control and manipulation of small volumes of fluids. Agglutination assays are rapid, single-step, low-cost immunoassays that use microspheres to detect a wide variety molecules and pathogens by using a specific antigen-antibody interaction. Agglutination assays are particularly suitable for the miniaturization and automation that two-phase microfluidics can offer, a combination that can help tackle the ever pressing need of high-throughput screening for blood banks, epidemiology, food banks diagnosis of infectious diseases. In this thesis, we present a two-phase microfluidic system capable of incubating and quantifying agglutination assays. The microfluidic channel is a simple fabrication solution, using laboratory tubing. These assays are incubated by highly efficient passive mixing with a sample-to-answer time of 2.5 min, a 5-10 fold improvement over traditional agglutination assays. It has a user-friendly interface that that does not require droplet generators, in which a pipette is used to continuously insert assays on-demand, with no down-time in between experiments at 360 assays/h. System parameters are explored, using the streptavidin-biotin interaction as a model assay, with a minimum detection limit of 50 ng/mL using optical image analysis. We compare optical image analysis and light scattering as quantification methods, and demonstrate the first light scattering quantification of agglutination assays in a two-phase ow format. The application can be potentially applied to other biomarkers, which we demonstrate using C-reactive protein (CRP) assays. Using our system, we can take a commercially available CRP qualitative slide agglutination assay, and turn it into a quantitative High Sensitivity-CRP test, with a lower detection limit of 0.5 mg/L using light scattering. Agglutination assays are an incredibly versatile tool, capable of detecting an ever-growing catalog of infectious diseases, proteins and metabolites. A system such as that presented in this thesis is a step towards being able to produce high throughput microfluidic solutions with widespread adoption.

Droplet generators are at the heart of many microfluidic devices developed for life science applications but are difficult to tailor to each specific application. The high fabrication costs, complex fluid dynamics, and incomplete understanding of multi-phase flows make engineering droplet-based platforms an iterative and resource-intensive process. First, we demonstrate the suitability of desktop micromills for low-cost rapid prototyping of thermoplastic microfluidic devices. With this method, microfluidic devices are made in 1 - 2 hours, have a minimum feature size of 75 μm, and cost less than $10. These devices are biocompatible and can accommodate integrated electrodes for sophisticated droplet manipulations, such as droplet sensing, sorting, and merging. Next, we leverage low-cost rapid prototyping to characterize the performance of microfluidic flow-focusing droplet generators. Specifically, the effect of eight design parameters on droplet diameter, generation rate, generation regime, and polydispersity are quantified. This was achieved through orthogonal design of experiments, a large-scale experimental dataset, and statistical analysis. Finally, we capitalize on the created dataset and machine learning to achieve accurate performance prediction and design automation of flow-focusing devices. The developed capabilities are captured in a software tool that converts high-level performance specifications to a device that delivers the desired droplet diameter and generation rate. This tool effectively eliminates the need for resource-intensive design iterations to achieve functional droplet generators. We also demonstrate the tool’s generalizability to new fluid combinations with transfer learning. We expect that our newly established framework on rapid prototyping, performance characterization informed by design of experiments, and machine learning guided design automation to enable extension to other microfluidic components and to facilitate widespread adoption of droplet microfluidics in the life sciences.

Cancer is a complex and dynamic disease that is difficult to treat due to its heterogeneous nature at multiple scales. Standard therapies such as surgery, radiation, and chemotherapy often fail, therefore superior therapies must be developed. Electroporation-based therapies offer an alternative to standard treatments, utilizing pulsed electric fields to permeabilize cell membranes to either enhance drug delivery (electrochemotherapy) or induce cancer cell death (irreversible electroporation). Electroporation treatments show promise in the clinic, however, are limited in the size of tumors that they can safely treat without increasing the applied voltage to an extent that induces thermal damage or muscle contractions in patients. A method to increase ablation size safely is needed. To make this advancement and to advance other cancer treatments as well, better in vitro tumor models are needed. Heterogeneity not only makes cancer difficult to treat, but also difficult to recapitulate in vitro. This dissertation addresses the complementary need to develop both better cancer therapies and more physiologically relevant in vitro tumor models. My results demonstrate that by using a calcium adjuvant with irreversible electroporation treatment, ablation size can be increased without using a higher applied voltage. Additional mechanistic studies identified signaling pathways that were differentially dysregulated under calcium and no calcium conditions, impacting cell death. Finally, I have successfully encapsulated cells in fibrin microgels which may enable the creation of more physiologically relevant and complex 3D in vitro and ex-vivo platforms to investigate IRE as well as other tumor therapies. / Doctor of Philosophy / Cancer is a complex and dynamic disease. Heterogeneity exists at the single cell, tumor, and patient levels making it difficult to establish a unified target for therapy. Standard therapies such as surgery, radiation, and chemotherapy often fail for this reason, therefore superior therapies must be developed. Electroporation-based therapies offer an alternative to standard treatments, utilizing pulsed electric fields to permeabilize cell membranes to either enhance drug delivery (electrochemotherapy) or induce cancer cell death (irreversible electroporation). Electroporation treatments show promise in the clinic, however, are limited in the size of tumors that they can safely treat without increasing the applied voltage to an extent that induces thermal damage or muscle contractions in patients. A method to increase ablation size safely is needed. To make this advancement and to advance other cancer treatments as well, better tumor models are needed. Many of the same challenges in treating cancer serve as challenges in creating physiologically relevant tumor models. In this dissertation, I have developed a simplified platform to test whether using a calcium additive with irreversible electroporation therapies enhances ablation size. My results demonstrate that by using a calcium additive with irreversible electroporation treatment, ablation size can be increased without using a higher applied voltage. In addition, the biological pathways responsible for cell death in irreversible electroporation treatment with and without calcium were studied. Finally, I have successfully encapsulated cells in fibrin microgels that can be used to create better tumor models that encompass the heterogeneity of tumors found in the body.