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Martino, Chiara. "Droplet-based microfluidic platforms for protein investigations." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4005/.
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In the last two decades, the integration of life science and micro-engineering has developed systems which are able to perform laboratory functions on devices only 10-100 μm in size. These microfluidic systems, called lab-on-chip (LOC), show promising capabilities in reducing both the time and the cost of a wide range of chemical and biological processes. More recently, the creation of microfluidic systems which are able to form and control sub-nanolitre droplets, comprising two phase emulsions, have been developed to deliver new experimental platforms. Such systems, also known microdroplets or segmented flow platforms, consist of stable liquid droplets, suspended in a second immiscible phase, with volumes on the nanolitre to the femtolitre scale. The potential of these systems within chemical and biological sciences has already been clearly demonstrated in the literature and commercial platforms are now becoming available. Some of the appealing features that these systems allow are the compartmentalization, the ultra-high throughput experimentation, the imitation of cellular conditions in terms of volumes and chemical composition. The aim of this research project is to exploit the droplet-based LOC systems using the two phase segmented flow to create platforms where proteins can be investigated and even expressed within the droplet chassis. Initial work used a single emulsion strategy (i.e. droplet of water in oil) to selectively capture cellular proteins from a cell suspension, which was directly processed on chip. It was observed that proteins remain active in these systems. In addition, a complexity of conventional laboratory procedures for protein quantification assays was reduced, both in terms of investigation times and amounts of valuable biological samples used. The obtained results demonstrated that this system has the potential to provide the same level of quantitative information obtained using standard biological techniques (i.e. Western blot) at a lower cost. The research has been moving over the development of artificial cell models, nanolitre sized watery droplets comprising a membrane separating the inner from the external environment. Within these systems, realised using a double emulsion strategy (i.e. droplet of water with a droplet of oil or other immiscible phase surrounded by another watery phase), proteins have been expressed using cell-free protein expression systems. The technology adopted lies broadly within the field of synthetic biology involving the transformation of microorganism’s DNA for the production of the desired proteins. Fluorescent proteins were designed and expressed within the artificial cell and fluorescence assays, implemented within the microfluidic format, confirmed not only the functionality of the expressed protein which behaved like in vivo, but also the possibility of its controlled release. Protein release was possible through the use of polymers, within the double emulsion format, which represent a good class of material for the production of nanometre thickness shells. Future developments in this research will aim to (i) expand the capabilities of the single emulsion system for the capture of multiple proteins from a cell lysate and (ii) use different class of polymers to enrich the artificial cell membrane with membrane proteins aiming towards the natural cell mimicking. Emulsions capable to mimic some aspects of the living cell, like the protein synthesis, represent a great opportunity to be used as tools for protein investigations.
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Guermonprez, Cyprien. "Droplet-based Microfluidic Platform for Quantitative Microbiology." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLX106/document.
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Développement d'une plateforme microfluidique pour la microbiologie quantitative. La plateforme permet la culture de milliers de colonies en parallèle dans des micro-gouttes. L'utilisation de tableau statique pour stocker les gouttes permet non seulement leur observation dans le temps pour des analyses dynamiques mais également la récupération de n'importe quelle goutte pour des études complémentaires. Nous avons également développé un outil permettant de soumettre les gouttes à des gradients chimiques directement sur la plateforme dont nous présentons les mécanismes physiques. Nous avons développé un software d'analyse des données générées par la plateforme pour l'étude de modèles de croissance bactérienne ainsi que l'impact des antibiotiques sur leur proliférationDevelopment of a microfluidic chip for quantitative microbiology. The chip allow for parallel culture of thousands bacterial colonies in micro-droplets stored in static array. The 2D-array enable not only the visualisation of each colonies in timelapse experiment but also the extraction of any of them out of the chip at any time for further analysis (PCR, re-culture,...). The platform is adaptable to a concentration gradient producer, for which we present the physical understanding of working mechanism, that can apply different chemical environments to each colony. We developed in parallel a software that perform the analysis of the data generated by the platform to adress bacteria growth studies as well as the impact of antibiotics on bacteria proliferation
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Schulz, Martin [Verfasser], and Felix von [Akademischer Betreuer] Stetten. "Microfluidic system integration for droplet based digital nucleic acid testing." Freiburg : Universität, 2020. http://d-nb.info/1229349278/34.
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Cavazos, Omar. "Microfluidic-Based Fabrication of Photonic Microlasers for Biomedical Applications." Thesis, University of North Texas, 2019. https://digital.library.unt.edu/ark:/67531/metadc1609066/.
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Optical microlasers have been used in different engineering fields and for sensing various applications. They have been used in biomedical fields in applications such as for detecting protein biomarkers for cancer and for measuring biomechanical properties. The goal of this work is to propose a microfluidic-based fabrication method for fabricating optical polymer based microlasers, which has advantages, over current methods, such us the fabrication time, the contained cost, and the reproducibility of the microlaser's size. The microfluidic setup consisted of microfluidic pumps and a flow focusing droplet generator chip made of polydimethylsiloxane (PDMS). Parameters such as the flow rate (Q) and the pressure (P) of both continuous and dispersed phases are taken into account for determining the microlaser's size and a MATLAB imaging tool is used to reduce the microlaser's diameter estimation. In addition, two applications are discussed: i) electric field measurements via resonator doped with Di-Anepps-4 voltage sensitive dye, and ii) strain measurements in a 3D printed bone-like structure to mimic biomedical implantable sensors.
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Leung, Kaston. "A programmable droplet-based microfluidic device for multiparameter single-cell analysis." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/43855.
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Bhattacharjee, Biddut. "Study of droplet splitting in an electrowetting based digital microfluidic system." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43314.
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This thesis focuses on the symmetric and asymmetric splitting of droplets, which is a prominent fluidic operation in a digital microfluidic system (DMFS). The prerequisite part of the investigation of droplet splitting is to understand the electrowetting-on-dielectric (EWOD) based droplet actuation. This thesis demonstrates that not only the EWOD actuation is a self-feedback system - implying that the actuation force depends on the position of the droplet, but also the size of the droplet affects the magnitude of actuation force. However, a sensing mechanism is essential for complex operations, e.g. dispensing and splitting. One contribution of this thesis is a novel method of sensing the droplet position that requires connections to the two adjacent electrodes in the lower plate only.
For the fabrication of prototype DMFS, a new polymeric material, cyanoethyl pullulan (CEP), is proposed as the dielectric layer resulting in a simple and low-cost fabrication of DMFS. The required voltage for droplet manipulation is drastically reduced owing to high relative permittivity of CEP.
Droplet splitting is investigated both numerically and experimentally. Numerical investigation of droplet splitting in FLOW-3D®, a commercial computational fluid dynamics software, revealed that the strength of viscous forces relative to the surface tension force determines the success of splitting. For successful asymmetric splitting, performed by applying voltages of unequal magnitude to left-hand and right-hand sides of the droplet, there exists a minimum voltage for the low-voltage side that guarantees splitting. This minimum voltage increases if the aspect ratio (i.e., diameter to height) of the droplet is reduced while keeping the diameter of the droplet constant. Investigation of the asymmetric splitting with different ratios of applied voltage revealed that the ratio between the volumes of accumulated liquid on either sides increases with voltage ratio. The feasibility of asymmetric splitting as well as the effects of different ratios of applied voltages were studied in prototype DMFS. The results verify the existence of a minimum voltage for successful splitting. The ratio between the volumes of the sister droplets increases with that of the applied voltages. Moreover, the general characteristics of flow-rates and liquid accumulation were found to be similar to those in simulations.
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Lu, Heng. "Development of droplet-based microfluidic tools for toxicology and cancer research." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCB064.
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Ce projet de thèse portait sur le développement d’outils microfluidiques pour la toxicologie et la recherche contre le cancer. En permettant l’analyse simultanée d’un très grand nombre de réactions biologiques ou chimiques réalisés dans des compartiments indépendants (ie. gouttelettes), la microfluidique de gouttes offre une sensibilité de détection et une précision sans précédent pour l’analyse de molécules biologiques, telles que l’ADN ou les Anticorps, en comparaison des expériences réalisées conventionnellement en tubes ou en microplaques (essais en « bulk » ou volume). Ce format permet également de réaliser des expériences à très haut débit et est particulièrement pertinent pour la toxicologie, où des analyses robustes de l’effet des médicaments sont nécessaires. De même, ces procédures sont également très adaptées à l’analyse de cellules uniques pour le séquençage ADN ou ARN et l’épigénomique. Tout cela fait de la microfluidique en goutte un outil puissant pour la toxicologie et la recherche sur le cancer. En premier temps, une méthode du comptage précise des cellules encapsulée dans des microgouttelettes, nommée « hémocytométrie microfluidique », a été développée. Un nouvel algorithme de comptage a été proposé. Des cellules bactériennes (Escherichia Coli) et des cellules de 2 lignées humaines différentes (HL60 and H1975) ont été testées. Le nombre de chaque type de cellules a été déterminé avec une haute corrélation entre la théorie (basée sur la distribution de Poisson) et les résultats expérimentaux. Avec ces résultats robustes, un protocole de microfluidique en goutte a été mis en place pour interroger la viabilité cellulaire et la prolifération des 2 lignées humaines. Ces résultats sont en concordance avec ceux de la littérature. Pour la toxicologie, 3 différents modèles, y compris des microsomes (extrait de cellules d’insectes infectées par un baculovirus exprimant le cytochrome P450 3A4 humain, CYP3A4), HepG2-CYP3A4 (modifiée génétiquement pour exprimer le gène CYP3A4 humain), et HepaRG, une lignée hépatique, ont été évaluées pour l’activité enzymatique du CYP3A4, une enzyme largement utilisée en routine pour le criblage de médicament candidat. Les microsomes ont permis de développer un essai fluorogénique permettant de mesurer l’inhibition du CYP3A4. Cependant, ni l’utilisation des microsomes ni des cellules HepG2 exprimant CYP3A4 n’a donné de résultats satisfaisants en microgouttelettes. L’utilisation des cellules HepaRG, une lignée cellulaire qui conserve la majorité de l’expression des cytochromes P450 et des récepteurs nucléaires nécessaire à leur expression, a montré des résultats encourageant à la fois sur les tests de mesure de l’activité enzymatique et d’analyse de l’induction du CYP3A4. Pour la recherche sur le cancer, 4 essais originaux de PCR digitale en gouttes ont été mis en place pour la détection et la quantification de mutations (NRAS, DNMT3A, SF3B1 and JAK2) importante pour les syndromes myélodysplasiques, un groupe hétérogène de maladies touchant les cellules souches hématopoïétiques caractérisées par une hématopoïèse inefficace et des cytopénies périphériques. Finalement, un essai de PCR sur cellule unique encapsulées au sein de billes agarose a été proposéThis thesis project consists in developing droplet-based microfluidic tools for toxicology and cancer research. Owing to its large numbers of discretized volumes, sensitivity of detection of droplet-based microfluidics for biological molecules such as DNA and antibody is much higher than bulk assays. This high throughput format is particularly suitable for experiments where a robust dose-response curve is needed, as well as for single cell analysis with applications in genomic or sequencing and epigenetics. All above makes droplet-based microfluidics a powerful tool for toxicology and cancer research. In a first part of the work, an accurate cell counting method, named “microfluidics hemocytometry”, has been developed. A new counting algorithm was proposed to count the cells within each droplet. Escherichia Coli and two different human cell lines (HL60 and H1975) were used to validate our strategy. The number of each type of cells in droplets was determined with a high consistency between theory (Poisson distribution) and experimental results. With these robust results, a droplet-based microfluidic protocol has then been established to inquiry both cell viability and proliferation for the two human cell lines. The results are in good agreement with the one of the literature. For the toxicology, 3 different biological models, including microsomes (extracted from baculovirus-infected insect cell expressing human CYP3A4), HepG2-CYP3A4 (genetically modified to express the human CYP3A4 gene) and HepaRG liver cells lines were evaluated for enzymatic activity of cytochromes P450 (CYP3A4), a routinely used enzyme for drug candidate screening. Microsome-based assays were used to validate a fluorogenic inhibition assay. However neither microsome-based assay nor the assay using CYP3A4 expressing HepG2 gave satisfying results in droplet-based format. However, HepaRG cells, a hepatic function-conserved cell line with most cytochrome and related nuclear receptors, demonstrated high relevance both for enzymatic activity testing and CYP3A4 expression induction study. For cancer research, 4 different picoliter droplet-based PCR assays were developed for the detection and quantification of mutations (NRAS, DNMT3A, SF3B1 and JAK2) present in Myelodysplastic syndromes, a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Furthermore, a single cell multistep PCR assay using encapsulation of target DNA in agarose droplets was proposed
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Pekin, Deniz. "Development of novel droplet-based microfluidic strategies for the molecular diagnosis of cancer." Phd thesis, Université de Strasbourg, 2013. http://tel.archives-ouvertes.fr/tel-00856594.
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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.
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Cho, Soongwon David. "An integrated droplet based microfluidic platform for high throughput, multi-parameter screening of photosensitiser activity." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/18482.
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With rapid advances in the field of cellomics, genomics, and proteomics, the demands for development of enabling technologies for performing high throughput biological experimentation are ever increasing. Droplet based microfluidic systems have recently been developed to perform high throughput experimentations. With the ability to generate droplets over 1 kHz frequency and perform combinatorial experiments via various passive and active manipulating techniques, microdroplet technology provides an ideal platform for combinatorial biological experiments whilst consuming minimal amount of reagent. As it is possible to generate droplets, manipulate them, and characterise droplets using highly sensitive on-line detection systems, it is now crucial to bring various functionalities together to create a micro total analysis system capable of performing complex biological experiments within microfluidic devices. As such, an integrated droplet based microfluidic platform was developed to assess the efficacy of photodynamic therapy against microbial organisms. Photodynamic therapy is an alternative efficacious treatment method for the treatment of localized microbial infections with several favourable features such as broad spectrum of action, efficient inactivation of multidrug-resistant bacteria, and low mutagenic potential. In order to perform the photosensitiser cytotoxicity screening, various microfluidic modules such as droplet generation, chamber based microdroplet storage and light irradiation, droplet reinjection, electrocoalescence and on-chip viability scoring of cells within droplets using a combination of carboxyfluorescein diacetate and propidium iodide were developed and integrated within the microfluidic platform. The microfluidic system was then used to screen the cytotoxicity of TBO against E.coli cells and the results were validated against conventional colony forming unit assays. Finally, the integrated system was used to assess the effects of several parameters on E.coli viability such as dark toxicity, photosensitiser concentration, light dose and poor oxygenation condition.
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Ghifari, Najla. "Microfluidic-based colloidal ZnO microcapsules : synthesis, structure,organization and first applications." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPAST066.
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Ce travail se concentre sur le développement d’une approche microfluidique originale à base degouttelettes pour générer des sphères de ZnO de taille micrométrique très monodispersées avec une taille et une morphologie bien contrôlées. Cette approche est simple et prometteuse non seulement pour la fabrication de microcapsules de ZnO de taille uniforme, avec une taille ajustable et un contrôle précis à l’échelle micrométrique, mais aussi pour acquérir de nouvelles connaissances sur la compréhension des processus de croissance colloïdale et l’auto-organisation des nanoparticules de ZnO par la voie microfluidique. En outre, ces microparticules peuvent trouver des applications intéressantes dans de nombreux domaines tels que la photonique, le photovoltaïque ou la biomédecine. Ce travail porte sur l’effet des paramètres de fabrication sur la formation de gouttelettes, la taille et la stabilité des microsphères résultantes, ainsi que sur l’étude de leurs propriétés optiques et électriques, encouplant les travaux expérimentaux et théoriques. Nous avons montré la synthèse, dans une gamme micrométrique allant de 10 µm à 30 µm, de microcapsules de ZnO mésoporeuses à enveloppe fine et flexible. Nous étudions la caractéristique polaire des nanoparticules de ZnO et leur auto-organisation interfaciale. En outre, nous révélons que les charges électriques portées par lesunités primaires de ZnO jouent un rôle crucial dans la stabilité des gouttelettes en présence et en absence de molécules chargées. Elle joue également un rôle clé tout au long du processus d’assemblage, de la création des nanoparticules colloïdales de ZnO aux microgouttelettes, et enfin aux microsphères. Nous rapportons, pour la première fois, l’auto-organisation de microgouttelettes de ZnO liquide dopé en réseaux carrés. Nous démontrons qu’un tel résultat révèle l’aspect polaire des microgouttelettes de ZnO et corrobore un changement d’équilibre entre les forces motrices contrôlant l’organisation des nanoparticules de ZnO à l’échelle nanométrique. Nous avons développé différents modèles, en très bon accord avec le champdipolaire et les mécanismes de forces interfaciales, pour étayer les résultatsexpérimentaux mis en avant, et pour expliquer l’organisation interfaciale des nanoparticules de ZnO/RhB sur la base des propriétés d’organisation des gouttelettes de ZnO. À partir de nos résultats et de la dépendance constatée de la taille des microcapsules, de l’épaisseur de la coquille et de la densité de surface des nanoparticules par rapport à la taille des gouttelettes, nous fournissons un modèle original de la contribution des facteurs impliqués dans le mécanisme de formation de la coquilleThis work focuses on the development of an original droplet-based microfluidics approach to generate highly monodisperse micrometer-sized ZnO spheres with well-controlled size and morphology. This approach is straightforward, and promising not only for the fabrication of uniform-sized ZnO microcapsules, with adjustable size and precise control at the microscale, but also for gaining new insights into the understanding of colloidal growth processes and self-organization of ZnO nanoparticles by the microfluidic route. In addition, such microparticles may find interesting applications in many areas such as photonics, photovoltaics, or biomedecine. This work deals with the effect of handling parameters on droplet formation, size, and stability of the resulting microspheres, as well as the study of their optical and electrical properties coupling experimental and theoretical works. We have shown the synthesis, in a micrometric range from 10 mm to 30 mm, of mesoporous ZnO microcapsules with a thin and flexible shell. We investigate the polar feature of ZnO nanoparticles and their interfacial self-organization. Besides, we reveal that the electric charges carried by ZnO primary units play a crucial role in the stability of the droplets in the presence and in the absence of charged molecules. It also plays a key role throughout the assembly process from the creation of the colloidal ZnO nanoparticles to the microdroplets, and finally the microspheres. We report, for the first time, the selforganization of doped-ZnO liquid microdroplets in square arrays. We demonstrate that such a result discloses the polar aspect of ZnO microdroplets and corroborate a shift in the balance between the driving forces controlling the ZnO nanoparticles organization at the nanoscale. We have developed different models, in very good agreement with the dipole-field and interfacial forces mechanisms, to support the experimental results put forward, and to explain the ZnO/RhB nanoparticles interfacial organization based on ZnO droplets organization properties. Based on our findings, and on the stated dependence of the microcapsules size, shell thickness, and nanoparticles surface density versus the droplets size, we provide an original model for the contribution of the involved factors in the shell formation mechanism
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Doineau, Raphaël. "Development of droplet-based microfluidic technology for high-throughput single-cell phenotypic screening of B cell repertoires." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC263/document.
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Le système immunitaire adaptatif joue un rôle de premier plan dans la défense contre les infections. La réponse humorale, impliquant la production d'anticorps, est un élément important de la réponse immunitaire adaptative. Au cours d'une infection, des cellules B spécifiques du système immunitaire prolifèrent et libèrent de grandes quantités d'anticorps qui se lient sélectivement à la protéine cible (antigène) trouvée sur le pathogène invasif, induisant la destruction du pathogène.Cependant, le système immunitaire ne répond pas toujours suffisamment efficacement pour détruire les agents pathogènes, et les mécanismes de tolérance empêchent la génération d'anticorps contre les protéines humaines - comme les marqueurs de surface cellulaire sur les cellules cancéreuses ou les cytokines impliquées dans des maladies inflammatoires et auto-immunes - qui pourraient être des cibles thérapeutiques importantes. Par conséquent, il existe un grand intérêt pour la recherche et le développement d'anticorps spécifiques qui peuvent être utilisés pour le traitement des patients par immunothérapie. En raison de leur grande affinité et de leur liaison sélective aux antigènes, les anticorps monoclonaux (mAbs) sont apparus comme des agents thérapeutiques puissants. Les anticorps monoclonaux dérivés de cellules B individuelles ont une séquence unique et présentent une affinité de liaison pour un antigène spécifique. Cependant, jusqu'à maintenant, la découverte des mAbs a été limitée par l'absence de méthodes à haut débit pour le criblage direct et à grande échelle de cellules B primaires non immortalisées pour découvrir les rares cellules B qui produisent des anticorps spécifiques d'intérêt clinique. Ceci est maintenant possible avec l'émergence et l'amélioration des méthodes de compartimentation in vitro pour l'encapsulation et le criblage de cellules uniques dans des gouttelettes picolitriques. Dans mon projet de doctorat, je décris le développement d'immunodosages et de dispositifs microfluidiques pour le criblage phénotypique direct de cellules individuelles à partir de populations de cellules B enrichies. Ce développement a permis une analyse détaillée de la réponse immunitaire humorale, avec une résolution à l’échelle de la cellule unique. C’est aussi un élément essentiel d'un pipeline de détection d'anticorps couplant le criblage phénotypique de cellules individuelles au séquençage d'anticorps sur cellules uniques. Il est maintenant possible, pour la première fois, de cribler des millions de cellules B individuelles en fonction de l'activité de liaison des anticorps sécrétés et de récupérer les séquences d'anticorpsThe adaptive immune system plays a leading role in defense against infection. The humoral response, involving the production of antibodies, is an important component of the adaptive immune response. During an infection, specific B cells of the immune system proliferate and release large amounts of antibodies which bind selectively to the target protein (antigen) found on the invading pathogen, inducing destruction of the pathogen. However, the immune system does not always respond efficiently enough to destroy pathogens, and tolerance mechanisms prevent the generation of antibodies against human protein - such as cell surface markers on cancer cells or cytokines involved in inflammatory and autoimmune disease - that could be important therapeutic targets. Hence, there is great interest in research and development of specific antibodies that can be used for immunotherapy of patients. Due to their high affinity and selective binding to antigens, monoclonal antibodies (mAbs) have emerged as powerful therapeutic agents. Monoclonal antibodies derived from single B cells have a unique sequence and display binding affinity for a specific antigen. However, until now, the discovery of mAbs has been limited by the lack of high-throughput methods for the direct and large-scale screening of non-immortalized primary B cells to uncover rare B cells which produce the specific antibodies of clinical interest. This is now becoming possible with the emergence and improvement of in vitro compartmentalization methods for single-cell encapsulation and screening in picoliter droplets. In my PhD project, I describe the development of binding immunoassays and microfluidic devices for the direct phenotypic screening of single-cells from enriched B cell populations. This development has enabled detailed analysis of the humoral immune response, with single-cell resolution and is an essential component of an antibody-discovery pipeline coupling single-cell phenotypic screening to single-cell antibody sequencing. It is now possible, for the first time, to screen millions of single B cells based on the binding activity of the secreted antibodies and to recover the antibody sequences
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Vitor, Micaela. "Droplet-based microfluidic systems to incorporate nucleic acids into cationic liposomes and to transfect mammalian cells in vitro." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLX020/document.
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Ce travail consiste à utiliser deux systèmes microfluidiques de gouttes pour incorporer d'une part des acides nucléiques dans des liposomes cationiques et d'autre part étudier la dynamique de transfection dans des cellules mammifères. La première micropuce permet d'insérer de l'ADN dans des liposomes cationiques afin d'obtenir de manière reproductible des lipoplexes appropriés à la transfection de cellules dendritiques (DC). Plusieurs paramètres expérimentaux sont tout d'abord étudiés, tels que les débits d'entrée, l’entretien des propriétés des liposomes après leur traitement dans des micro-gouttes, les caractéristiques des lipoplexes (taille, polydispersité et charge) en fonction du rapport molaire de charge (R+/-) et de la géométrie de la puce. Ensuite, les lipoplexes produits dans des conditions optimisées: une micropuce avec un grand canal en serpentin et une région de division des gouttes qui diminuent la polydispersité des lipoplexes, fonctionnant à un rapport de débit eau/huile 0,25 et R+/- 1,5; 3; 5; 7 et 10; sont utilisés pour transfecter des DCs in vitro. Tous les lipoplexes transfectent les DCs, tout en offrant une activation des DCs. La seconde étape consiste à utiliser une micropuce à l'échelle de la cellule unique afin de contrôler les conditions de transfection et d'optimiser le rendement de production de protéines recombinantes. Ainsi, des cellules ovariennes de hamster Chinois (CHO-S) sont transfectées dans la micropuce avec différents types de lipoplexes (R+/- 1,5; 3; 5) dont la dynamique de transfection est suivie par la production de protéines vertes fluorescentes (GFP) et par la viabilité cellulaire. Cette micropuce a permis d'évaluer l’hétérogénéité des cellules transfectées, révélant la présence d'une sous-population produisant des niveaux particulièrement élevés de GFP. Ces hautes productrices (HP) ont une taille cellulaire plus importante que celle de la population moyenne. La charge des lipoplexes montre un rôle important pour transfecter CHO-S, puisque l’unique lipoplex chargé positif R+/- 5 produit plus de HPs. La quantité d’ADN délivrée influe sur la production de protéine, puisque R+/- 1,5 avec plus d’ADN augmente la productivité spécifique de GFP des HPs. Cette thèse est réalisée dans le cadre d'un programme de co-tutelle entre l'Université de Campinas, au Brésil, et l'École Polytechnique, en France. Ce travail a principalement contribué aux domaines de microfluidique et de délivrance de gènesThis work aims to use one droplet-based microfluidic systems to incorporate nucleic acids into cationic liposomes and another one to study the mammalian cell transfection process. For this, the first step uses a droplet-based microfluidic system to complex cationic liposomes with pDNA in order to obtain reproducible and suitable lipoplexes to dendritic cells (DCs) transfection. For this purpose, some experimental parameters are investigated, such as inlet flow rates, the maintenance of liposomes’ properties after microfluidic processing, lipoplex characteristics (size, polydispersity and zeta potential) as function of molar charge ratio (R+/-) and microchip design. Then, lipoplexes produced in selected conditions: a microchip with large serpentine channel and split region, which decreases lipoplex polydispersity, operating at ratio aqueous/oil flow rate 0.25 and R+/- 1.5, 3, 5, 7 and 10; are used to transfect DCs in vitro. All lipoplexes transfect DCs while providing cells activation. The second step uses a single-cell microfluidic platform to investigate and control over the transfection conditions, in view of optimizing the recombinant protein production by transfected cells. In this context, Chinese hamster ovary cells (CHO-S) are transfected in microchip with different types of lipoplexes (R+/- 1.5, 3, 5) and monitored by green fluorescent protein (GFP) production and cell viability. The single-cell platform enables to assess the heterogeneities of CHO-S population, revealing the presence of a subpopulation producing significantly high levels of GFP. These high producers (HP) show increased cell size in comparison to the average population. Moreover, the charge of lipoplexes shows an important role to transfect CHO-S, since the unique positive charged lipoplex R+/- 5 produces more HPs. Additionally, the amount of pDNA delivered affects the protein production, since R+/- 1.5 with more pDNA increase GFP specific productivity of HPs. This thesis is a co-supervised program between University of Campinas, Brazil and École Polytechnique, France. In general, this work contributes to microfluidics and gene delivery areas
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Weiss, Marian [Verfasser], and Joachim [Akademischer Betreuer] Spatz. "Microfluidic Approaches for the Sequential Bottom-up Assembly of Droplet-based Minimal Synthetic Cells / Marian Weiss ; Betreuer: Joachim Spatz." Heidelberg : Universitätsbibliothek Heidelberg, 2018. http://d-nb.info/1177691485/34.
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Weiss, Marian [Verfasser], and Joachim P. [Akademischer Betreuer] Spatz. "Microfluidic Approaches for the Sequential Bottom-up Assembly of Droplet-based Minimal Synthetic Cells / Marian Weiss ; Betreuer: Joachim Spatz." Heidelberg : Universitätsbibliothek Heidelberg, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:16-heidok-240369.
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Frey, Christoph [Verfasser], and Joachim P. [Akademischer Betreuer] Spatz. "Development of functional droplet based microfluidic systems for synthetic biology and biomedical high-throughput applications / Christoph Frey ; Betreuer: Joachim P. Spatz." Heidelberg : Universitätsbibliothek Heidelberg, 2021. http://d-nb.info/1225184797/34.
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Vigne, Aurélie. "Microfluidic tools for the engineering of enzymes of therapeutic interest." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0391/document.
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Cette thèse concerne le développement d’outils microfluidique pour l’ingénierie d’enzymes d’intérêt thérapeutique. La microfluidique à base de gouttelettes présente un énorme potentiel dans le domaine de la biologie quantitative. Nous développons des outils microfluidiques pour l’évolution dirigée de l’enzyme L-asparaginase, enzyme utilisée comme traitement de laleucémie lymphoblastique aiguë. Ce traitement est basée sur une enzyme d’origine bactérienne,ce qui conduit à déclencher des réactions immunitaires qui se traduit par l’interruption du traitement, souvent fatale pour le patient. Cependant, une version humaine de l’enzyme L-asparaginase, qui est moins immunogénique, n’est à l’heure actuelle pas suffisamment active pour être utilisée. L’objectif principal de cette thèse est d’alors d’analyser et de cribler des banques de mutants d’enzymes en utilisant des méthodes classiques de mutagenèse et d’analyser chaque mutant individuellement par le biais de la microfluidique. Pour cela, plusieurs systèmes microfluidiques ont été développés et optimisés afin de répondre à différents critères de sélection pour l’analyse et la sélection de l’enzyme L-asparaginase. La version bactérienne a servi de contrôle positif pour l’optimisation des systèmes microfluidiques afin de pouvoir analyser et de cribler des banques de mutants de la version humaine de l’enzyme L-asparaginaseThis thesis deals with the development of microfluidic tools for the engineering ofenzymes of therapeutic interest. Droplet microfluidics has enormous potential in the field ofquantitative biology. We are developing microfluidic tools based on the directed evolutionof the enzyme L-asparaginase, an enzyme used to treat acute lymphoblastic leukemia. Thistreatment is based on an enzyme of bacterial origin, which leads to immune reactions thatresult in the interruption of treatment, often fatal for the patient. However, a human version ofthe enzyme L-asparaginase, which is less immunogenic, is currently not sufficiently active to beused. The main objective of this thesis is to analyze and screen enzyme mutant libraries usingstandard mutagenesis methods and to analyze each mutant individually through microfluidics.For this, several microfluidic systems have been developed and optimized for different selectioncriteria for the analysis and selection of the enzyme L-asparaginase. The bacterial versionserving as a positive control for the optimization of microfluidic workflows to analyze andscreen mutant libraries of the human version of the enzyme L-asparaginase
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Trouchet, Amandine. "PCR digitale pour la détection et la caractérisation de micro-organismes pathogènes au niveau de la cellule unique." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC170/document.
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Nous avons pour but de développer un système microfluidique en gouttes, capable, à l’échelle de la cellule/bactérie unique, de détecter et de co-localiser plusieurs marqueurs génétiques, en utilisant une version digitale et multiplexée de la réaction de polymérisation en chaîne (PCR). Les systèmes de PCR digitale actuellement commercialisés ne le permettent toujours pas. Un tel prototype garantira la présence de multiples marqueurs à l’intérieur d’un même génome, ce qui permettra l’identification du pathogène avec précision et un taux de faux-positifs proche de zéro. Comme première application, nous démontrerons la possibilité de co-localiser quatre gènes de virulence de la souche O157:H7 d’Escherichia coli, un pathogène majeur, qui est détecté dans des échantillons alimentaires ou provenant de fèces cliniques pouvant aussi contenir des E. coli non pathogènes porteuses d’une partie des gènes de virulence. Avant de procéder à des tests TaqMan multicolores en point final, E. coli sera d’abord encapsulée dans des gouttes micrométriques et lysée par la chaleur in situ. Notre objectif est de démontrer que ce test peut être appliqué avec succès à un petit ensemble d’échantillons cliniques ou alimentairesWe aim to develop a prototype of droplet-based microfluidic system capable of detecting and colocalizing multiple genetic markers at the single cell/bacteria level, using the Polymerase Chain Reaction (PCR) in a digital multiplexed version. This cannot be achieved using current commercial digital PCR systems, and should increase the sensitivity and reliability of the detection of pathogens. Importantly, the system will guarantee the presence of multiple markers within the same genome and enable accurate identification, and bring the false positive rate close to zero. As a first application, we will demonstrate the possibility to co-localize 3 virulence genes in the E.coli strain O157:H7, a major foodborne pathogen, which has to be detected in clinical feces samples or food samples, which may also contain non pathogenic E. coli carrying only a subset of these virulence genes. E. Coli will be encapsulated in micrometric droplets, lysed by heating in situ prior performing a multicolour end-point Taqman assay. Our objective is to demonstrate that this test can be successfully applied to real clinical or food samples
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Mazutis, Linas. "Droplet-based microfluidics for protein evolution." Strasbourg, 2009. http://www.theses.fr/2009STRA6178.
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La compartimentalisation de la "soupe primordiale" dans des vésicules est considérée comme l'un des principaux facteurs ayant permis l'émergence de la vie. Ces gouttelettes de quelques micromètres créent un lien entre génotype et phénotype, et grâce à la division, un mécanisme pour l’hérédité et l’évolution, qui a conduit à l'émergence des cellules actuelles. De tels microcompartiments, peuvent être créés au laboratoire sous la forme d’une émulsion composée de millions de gouttelettes contenant des gènes et tous les ingrédients nécessaires pour leur expression in vitro. Ces émulsions miment ainsi des populations de cellules artificielles qui peuvent être sélectionnées pour un phénotype donné sous des conditions strictement contrôlées non réalisables dans des systèmes in vivo. Cette thèse de doctorat présente le développement de systèmes microfluidiques pour l’évolution dirigée. Les résultats obtenus montrent qu’il est possible de produire des gouttelettes hautement monodisperses pouvant être manipulées de manière précise et contrôlable, ce qui était jusqu’à présent impossible pour des émulsions réalisées par des méthodes classiques. En utilisant un ensemble de nouveaux dispositifs microfluidiques et une composition adéquate d’huile porteuse, des gènes uniques ont été amplifiés et leur expression in vitro mesurée en microgouttelettes. Ces dispositifs ont ensuite été utilisés pour réaliser et analyser des réactions biologiques complexes et multi-étapes. Une technique originale de fusion passive de paires de gouttelettes a également été développée. Ces travaux constituent les premiers pas vers la création de plate-formes microfluidiques intégrées et totalement in vitroThe compartmentalization of the primordial soup into vesicles is thought to be one of the key features in the early emergence of life. These tiny micrometer-sized droplets provided a linkage between phenotype and genotype, and through division, a mechanism for heredity and evolution, which gave rise to modern cells. Man-made compartments, in the form of an emulsion, can also provide a tool of linking genotype to phenotype. Composed of millions of droplets containing genes with all ingredients necessary for in vitro expression, emulsions mimic populations of artificial cells that can be selected for a particular phenotype under strictly controlled conditions not feasible in living systems. The research described in this doctoral thesis focuses on the development of droplet-based microfluidics for protein evolution and presents the first steps toward an integrated and completely in vitro microfluidics platform. The results obtained in this work show that it is possible to produce highly monodisperse picoliter volume droplets (CV<1%) that can be manipulated in a precise and controllable manner, previously impossible in bulk emulsions. Using a set of novel microfluidic devices and an adequate composition of carrier oil single genes in droplets were amplified and their in vitro expression measured. The same microfluidic system was also used to perform multiple operations in order to analyze complex and sequential biological reactions in droplets. Moreover, a new passive droplet fusion technique has been developed, which can be used for preparation of monodisperse emulsions composed of pairwise fused droplets
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Mohammadi, Kimia. "Synthetic biology in droplet-based microfluidics." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7596/.
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Droplet microfluidics is an active multidisciplinary area of research that evolved out of the larger field of microfluidics. It enables the user to handle, process and manipulate micrometer-sized emulsion droplets on a micro- fabricated platform. The capability to carry out a large number of individual experiments per unit time makes the droplet microfluidic technology an ideal high-throughput platform for analysis of biological and biochemical samples. The objective of this thesis was to use such a technology for designing systems with novel implications in the newly emerging field of synthetic biology. Chapter 4, the first results chapter, introduces a novel method of droplet coalescence using a flow-focusing capillary device. In Chapter 5, the development of a microfluidic platform for the fabrication of a cell-free micro-environment for site-specific gene manipulation and protein expression is described. Furthermore, a novel fluorescent reporter system which functions both in vivo and in vitro is introduced in this chapter. Chapter 6 covers the microfluidic fabrication of polymeric vesicles from poly(2-methyloxazoline-b-dimethylsiloxane-b-2-methyloxazoline) tri-block copolymer. The polymersome made from this polymer was used in the next Chapter for the study of a chimeric membrane protein called mRFP1-EstA∗. In Chapter 7, the application of microfluidics for the fabrication of synthetic biological membranes to recreate artificial cell- like chassis structures for reconstitution of a membrane-anchored protein is described.
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Bartolo, Jean-François. "Développement de sondes et de systèmes microfluidiques pour la détection de nouveaux biomarqueurs spécifiques." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAF052/document.
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L’efficacité des traitements contre diverses pathologies dépend dans bien des cas de la précocité de la prise en charge des patients. Ce contexte pousse de nos jours les chercheurs à élaborer de nouvelles méthodes de diagnostic, généralement basées sur la détection de biomarqueurs spécifiques, permettant d’établir une corrélation entre un dérèglement moléculaire de l’organisme et la survenue d’une maladie. L’objectif de ces travaux était, par l’utilisation de la microfluidique digitale en gouttelettes, d’établir de nouvelles procédures simples et reproductibles, témoignant d’une sensibilité importante afin de déterminer d’infimes variations de l’état moléculaire de l’organisme à travers la recherche de biomarqueurs spécifiques. Pour cela nous avons élaboré une nouvelle gamme de tensioactifs fluorées adaptés aux applications biologiques en microfluidique digitale, ainsi que différentes stratégies d’étude des variations de l’expression de microARN extrait d’échantillons biologiques, basées respectivement sur les réactions induites par hybridation nucléotidique et sur la réaction de RT-PCR digitaleEfficiency of treatments for various diseases depends in many cases in precocity of patient management. Nowadays, this context urges researchers to develop new methods of diagnosis, generally based on the detection of specific biomarkers. These new methods allowing to establish correlations between physiological disorders and arisen of diseases states.The aim of this study was, by the use of droplet-based microfluidic, to work out a simple and reproducible procedure, with an increased sensitivity, to determine tiny variations of physiological state through the detection of specific biomarkers. Thus, we developed a new range of fluorinated surfactants fitted to biological applications in droplet-based microfluidics as well as various strategies to study variations of microRNA expressions in a biological sample. These methods, based on DNA-template reaction and digital PCR reaction, allows performing a substantial number of simultaneous reactions in micro-compartments (microdroplets) of picolitre volumes
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Bauer, Wolfgang-Andreas Christian. "Complex emulsion systems via droplet-based microfluidics." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610619.
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Chacon, Orellana Laura A. "Fluorinated pickering emulsions for droplet-based microfluidics technology." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0121/document.
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Les émulsions fluorées de Pickering sont étudiées et mises au point dans la technologie demicrofluidique en gouttes pour des applications d’études sur des cellules adhérentes isolées.Les principaux résultats de ce projet sont : l’établissement d’un lien entre la couverture desurface des nanoparticules et la fluidité de l’émulsion de Pickering ; l’établissement deslignes directrices pour la stabilisation des gouttes avec un débit de production élevé et unminimum de déchets de particules ; et la mise en oeuvre d’une plateforme technologiquecomplète pour l’étude des cellules RPE, pour mesurer leur hétérogénéité phénotypique auniveau de la cellule individuelleFluorinated Pickering emulsions are studied and engineered within droplet-based microfluidicstechnology for adherent-cell studies applications. The main findings of this projectinclude: linking the nanoparticles surface coverage to the bulk flowability of the Pickeringemulsion; deriving guidelines for droplet stabilization with high production throughput andminimal particle waste; and implementing the full technological platform for the study ofRPE cells, while unraveling their phenotypic heterogeneity at the single cell level
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Theberge, Ashleigh Brooks. "Droplet-based microfluidics for chemical synthesis and integrated analysis." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609687.
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Pan, Jie. "Droplet-based microfluidics for the development of microalgal biotechnology." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648230.
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El, Debs Bachir. "Functional single-cell hybridoma screening using droplet-based microfluidics." Strasbourg, 2011. http://www.theses.fr/2011STRA6182.
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µm de diamètre) pour la culture de cellules mammifères. On a utilisé ce système pour sélectionner spécifiquement des cellules hybridomes sécrétant des anticorps inhibant l’Enzyme de Conversion de l’Angiotensine. -1 (ECA-1). L’émulsion contenant les cellules encapsulées dans les gouttes a été incubé pendant 6 heurres pour obtenir une quantité considérable d’anticorps avec l’ECA-1. Ensuite, cette émulsion a été réinjectée dans une puce microfluidique, fusionnée avec des gouttes contenant un mélange réactionnel permettant l’obtention d’un signal fluorescent d° à l’activité de l’ECA-1. Les gouttes ayant une faible intensité de fluorescence ont été triées. Une variance considérable dans le taux d’expression d’anticorps a été constatée au niveau mono-cellulaire au sein d’une même lignée de cellules hybridomes o_ les cellules exprimant un taux élevé d’anticorps ont été isolées et cultivées. Ce système permet le criblage de 5_104 cellules par heure et pourra être utiliser pour le criblage de lymphocytes B non immortaliséesThis thesis describes a microfluidic platform allowing the functional screening of hybridoma cells on the single-cell level. In this system, individual cells from a heterogeneous population are encapsulated into aqueous microdroplets of a water-in-oil emulsion and assayed directly for the release of antibodies inhibiting drug targets. The microfluidic setup comprises a novel fully integrated chip which allows reinjection, fusion and sorting of droplets sufficiently large (~100 µm in diameter) for the cultivation of mammalian cells. We successfully used this device for the specific selection of hybridoma cells releasing antibodies inhibiting angiotensin converting enzyme-1 (ACE-1). After cell encapsulation, the resulting emulsion was incubated off-chip for 6h to obtain significant antibody concentrations. Subsequently, the droplets were reinjected into another chip, fused with a second droplet species containing all components of a fluorescence assay for ACE-1 activity, and droplets with low fluorescence intensity (indicating ACE-1 inhibition) were sorted. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be sorted and recultivated. The approach enabled screening more than 5_104 cells per hour and should even be applicable to non-immortalized primary B-cells, as no cell proliferation is required
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Yashina, Alexandra. "Droplet-based microfluidics for the study of CaCO3 crystallisation." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/10165.
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Research into the crystallisation of calcium carbonate traverses many disciplines, including chemistry, physics, biology and materials science. Control over the polymorph, hierarchical assembly, orientation, size and shape has been the focus of significant interest in developing new applications. Current synthetic techniques offer limited control over the crystallisation process and often require high temperatures and/or pressures and the use of organic additives and functionalised templates. Microfluidic systems offer superior control over reaction conditions when compared to traditional macroscale methods and hence provide an alternative approach for the synthesis of particles. Not surprisingly, the literature includes hundreds of papers reporting the microfluidic synthesis of nanomaterials. The control provided by microfluidics allows the arrest of a reaction at any particular time, which is hugely advantageous when studying the nucleation and crystallisation of such particles. Moreover, since most biochemical reactions occur in aqueous media, droplet-based microfluidic devices are excellent tools for the miniaturisation of reaction volumes and the simulation of natural or synthetic processes. In the first part of this thesis, custom built droplet-based microfluidic systems are applied to the study of the precipitation of calcium carbonate under highly controlled conditions. Reactions performed in pL-volume droplets dispersed within a continuous carrier fluid afford reproducible control over crystal polymorph, such that pure calcite, pure vaterite or a mixture of calcite and vaterite can be preferentially precipitated by varying the concentration of reagents. This contrasts with poor reaction control on the macroscale. In the second part of this thesis the early stages of the nucleation of amorphous calcium carbonate are studied in similar droplet-based formats. The precipitation of calcium carbonate is arrested at various time points, with a maximum residence time of 2 minutes, and amorphous calcium carbonate is analysed over different growth periods. Additionally, the effect of poly(styrene sulphonate) on the precipitation and growth of amorphous calcium carbonate is investigated. Results show that in the presence of polymer, calcium carbonate growth occurs by mesoscale assembly rather than by classical nucleation.
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Xu, Xuejiao. "Droplet-based microfluidics for supramolecular synthesis and biological kinetics assay." Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708115.
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Lieber, Diana. "Functional assays for screening antibody activity in droplet-based microfluidics." Strasbourg, 2010. https://publication-theses.unistra.fr/public/theses_doctorat/2010/LIEBER_Diana_2010.pdf.
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Les méthodes de criblage haut-débit sur des cellules nécessitent de petits volumes afin de réduire les coûts et permettre une manipulation rapide des échantillons. En effet, une miniaturisation supplémentaire des technologies classiques de criblage haut-débit en plaques devient problématique à cause de l’évaporation et des forces capillaires. Pour surmonter ces limitations, on a développé des systèmes basés sur la microfluidique en gouttes où des cellules sont cultivées dans des microcompartiments aqueux indépendants entourés d’huile perfluorée inerte. Un tel système permet d’effectuer des analyses automatisées à l’échelle individuelle de chaque compartiment suite à un certain temps d’incubation comme le requiert les tests cellulaires à haut-débit. Nous nous sommes focalisés également sur le développement de tests fonctionnels pour le criblage d’anticorps comme par exemple les anticorps neutralisants le VIH (Virus de l’Immunodéficience Humaine) ou inhibant l’ECA (Enzyme de Conversion de l’Angiotensine). Les approches haut-débit pour la sélection d’anticorps utilisent le « phage display » ou des hybridomes. « Phage display » est une méthode efficace mais permet de sélectionner les anticorps pour leur activité de liaison et non pas de neutralisation. Les hybridomes permettent d’effectuer une sélection en fonction de l’activité neutralisante des anticorps, mais dans cette approche on est limité par le nombre de clones pouvant être sélectionnés. Le but de ce projet était d’établir un nouveau test permettant de sélectionner, au niveau de cellules uniques, des cellules sécrétrices d’anticorps (hybridomes). Ce système pourra être utilisé également pour le criblage de cellules B. Une fois établi, ce système pourrait être utilisé pour le criblage/sélection de beaucoup d’autres anticorps thérapeutiquesHigh-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturisation of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we developed droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Furthermore, this system allowed for an automated analysis of individual compartments subsequent to an incubation period as required for high-throughput, cell-based assays. In addition, we focused on the development of functional assays for screening antibody activity, e. G. Neutralisation of HIV or the inhibition of ACE-1. Common high throughput approaches for antibody screening use phage display or hybridoma cells. Phage display is powerful but based on binding properties rather than neutralising effects. Hybridoma cells allow for direct screening of neutralising activity, but are very restricted in the number of clones that can be screened due to their generation and proliferation as required for conventional screens. The aim of this study was the development of novel screening technology with the ultimate goal of screening single antibody-releasing cells (hybridoma cells). This system should also allow for direct B-cell screening. Once established, this technology could be used for the screening/ selection of many more therapeutic antibodies
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Frenz, Lucas. "Development, integration and application of modules for droplet-based microfluidics." Strasbourg, 2009. http://www.theses.fr/2009STRA6243.
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La miniaturisation est devenue un concept puissant influençant la plupart des disciplines scientifiques. Révolutionnant au départ l’électronique et l’informatique, elle s’est alors étendue aux domaines de la microélectromécanique et plus récemment à ceux de la microfluidique. Le travail réalisé dans cette thèse touche aux trois principaux domaines de recherche de la microfluidique en gouttes: la physique, la science des matériaux ainsi que les applications de criblage. Des modules et des principes novateurs pour la manipulation de gouttes ont été développés et caractérisés. Un module permet de trier des gouttelettes suivant leur différence de taille plutôt que selon leur contenu. Un autre développement concerne un module original de synchronisation de gouttes qui peut créer des paires de gouttes avec une précision quasi parfaite. Le module présentant le plus large intérêt est probablement le développement d’une ligne de délai pour une incubation sur puce. Grâce à ces efforts, il a donc été possible d’intégrer plusieurs modules en gouttes fonctionnant l’un avec l’autre sur une puce unique, afin de créer des dispositifs complexes utiles pour les applications de criblage comme par exemple, l’évolution dirigée d’enzymes. Un autre développement concernant les applications de criblages est un système de dilution permettant de générer plusieurs ordres de grandeur de concentrations d’un composé pour réaliser des criblages à haut débit avec des données statistiques de qualité largement supérieure aux méthodes conventionnelles. En plus la microfluidique en gouttes a été utilisée pour synthétiser des nano-particules d’oxyde de fer via une réaction très rapide et aisément contrôlableMiniaturization has become a powerful concept influencing almost every scientific discipline. Initially revolutionizing electronics and computing, it has soon expanded into the microelectromechanical and more recently microfluidic fields. Here, channels which are often thinner than a human hair are used to manipulate micro- to picoliter amounts of reagents to reduce costs and increase sensitivity by the special mechanisms present at these size scales. The work performed within this thesis involves physics, material sciences and screening applications. Novel droplet manipulation modules and principles have been developed and characterized. One module enables to sort droplets by size differences rather than on its content. Another development concerns a novel droplet synchronization module which can create droplet pairs with an almost perfect accuracy. Probably the most broadly useful module is the development of an on-chip incubation delay-line. Due to these efforts it was possible to integrate several dropletbased modules functionally with each other on a single chip, in order to create complex devices useful for screening applications as e. G. Directed evolution of enzymes. Another development concerning screening applications is a dilution system enabling to adjust and ramp concentrations of a compound over several orders of magnitude, allowing to perform quantitative high-throughput screening with a statistical data quality far in excess of conventional methods. Additionally to these biological applications the microfluidic droplets have been used to synthesize superparamagnetic iron-oxide nano-particles in a very fast and well controllable reaction
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Kim, Jin-Young. "Hybrid devices for lab-chip chromatography and droplet-based microfluidics." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/11088.
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Recent years have seen considerable interest in the use of microfabricated systems in chemical and biological due to their significant advantages in terms of speed, analytical throughput, yield, unit cost, footprint, reagent requirements and control. Inevitably this has led to a growing interest in transferring chromatographic methods to planar chip formats since techniques using high performance LC play such a prominent role in modern bioanalysis. In addition, the manipulation of multiphase (or segmented) flows within microfluidic channels has been recently investigated as a promising approach for large-scale experimentation in biology and chemistry. Importantly, flow segmentation allows for the compartmentalisation of reagent volumes ranging from a few femtolitres to hundreds of nanolitres within a continuous and immiscible fluid, the production of monodisperse droplets at high frequencies, the accurate control of droplet contents and the ability to perform kinetic analysis with high precision. Accordingly the integration of droplet-based microfluidics with HPLC has the potential to dramatically reduce dispersion and minimise dead volume effects by using droplets to collect fractions of the column effluent. This basic progress preserves the chemical identity of each fraction allowing further analysis downstream. In this work, microfluidic devices were fabricated using thermoset polyester (TPE) to operate under high pressure which is required for LC separation and high frequency droplet generation. The optical characteristics of the fabricated devices were assessed for feasibility of optical detections for droplets. Substrate resistance to pressure also was investigated for droplet generation with high frequency. Lastly, droplets were generated under various conditions by adjusting flow-rates and the oil viscosity. Secondly LC separation columns were formed in TPE channels using two different column materials: particulate and polymer monolithic columns. The packed channels were investigated by SEM. In addition, permeability was calculated from back25 pressures measured as a function of flow-rates and compared with columns. Neurotransmitters were separated by the columns to estimate performance. Thirdly, the both operations, LC separation and droplet-based microfluidics, were combined in a single planar format. Sequential operations of separation, compartmentalisation and concentration gradient generation were integrated on a single chip and characterised using confocal laser-induced fluorescence detection. Finally, a preliminary investigation is reported into the possibility of the indirect electrochemical detection as a universal detection that can monitor electrochemically detectable samples as well as non- or less-electroactive bio samples. Amino acids were separated by a commercial RPHPLC column and detected indirectly.
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Ma, Shaohua. "Routes to controlled microenvironments for cell culture using droplet-based microfluidics." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648213.
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Chauvin, Dany. "Droplet-based microfluidics for the genotype-phenotype mapping of model enzymes." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC192/document.
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La relation qui lie la séquence d'une protéine à sa fonction nous échappe toujours en grande partie, pourtant elle est essentielle à la compréhension de l'évolution moléculaire.La microfluidique permet de remplacer les traditionnels tubes à essais par des micro-gouttelettes afin de tester séparément des mutants d'enzyme à des fréquences de l'ordre du kilohertz. Cette technique fournit un moyen de coupler le génotype et le produit de l'activité enzymatique (phénotype). Sélectionner et récupérer les gouttelettes sur demande et séquencer leur contenu permet d'effectuer la cartographie génotype-phénotype de millions de mutants d'enzymes en une seule expérience.Au cours de cette thèse, j'ai tout d'abord développé un système microfluidique basé sur l'expression de protéines in vitro afin de pouvoir réaliser la cartographie génotype-phénotype de Streptomyces griseus aminopeptidase (SGAP). Des gènes mutants de l'enzyme SGAP sont encapsulés (un par gouttelette au maximum) amplifiés, exprimés et testés contre un substrat fluorogénique. Des incompatibilités entre les étapes d'amplification, d'expression et d'essai enzymatique en gouttelettes obligent à réaliser chacune de ces étapes séparément et successivement, afin de diluer le produit de chaque réaction par l'électro-coalescence des gouttelettes. Je montre qu'un work-flow microfluidique dans lequel (i) les gènes sont encapsulés et amplifiés dans des gouttes de 0.2 pL, (ii) exprimés in vitro, (iii) testés contre un substrat fluorogenique dans des gouttelettes de 20 pL, permet de mesurer l'activité de variants de SGAP avec un contraste important. Afin d'optimiser l'essai enzymatique en gouttelettes de SGAP, j'ai aussi développé, en collaboration avec Dr. Johan Fenneteau (Laboratoire de Chimie Organique, ESPCI Paristech), un nouveau substrat fluorogénique basé sur une rhodamine hydrophile. Cette sonde est caractérisée par un échange limité de la rhodamine entre les gouttelettes.J'ai ensuite développé un work-flow microfluidique in vivo, pour Ratus norvegicus trypsin (la trypsine du rat), dans lequel les capacité de sécrétion de Bacillus subtilis sont utilisées afin de simplifier les expériences. Des cellules uniques de B. subtilis sont encapsulées dans des gouttelettes de 20 pL où elles sécrètent des mutants de la trypsine en protéine de fusion avec un rapporteur permettant de mesurer le niveau d'expression. Les mutants sont testés par électro-coalescence avec des gouttelettes de 2 pL contenant un substrat fluorogénique de la trypsine. En normalisant l'activité totale détectée par la fluorescence du rapporteur du niveau d'expression, l'efficacité catalytique peut être directement mesurée en gouttelettes. C'est la première fois qu'un système expérimental d'essai enzymatique haut-débit fournit l'opportunité de mesurer directement l’efficacité catalytique de mutants d'une enzyme à une fréquence de l'ordre du kilo Hertz. Une méthode afin de réaliser la mutagenèse saturée (tous les simples mutants) du gène de la trypsine du rat a aussi été développée. Combinée au séquençage nouvelle génération, la méthode microfluidique développée permettra de réaliser la première cartographie génotype-phénotype de tous les simples mutants de la trypsine du ratThe question of how sequence encodes proteins' function is essential to understand molecular evolution but still remains elusive.Droplet-based microfluidics allows to use micro-metric droplets as reaction vessels to separately assay enzyme variants at the kHz frequency. It also provides an elegant solution to couple the genotype with the product of the catalytic activity of enzymes. Sorting droplets on demand and sequencing their content enables to map the genotype of millions of enzyme variants to their phenotype in a single experiment.First, I developed a cell-free microfluidic work-flow to carry out genotype-phenotype mapping of Streptomyces griseus aminopeptidase (SGAP). Single enzyme variant genes are encapsulated and amplified in droplets, expressed, and assayed against a fluorogenic substrate. Incompatibilities between gene amplification, expression and assay reactions, constrain to execute each one of those steps successively and to dilute the product of each reaction by droplet electro-coalescence. I show that a work-flow in which (i) genes are encapsulated and amplified into 0.2 pL droplets, (ii) expressed using cell-free expression reagents in 2 pL droplets and (iii) assayed with a fluorogenic substrate in 20 pL droplets, allows to measure SGAP variants activity with high contrast. To optimize the SGAP droplet assay, I also developed in collaboration with Dr. Johan Fenneteau (Laboratory of Organic Chemistry, ESPCI Paristech), a hydrophilic rhodamine based substrate, characterized by limited exchange of the released fluorophore between droplets.Second, I developed an in vivo microfluidic work-flow on Ratus norvegicus trypsin (rat trypsin), in which Bacillus subtilis secretion abilities are used to simplify the microfluidic work-flow. Single B. subtilis cells are encapsulated in 20 pL droplets where they secrete trypsin variants as fusion proteins with a fluorescent expression-level reporter. The variants are assayed by droplet electro-coalescence with 2 pL droplets containing a trypsin fluorogenic substrate. Trypsin variants catalytic efficiency can be directly measured in droplets, by normalizing the total trypsin activity by the expression-level reporter fluorescence. This is the first time a high-throughput protein assay work-flow gives the opportunity to directly measure the catalytic efficiency of enzyme variants at the kHz frequency. A method to carry out saturated mutagenesis on the rat trypsin gene was also developed. Together with deep sequencing, the developed experimental work-flow will allow to perform the first quantitative genotype-phenotype mapping of all single point mutants of the rat trypsin protein
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Hassan, Sammer-Ul. "Droplet-based microfluidics for continuous chemical sensing and quantitative high-throughput separations." Thesis, University of Southampton, 2016. https://eprints.soton.ac.uk/400444/.
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By translating bioanalytical testing from the laboratory into the clinic or at home, point-of-care (POC) diagnostics enables timely diagnosis, monitoring, and treatment of patients. Fundamental to the development of POC diagnostics has been the use of microfluidics, which offers small sample volume consumption and can combine multiple sample-processing steps into a single device. However, microfluidics has provided a limited throughput due to the multiple microvalve switching of samples, slow mixing, sample dispersion and band-broadening in microfluidic channels which result in decreasing the temporal resolution of the system. Droplet-based microfluidics has emerged as an alternative technique to manipulate and analyse sample droplets in a high-throughput format. Samples are compartmentalized in nanolitre sized droplets which eliminate dispersion or band-broadening and hence increase the temporal resolution of the system. The development of high-throughput, quantitative and rapid microfluidic-based separations has been a long-sought goal for applications in proteomics, genomics, biomarker discovery and clinical diagnostics. Using droplet-interfaced microchip electrophoresis (MCE) techniques, a novel parallel MCE platform, based on the concept of Slipchip and a newly developed ‘Gelchip’ has been presented in this Thesis. The platform consisted of two “slipping” plastic plates, with droplet wells on one plate and separation channels with preloaded/cured gel on the other. A single relative slipping of the plates enabled generation and then injection of multiple droplets in parallel into the separation channels, allowing to analyse sample droplets in parallel and in a high-throughput format. As proof-of-concept applications, the separation of 30 sub-nanolitre sample droplets containing fluorescent dyes was performed. Theoretical plates were calculated to be 7560/s at a distance of 3.5 cm. We further determined the effect of droplet sizes on the separation efficiency and found that theoretical plates were higher (2220/s) for smallest droplets (320 picolitres) and lower (1480/s) for largest droplets (1750 picolitres) at a distance of 8 mm. For the quantitative determination of fluorescent molecules, the change in peak areas of different concentrations of the fluorescent molecule (Fluorescein-isothiocyanate) was found to be linear with a small error of 3.6% RSD. Furthermore, separation of DNA step ladder at a distance of 1.3 cm was also achieved with theoretical plates of 79800/s which is one order of magnitude higher than our previously reported droplet-interfaced platform (Niu et al. 2013). To facilitate ‘non-microfluidic users’, new protocols were also developed to pre-cure separation gels (e.g. agarose and polyacrylamide) in open channels forming a ‘Gelchip’ that can be prepared in batch and used off-the-shelf. Droplet-based microfluidics is ideally suited to continuous biochemical analysis, requiring small sample volumes and offering a high temporal resolution. Many biochemical assays are based on enzymatic reactions, the kinetics of which can be obtained by probing droplets at multiple points over time. Real-time and continuous chemical monitoring of traumatic brain injury (TBI) or liver transplant in intensive care unit (ICU) could be life-saving and protect from further damage to the tissue. Current systems to monitor these patients are bulky and are placed at a distance from the patient (typically 30-40 cm) which introduces sample dispersion and limits the temporal resolution of the system. A droplet-based portable continuous chemical sensing device capable of generating droplets-on-demand and analysing glucose or lactate from near patient has been developed. The primary task of the project was to calibrate the performance of the device by generating droplets by a novel screw-driven pump, calibrating droplet sizes and detecting different droplet concentrations in an absorbance based flow cell. To measure the reactions in droplets calorimetrically, a miniaturised 7-detector flow cell to analyse enzyme kinetics, with an example application of continuous glucose assay in droplets has been developed. Reaction rates and Michaelis-Menten kinetics were quantified for each individual droplet, and unknown glucose concentrations were accurately determined (errors < 5%) with the lowest quantified concentration of 0.5 mM. Droplets can be probed continuously giving short sample-to-result time (~30 s) measurement. In contrast to previous reports of multipoint droplet analysis (all of which used bulky microscope-based setups) the flow cell presented here has a small footprint (45 by 10 by 15 mm) and uses low-powered, low-cost components, making it ideally suited for use in field-deployable devices. The flow cell was further upgraded via fabrication of precise micromilling of the cartridge and successfully applied for detection of glucose and lactate droplets (lowest quantified concentrations of 0.175 mM and 0.1 mM respectively) for clinical applications. Another significant development of the optical flow cell is dual light paths for accurately quantifying the size and velocity of the continuously flowing droplets, as well as quantifying the composition of the droplet. The flow cell was capable of measuring droplet lengths with errors of < 5%. With all these features, a fully functional optical flow cell is built that can be readily used in wearable/portable microfluidic devices.
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Granieri, Lucia. "Droplet-based microfluidics and engineering of tissue plasminogen activator for biomedical applications." Strasbourg, 2009. http://www.theses.fr/2009STRA6162.
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Le phage display est une méthode généralement utilisée pour l'évolution dirigée de protéines, elle permet de produire une immense diversité de variants de protéines exprimés sur la membrane de particules virales. Ces variants de protéines peuvent être sélectionnés soit pour leur affinité de liaison (p. Ex. Les anticorps) soit pour leur activité catalytique (p. Ex. Les enzymes). La sélection d'enzymes pour leur activité catalytique requiert néanmoins l'utilisation de substrats et/ou produits immobilisés, ce qui empêche la sélection de protéines sur plusieurs cycles catalytiques. Le but de ce projet de thèse était de développer une méthode nous permettant d'outrepasser ces limites : la compartimentation dans des systèmes microfluidiques de particules virales exprimant des variants de protéines unique sur leur surface. L'encapsulation de ces particules dans des gouttes nous a permit d'utiliser des substrats/produits solubles et donc la sélection pour de multiples cycles catalytiques a été possible. L'utilisation d'un système d'expression mammifère a permit aux protéines de subir les modifications post-traductionnelles (ponts disulfure, glycosylations) nécessaires. Ce système permet de monitorer l'activité enzymatique, de variants de protéines uniques d'une banque, exprimés sur la membrane de particules virales. En plus, nous avons développé un système nous permettant de trier des virus en utilisant comme critère la présence ou l'absence d'activité enzymatiquePhage display is a widely used method for directed evolution of proteins, allowing the generation of an enormous diversity of protein variants displayed on the viral particles (library diversity <1012). These protein variants can then either be selected for binding affinity (e. G. Antibodies) or for catalytic activity (e. G. Enzymes). However, since selection for catalytic activity requires immobilized substrates and/or products, selection for multiple turnover or maximum rate acceleration remains challenging. To overcome these limitations a new method has been developed: Microfluidic-based compartmentalisation of viral particles displaying single protein variants on their surface. Encapsulation of these particles into picoliter drops allows the use of soluble substrates/products and therefore the selection for multiple turnover. The model system used here is based on retroviral particles displaying tPA (tissue plasminogen activator), a protein used in current emergency therapies of myocardial infarction and stroke, and a non-related control protein (neuraminidase, NA, inactive particles). Single particles displaying tPA and NA variants were encapsulated into aqueous droplets and the enzymatic activity was monitored using a fluorescence assay. Active variants could be sorted from a mixture of active and inactive variants
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Hübner, A. M. "Microfluidic droplets : cell-based and (bio-)chemical assays on the micro-scale." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603910.
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The first chapter of this thesis introduces the reader into the field of microdroplets. A brief literature review is given summarising the achievements in biochemistry and biology using microfluidic droplets. In Chapter 2 the techniques that were applied to fabricate devices and to generate droplets are highlighted. Chapter 3 demonstrates that fluorescently labelled single cells can be controllably compartmentalised within aqueous microdroplets. A high-throughput screening was performed by detecting the expression of the fluorescent protein in individual cells with simultaneous measurement of droplet size and cell occupancy. Chapter 4 comprises a cell-based enzymatic assay performed inside picolitre droplets. Comparing studies on enzymatic activity of wild type and a mutant enzyme successfully demonstrate the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements. Chapter 5 describes the design, fabrication and use of a single layered poly(dimethylsiloxane) microfluidic structure for the entrapment and release of microdroplets in a plate reader format controlled entirely by liquid flow. Such an array-based approach is used to characterise droplet shrinkage, aggregation of encapsulated E. coli cells and enzymatic reactions. Chapter 6 and 7 widens the approach described in Chapter 5 by entrapping droplets next to each other. Those entrapped droplets could be fused by means such as lasers and electric fields. With such an approach the formation of a chemical complex was monitored with sub-millisecond time resolution. Furthermore, depending on the surfactant that was chosen leakage and cross-contamination of an encapsulated solute between two adjacent droplets could be investigated. Chapter 8 presents an approach of droplets incubation for several minutes up to almost an hour in a continuously operating device. Chapter 9 lists all methods that were applied to generate, manipulate and utilise the droplets in each individual experiment.
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Nögel, Lukas [Verfasser], and Christoph [Akademischer Betreuer] Merten. "Multiplexed combinatorial drug screening using droplet-based microfluidics / Lukas Nögel ; Betreuer: Christoph Merten." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/120395851X/34.
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Godina, Alexei. "In vivo and in vitro directed evolution of enzymes using droplet-based microfluidics." Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAF061/document.
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L’ingénierie des protéines fonctionnelles est un processus d’amélioration des propriétés physiques ou catalytiques d’enzyme au travers d’approches rationnelles et d'évolution dirigée, aussi bien que la combinaison des deux méthodes. Malgré le progrès de la modélisation moléculaire des protéines, les méthodes de prédiction restent aléatoires et un grand nombre de variantes restent à tester. De ce fait, le développement et l’utilisation d’un système de criblage d’activité de protéines à très haut débit, comme la microfluidique en gouttes, est indispensable. Cette thèse de doctorat présente trois projets d’évolution dirigée de protéines en trois approches différentes avec expression d’enzyme in vitro et in vivo. Les plateformes microfluidiques ont été développées et validées pour chaque projet. De plus, plusieurs banques de variants ont été criblées avec, dans certains cas, isolement de molécules 5-10 fois que le clone parentalThis work describes the development of high-throughput droplet microfluidic platforms fine-tuned for protein of interest and their employment in directed evolution experiments. When not available, fluorogenic assay for monitoring desired enzyme activity (-ies) in droplets was developed. Moreover, the in vivo expression allowed the successive integration of microfluidic modules on the same chip. After a couple of evolution rounds the initial retro-aldolase variant was significantly improved. In other project, to meet industrial requirements a high-throughput screening platform for protease evolution in detergent has been assembled and validated. Two evolution rounds showed the accumulation of a certain pool of beneficial mutations over the selection rounds. The research described in this work highlighted that in vitro expression systems are sensitive to the amount of supplied DNA and reaction conditions. This observation led to the development of a multistep completely in vitro microfluidic platform
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Thibault, Derek M. "Applications of droplet-based microfluidics to identify genetic mechanisms behind stress responses in bacterial pathogens." Thesis, Boston College, 2016. http://hdl.handle.net/2345/bc-ir:106985.
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Thesis advisor: Michelle MeyerThe primary bacterial targets for most antibiotics are well known. To survive the stress of an antibiotic a bacterium must decrease the antibiotic to target binding ratio to escape from harmful effects. This can occur through a number of different functions including down-regulation of the target, mutation of the binding site on the target, and decreasing the intake or increasing the efflux of the antibiotic. However, it is becoming more evident that an antibiotic stress response influences more than just the primary target, and that a wave of secondary responses can be triggered throughout the bacterium. As a result resistance mutations may arise in genes that are indirectly affected by the initial interaction between the antibiotic and target. These indirect responses have been found to be associated with metabolism, regulation, cell division, oxidative stress, and other critical pathways. One technique recently developed in our lab, called transposon insertion sequencing (Tn-seq), can be used to further understand the complexity of these indirect responses by profiling growth rates (fitness) of mutants at a genome-wide level. However, Tn-seq is normally performed with large libraries of pooled mutants and thus it remains unclear how this may influence fitness of some independent mutants that may be compensated by others in the population. Additionally, since the original method has only utilized planktonic culture, it is also not clear how higher order bacterial structures, such as biofilms or microcolonies, influence bacterial fitness. To better understand the dynamics of pooled versus individual mutant culture, as well as the effect of community structure in microcolony development on the influence of fitness, we adapted a droplet microfluidics-based technique to encapsulate and culture single mutants. We were able to successfully encapsulate at least 7 different species of bacterial pathogens, including Streptococcus pneumoniae, and culture them planktonically, or as microcolonies, in either monodisperse liquid or agarose droplets. These experiments, however, raised an important challenge: the DNA yield from one encapsulation experiment is insufficient to generate samples for sequencing by means of the traditional Tn-seq method. This led us to develop a novel Tn-seq DNA library preparation method, which is able to generate functional Tn-seq library molecules from picogram amounts of DNA. This method is not ideal yet because fitness data generated through the new method currently does not correlate well with data from traditional Tn-seq library preparation. However, we have identified one major culprit that should be easily solvable. We expect by modifying the binding site of the primer used for linear amplification of transposon ends that the new preparation method will be able recapitulate results from the traditional Illumina preparation method for Tn-seq. This will enable us to prepare robust Tn-seq samples from very small amounts of DNA in order to probe stress responses in single mutants as well as in microcolonies in a high-throughput manner
Thesis (MS) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology
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Illing, Rico. "Development of a Fluorescent Droplet Analyser for microbiological studies." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-232345.
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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.
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Bleier, Blake J. "Droplet-Based Approaches to Probe Complex Behavior in Colloidal Fluids with High Composition Resolution." Research Showcase @ CMU, 2018. http://repository.cmu.edu/dissertations/1173.
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In this work, microfluidic and millifluidic droplets are utilized to study and control complex fluid behavior with high composition resolution. Different techniques are used on two length scales to create unique approaches towards the same goal of merging droplet-based experiments with classical colloidal characterization experiments. First, a microfluidic dehydrating droplet device is characterized and a procedure established by concentrating a phase separating organic-inorganic system on chip and using geometric calculations to determine composition. The device is then expanded to a more complex, particle-polymer system to investigate suspension stability and interparticle behavior. A model system containing silica particles and PEO polymer is found to transition from a bridging flocculation mechanism to polymer-coated particle jamming based on the mass ratio of polymer to particle. Lastly, a phase separating particle-polymer system consisting of polystyrene particles and hydroxyethyl cellulose is concentrated on-chip. Interparticle interactions are controlled by varying particle size, polymer size, and polymer type and the effects on phase behavior are examined. Droplet experiments are scaled-up to millifluidic droplets and concentration gradients are used to produce high composition resolution in place of time, used in the dehydrating microfluidic experiments. A novel, millifluidic containment device is created to study aggregation and sedimentation in droplets containing carbon black and OLOA surfactant suspended in dodecane. A slow increase in stabilization behavior is observed as opposed to the previously observed sharp “on-off” effect. The droplet production technique is then improved to achieve more complex composition paths and the device is expanded for a small angle neutron scattering (SANS) application. SANS is performed on flowing droplets with varying concentration to map interparticle interactions and phase behavior of complex particulate systems. Feasibility of device is demonstrated and preliminary model systems of silica particles and polymer, salt, and surfactant are analyzed and characterized.
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Janiesch, Jan-Willi [Verfasser], and Joachim P. [Akademischer Betreuer] Spatz. "Development of Droplet-Based Microfluidics for Synthetic Biology Applications / Jan-Willi Janiesch ; Betreuer: Joachim P. Spatz." Heidelberg : Universitätsbibliothek Heidelberg, 2015. http://d-nb.info/1180609247/34.
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Wasson, Elisa Marie. "Engineered Platforms for the Development of Electroporation-based Tumor Therapies." Diss., Virginia Tech, 2020. http://hdl.handle.net/10919/104183.
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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.
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Lin, Gungun. "Multifunctional Droplet-based Micro-magnetofluidic Devices." Doctoral thesis, Universitätsbibliothek Chemnitz, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:ch1-qucosa-208797.
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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.
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Riechers, Birte [Verfasser], Jean-Christophe [Akademischer Betreuer] Baret, and Sarah [Akademischer Betreuer] Köster. "Dynamics of Surfactants at Soft Interfaces using Droplet-Based Microfluidics / Birte Riechers. Betreuer: Jean-Christophe Baret. Gutachter: Jean-Christophe Baret ; Sarah Köster." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2016. http://d-nb.info/108182039X/34.
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Grosselin, Kevin. "Cartographie épigénétique de cellules cancéreuses résistantes rares par microfluidique en gouttelettes." Thesis, Paris Sciences et Lettres (ComUE), 2018. http://www.theses.fr/2018PSLET015.
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La nature dynamique de la chromatine est un acteur majeur de la régulation de la transcription et est suspectée de contribuer à l'évolution tumorale. L'étude des modifications de la chromatine à l'échelle de la cellule unique est indispensable pour comprendre l'impact de la plasticité épigénétique au cours de la tumorigénèse.Dans ce manuscrit, je décris le développement d'un système basé sur la microfluidique en gouttelettes permettant d'obtenir la cartographie des modifications de la chromatine à l'échelle de la cellule unique.Le système a été évalué pour cartographier des modifications d'histones associées à un état transcriptionnel actif (H3K4me3) ou réprimé (H3K27me3) de cellules B et T humaines. Les données ont permis de classer >99% des cellules sur la base de leur profil épigénétique, définissant ainsi avec une grande précision des états de la chromatine propres à chaque type cellulaire.A partir de xénogreffes dérivées de patient atteint du cancer du sein et ayant acquis une résistance thérapeutique, le système a permis la détection de sous-populations rares de cellules parmi les tumeurs non-traitées, présentant un profil chromatinien similaire aux cellules cancéreuses résistantes.Cette étude démontre l'importance de l'hétérogénéité cellulaire sur la progression tumorale et met en évidence une signature épigénétique associée à la résistance et susceptible d'être la cible d'un traitement thérapeutiqueThe dynamic nature of chromatin and transcriptional features play a critical role in normal differentiation and are expected to contribute to tumor evolution. Studying the heterogeneity of chromatin alterations with single-cell resolution is mandatory to understand the contribution of epigenetic plasticity in cancer.In this thesis, I describe a droplet microfluidics approach to profile chromatin landscapes of thousands of cells at single-cell resolution, with an unprecedented coverage of 10,000 loci per cell.The system was evaluated to profile histone modifications associated with active (H3K4me3) and inactive transcription (H3K27me3) of human B cells and T cells, and revealed that >99% of the cells were correctly assigned to one cell type, defining distinct chromatin states of immune cells with high accuracy.In patient-derived xenograft (PDX) models of breast cancer with acquired drug resistance, the system enabled the detection of a rare subpopulation of cells in the untreated, drug-sensitive tumors with chromatin features characteristic of resistant cancer cells. These cells had lost chromatin marks (H3K27me3) associated with stable transcriptional repression for a number of genes known to promote resistance, potentially priming them for transcriptional activation.These results highlight the potential selection of cells with specific chromatin marks in response and in resistance to cancer therapy
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Chen-Jolly, Hongyu. "Conception d'un dispositif microfluidique de synthèse en continu du poly(acide acrylique) en milieu hétérogène eau/CO2 supercritique." Thesis, Bordeaux, 2014. http://www.theses.fr/2014BORD0233/document.
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Ce travail de thèse rend compte de la mise en oeuvre d’un système de synthèse en continu dupoly(acide acrylique) en milieu CO2 supercritique (15 MPa et 75 °C). Nous avons conçu undispositif microfluidique résistant aux hautes pressions permettant l’écoulement de gouttes desolution aqueuse de monomère dans une phase continue constituée d’un mélangesupercritique d’éthanol dans du CO2 et contenant l’amorceur azobisisobutyronitrile (AIBN).Nous avons déterminé par spectroscopie IR la répartition des différentes espèces chimiquesdu mélange en fonction de la pression et la température, puis caractérisé la décompositionthermique de l’amorceur selon la composition du milieu réactionnel par spectroscopie UVVis.Enfin, nous avons montré que les gouttes sont comparables à des réservoirs demonomère alimentant sans cesse la phase continue. En raison de ce transfert rapide vis-à-visde la conversion de l’AA en chaîne polymère, la réaction de polymérisation s’effectuecontinûment avec un rapport molaire monomère sur amorceur constant durant tout le tempsde séjour dans le microcanal (jusqu’à 41 min). Une gamme large de masses molaires avec desindices de polymolécularité faibles a été obtenue : de 20 000 à 120 000 g.mol-1 pour 1,35 à1,70, en variant simplement les concentrations de monomère de la solution aqueuse initiale.Les paramètres expérimentaux influençant les propriétés du poly(acide acrylique) obtenu,ainsi que le lieu de la polymérisation ont été étudiésIn this work, a continuous microfluidic device was developed to perform the synthesis ofpoly(acrylic acid) in supercritical CO2 (15 MPa and 75°C). This high pressure resistantdevice allows generating segmented flows in microcanal: an aqueous solution of monomerwas dispersed in a mixture of ethanol in CO2 containing initiator AIBN. The distribution ofdifferent components in this biphasic system has been determined by IR spectroscopyaccording to the pressure and the temperature. The thermal decomposition of AIBN indifferent reaction media has been investigated using UV-Vis spectroscopy. During thereaction, the droplets were used as reservoirs which insure the transfer of monomer to thecontinuous phase. Because of this rapid transfer compared to the reaction conversion, thepolymerization reaction was carried out continuously with a constant molar ratio betweenmonomer and initiator throughout the residence time (up to 41 min). It has been showed thata large range of molecular weights of poly(acrylic acid) (20 000 and 120 000 g.mol-1) withlow polydispersity index (1.35 à 1.70) can be obtained by just changing the initial monomerconcentration in the droplets. The effect of other parameters influencing the properties ofpoly(acrylic acid) as well as the locus of polymerization have been discussed
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Brosseau, Quentin Verfasser], Jean-Chrisophe [Akademischer Betreuer] Baret, Jörg [Akademischer Betreuer] Enderlein, Stephan [Akademischer Betreuer] [Herminghaus, Sarah [Akademischer Betreuer] Köster, Marcus [Akademischer Betreuer] Müller, and Philipp [Akademischer Betreuer] Vana. "Dynamics of soft interfaces in droplet-based microfluidics / Quentin Brosseau. Gutachter: Jörg Enderlein ; Jean-Chrisophe Baret ; Stephan Herminghaus ; Sarah Köster ; Marcus Müller ; Philipp Vana. Betreuer: Jean-Chrisophe Baret." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2014. http://d-nb.info/1050591895/34.
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Foulon, Sophie. "Développement du séquençage ARN ciblé sur cellules uniques en microfluidique de gouttes et applications." Thesis, Paris Sciences et Lettres (ComUE), 2019. http://www.theses.fr/2019PSLET037.
Full textAbstract:
Les technologies d'analyse à l'échelle de la cellule unique ont vu le jour il y a quelques années et sont depuis en constante évolution. Ces technologies permettent de mieux comprendre le fonctionnement d'ensemble de cellules très hétérogènes. Elles permettent par exemple de découvrir et suivre des sous types cellulaires, avec des applications en cancérologie ou encore en neurobiologie. Nous avons développé une technologie pour étudier le profil d'expression de gènes d'intérêt au niveau d'une cellule unique, en utilisant la microfluidique en gouttes. En limitant le nombre de gènes étudiés comparé aux technologies commerciales de transcriptome entier, l’approche ciblée a plusieurs avantages potentiels : gagner en profondeur de séquençage, augmenter le nombre de cellules étudiées, optimiser la détection pour les bas niveaux d’expression, tout en réduisant la complexité des données et des coûts. Le ciblage est parfois indispensable, notamment lorsque les ARNs ne portent pas de séquence d’amorce générique, comme dans le cas des ARNs viraux. Deux applications sont présentées : l'analyse de l'inflammation des cellules immunitaires du cerveau aux premières étapes du développement, ainsi que l'étude de la recombinaison génétique chez le virusSingle cells technologies were introduced a few years ago and have been dramatically evolving ever since. These technologies have revolutionized biology, making it possible to better understand how heterogeneous cell systems works. For example, they permit to discover and follow cell subtypes, with applications in oncology or neurobiology. We have developed a technology to study the expression profile of genes of interest at the level of a single cell, using droplet-based microfluidics. By limiting the number of genes studied compared to commercial whole-transcriptome technologies, the targeted approach has several potential benefits: gaining deeper sequencing, increasing the number of cells studied, optimizing detection for low levels of expression, while reducing the complexity of data and costs. Targeting is sometimes essential, especially when the RNAs do not carry a generic primer sequence, as in the case of viral RNAs. Two applications are presented: the analysis of inflammation of the immune cells of the brain in the early stages of development, as well as the study of genetic recombination in the virus
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Autour, Alexis. "Amélioration et criblages de propriétés d'ARN aptamères fluorogènes en systèmes microfluidiques." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ057.
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Les ARN (Acide RiboNucléique) remplissent de nombreuses fonctions clés dans le vivant. Ils peuvent être support de l'information génétique, régulateurs de celle-ci. Visualiser ces molécules au sein d'une cellule représenterait une étape importante vers une meilleure compréhension de la régulation de l'expression des gènes. Les ARN fluorogènes tels que Spinach et Mango sont des outils extrêmement prometteurs pour atteindre cet objectif. Cependant ces deux ARN fluorogènes présentent une brillance limitée. La Compartimentation in vitro assistée par microfluidique (µCIV) est un outil très prometteur dont notre groupe a démontré l’efficacité pour l’évolution d’ARN. Dans le cadre de cette thèse, la µCIV a été adaptée à la sélection d'aptamères d'ARN fluorogènes pour en améliorer les propriétés (surtout la brillance). De plus, l’utilisation conjointe du séquençage haut débit a permis l’optimisation très rapide et semi-automatisée à la fois d’aptamères mais aussi de biosenseurs fluorogènes. Ainsi, cette thèse a permis de mettre en place et d’exploiter des technologies de criblage robustes pour la découverte de nouveaux aptamères d'ARN et de biosenseursRNA is a key molecule in gene expression and its regulation. Therefore, being able to monitor RNA through live-cell imaging would represent an important step toward a better understanding of gene expression regulation. RNA-based fluorogenic modules are extremely promising tools to reach this goal. To this end, two light-up RNA aptamers (Spinach and Mango) display attractive properties but they suffer from a limited brightness. Since previous work in the group demonstrated the possibility to evolve RNA using microfluidic-assisted in vitro compartmentalization (µIVC), this technology appeared to be well suited to improve light-up aptamers properties by an evolution strategy. Therefore, the µIVC procedure was adapted to fluorogenic RNA aptamers to improve their properties (especially the brightness). Finally, using µIVC in tandem with high-throughput sequencing (NGS) allowed further developing the technology into a more integrated and semi-automatized approach in which RNAs and biosensors are selected by µIVC screening and the best variants identified by a bioinformatics process upon NGS analysis. To summarize, this thesis allowed establishing robust µIVC screening workflows for the discovery of novel efficient light-up RNA aptamers as well as metabolites biosensors
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"Constructing polymer phase diagram using droplet-based microfluidic system." Thesis, 2008. http://library.cuhk.edu.hk/record=b6074638.
Full textAbstract:
In Chapter 1, we briefly review the thermodynamics of polymer solutions, including the ideal solution based on the Flory-Huggins lattice chain model. The entropy change in the mixing of macromolecules with small solvent molecules is much smaller than that in the mixing of two kinds of small molecules. Therefore, the effect of the solution temperature is also smaller.In Chapter 2, we describe the basic difference between the complicate polymeric and simple Newtonian fluid and list some dimensionless numbers relevant to various physical phenomena, including the Reynolds number (Re) and the capillary number (Ca), respectively related to the inertial effect and the interfacial tension. As a fluid goes down to the micro scale, the inertial effect is usually negligible so that the flow becomes laminar. However, the interfacial tension starts to play a significant role, which leads to the development of some droplet-based (or digital) microfluidic systems. Using small droplets leads to the following advantages: (1) the reagents are limited within a small boundary of each droplet; and (2) no complex microfluidic device is required.
In Chapter 3, we use PNIPAM in water as a model system to detail how to construct a polymer phase diagram by using a microfluidic device, including the choice of the carrier fluid, the principle and experimental procedure of forming concentration-controllable PNIPAM droplets, the determination of PNIPAM concentration in each droplet by using a fluorescein probe, the effect of fluorescein on the phase transition, and the detection of the phase transition by dark field viewing. For comparison, we also did the normal LLS measurement of the phase transition of PNIPAM in water.
In Chapter 4, PS in cyclohexane is used as a model system to illustrate how to handle organic solvents because cyclohexane swells the PDMS channels. The swelling is much eliminated by directly loading the PS solution into the junction via glass capillaries. Since the addition of a fluorescence concentration probe dramatically influences the PS phase transition, we have to use a so-called parallel experimental method to produce concentration-controllable PS droplets. In this method, several PDMS chips from the same batch are used in the formation of small PS droplets. When the numbers of small PS droplets produced in the same procedure are similar to each other, the PS concentrations in different corresponding droplets are comparable. Therefore we are able to index the PS concentration in each droplet by comparing it with the droplets prepared by the same procedure, but with some added fluorescence probes.
In Chapter 5, on the basis of numerous experiments, we find inorganic salts play a significant role on the droplet forming. Thus we propose that droplet formation is a kinetics governed process when two immiscible liquids meet each other in microchannels.
In this thesis we have proposed and established a new method of constructing polymer phase diagrams. By employing the droplets-based microfluidic system, we are able to form an array of droplets of polymer solutions with several nanoliters in size. Each droplet has a controllable composition. The array of polymer droplets can be transferred and stored in a glass capillary; there the turbidity of each droplet due to the difference of scatted light immediately after the phase transition can be monitored under a microscope via dark field viewing, when the solution temperature changes. Therefore, we are able to construct a polymer phase diagram by simply combing each phase transition temperature with its corresponding compositions of polymer solution droplets.
This method has two distinguished advantages; namely, minimal sample consumption and much reduced experimental time required for the phase transition to reach its equilibrium at each given temperature. This is because the greatly increased surface-to-volume ratio allows rapid diffusion and fast heat transfer. To demonstrate the principle, we have chosen PS in cyclohexane with an upper critical solution temperature (UCST) and Poly(N-isopropylacrylamide) (PNIPAM) in water with a lower critical solution temperature (LCST) as two model systems. Primarily established phase diagrams of these two polymer solutions have demonstrated the feasibility of using droplets-based microfluidic system to construct polymer phase diagrams.
Shi, Feng.
Advisers: Chi Wu; Bo Zheng.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3532.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references.
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.