Cell encapsulation is a promising concept in regenerative medicine and stem cell treatment of diseases. Cells encapsulated in hydrogels have shown to yield better therapeutic outcome over cells in suspension. Microfluidic platforms have facilitated the process of cell encapsulation through the controlled mixing of aqueous cell solution and hydrogel with an immiscible liquid to yield a monodispersed population of microcapsules at a high throughput. However, given that the microfluidic process of placing cells in microcapsules is completely random, yielded samples are often riddled with empty microcapsules, raising the need for a post-encapsulation purification step to sort empty microcapsules from cell-laden ones. Sorting of microcapsules can be achieved through several techniques, most desirable of which are electrokinetic such as dielectrophoresis (DEP) and electrophoresis (EP). The advantages of DEP and EP techniques are that they support label-free sorting and yield a high throughput. However to achieve true effective DEP or EP sorting, there is a need to understand how empty microcapsules react to these electrokinetic forces versus occupied microcapsules. This study developed microfluidic devices for characterising the electrokinetic effects on microcapsules using DEP and EP. Results of both characterization techniques showed notable differences in the response of empty microcapsules versus cell-laden ones, reinforcing their potentials for sorting. Furthermore, this study proposed designs for microcapsules sorting devices that leverage EP and DEP.

Des phénomènes de fluidisation de billes magnétiques apparaissent à l'échelle micrométrique au sein du système de lit fluidisé microfluidique. On obtient un fonctionnement en flux continu à basse pression de travail avec un étroit contact liquide/solide et une recirculation constante des billes, des caractéristiques avantageuses pour des applications dédiées à la pré-concentration de cibles biologiques. La caractérisation du système physique a montré l'influence de paramètres tels que la géométrie de la chambre ou la distribution du champ magnétique, leur optimisation étant nécessaire pour obtenir des phénomènes de fluidisation à cette échelle et améliorer le mélange et la distribution des billes. De plus, le potentiel du lit fluidisé comme plateforme pour des bio-essais analytiques a été exploré avec succès lors d'applications biologiques: 1) la pré-concentration de bio-markers de la maladie d'Alzheimer et leur marquage in situ pour un future couplage avec des techniques de détection sensibles; 2) la détection de bactéries sans besoin de marquage préalable à travers une immuno-capture suivie d'une culture donnant lieu à des changements physiques du support fluidisé; 3) l'extraction d'ADN contenant un gène cible et son ultérieur amplification enzymatique sur la surface des billes, suivie d'une détection multiplexée des mutations présentes par un système de microarray. Ainsi, le lit fluidisé magnétique rend possible des applications au de-là d'un simple système de pré-concentration, permettant son utilisation comme une plateforme efficace de biologie moléculaire allant jusqu'à l'utilisation des propriétés autorégulatrices inhérentes au système comme mécanisme de détection. / With the use of an external magnetic field and magnetic microbeads, the microfluidic magnetic fluidized bed system enables fluidization phenomena at the microscale. This results in flow-through operations at low driving pressures with intimate liquid/solid contact and a continuous beads recirculation, interesting for efficient biological target preconcentration applications. The physical system has been characterized, showing the importance of chamber angle of aperture and height confinement as well as magnetic field distribution parameters, to obtain fluidization and further enhance mixing and maximize beads density. Further, the potential of the fluidized bed as a platform for analytical bioassays has been successfully explored with a series of biologically relevant applications: (1) the preconcentration of rare Alzheimer’s biomarkers together with their in situ fluorescence labeling for future enhanced detection with hyphenated techniques; (2) the label-free sensitive detection of bacteria in liquid food samples through the specific immunocapture and on-chip culture of these microorganisms and the resulting physical changes induced in the fluidized support; (3) the gene-specific extraction of DNA and its subsequent enzymatic amplification on the surface of the beads, coupled to a microarray detection system for a multiplexed detection of cancer-inducing mutations. These results show that the applications of the magnetic fluidized bed go beyond its initial conception as a dynamical affinity-based concentrator, serving as an efficient platform for molecular biology protocols and even making use of its inherent auto-regulating properties as a detection mechanism.

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

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No description available.

Une étude conjointe du CEA et d'AREVA la Hague a montré qu'une des voies d'amélioration majeure des usines de traitement/recyclage du combustible nucléaire usagé, actuelles et futures, concerné le domaine de l'analyse. En effet, le suivi et le pilotage des procédés déployés dans ces usines nécessitent de nombreuses analyses générant de grandes quantités d'effluents radioactifs. Réduire les volumes mis en jeu lors de ces analyses permettrait donc de réduire la nocivité des échantillons et des effluents et donc d'accroitre la sureté pour le personnel et de réduire l'impact sur l'environnement et le coût de fonctionnement des usines. Parmi toutes les analyses effectuées, la mesure d'acidité libre est la plus fréquente, car c'est un paramètre indispensable pour pouvoir piloter correctement le procédé. C'est pourquoi, ces travaux de thèse ont abouti à l'amélioration de la méthode de mesure via une réduction d'échelle de l'analyse et une automatisation du protocole de mesure. Deux voies ont été étudiées : - le titrage par injection séquentielle (SIA), qui est un dispositif de 25 L de volume et qui par rapport à la méthode d'analyse de référence, réduit 1000 fois le volume d'échantillon nécessaire à l'analyse, 8 fois le temps d'analyse et 40 fois le volume d'effluents générés. - le titrage ballist-mix emploie un dispositif microfluidique qui, après intégration et réduction des composants, peut atteindre un volume de 25 mL et offre des performances analytiques comparables à celles obtenues en SIA. La méthode par SIA a été validée sur des solutions chargées en uranium alors que la technologie utilisée pour développer les titrages ballist-mix est en cours de validation. Cependant le principe opératoire du titrage ballist-mix est plus avantageux puisqu'il simplifie le travail de développement analytique du fait de la possibilité de simuler en avance les phénomènes physicochimiques ayant lieu lors du titrage / A joint study between the CEA and Areva La Hague has shown that chemical analysis is a crucial parameter for achieving a better performance in present and future spent nuclear fuel reprocessing plants. In fact, each plant’s process monitoring and control require a significant amount of laboratory analysis leading in overall to a considerable amount of nuclear waste. Hence, reducing the sample’s required volume for analysis would reduce its toxicity and subsequent waste, therefore increasing personnel safety, decreasing the environmental impact and the plant’s operation cost. Among the process control analytical workload, the free acidity measurement has been identified as a key analysis due to its measurement frequency. For this reason, the main objective of this research has been focused in the improvement of a reference method for free acidity measurement. The following work has been divided in two main studies seeking for the reduction of the sample volume and the automation of the analytical method protocol: - Sequential Injection Analysis (SIA) titration, whose application requires the employment of a device occupying a 25 L space, and which reduces 1000 fold the sample volume per analysis, 8 times the analysis time and 40 fold the amount of waste generated when compared to the reference analytical method. - Ballist-mix titration, whose analytical performance is equivalent to the SIA titration, but whose implementation is done inside a microfluidic device occupying a volume as low as 25 mL after integration of all of the elements needed for analysis. At the present time, the SIA titration has been validated using nitric acid samples containing uranyl cations, whereas the ballist-mix titration is being validated with the same sample conditions. However, this last analytical technique features a simplified operating principle which allows the user to shorten the analytical development process by opening the possibility to simulate the process before any experimentation

Cette thèse propose d'intégrer les principes de la micro-robotique dans un laboratoire sur puce afin d'améliorer les performances du tri cellulaire par diélectrophorèse. Contrôler la trajectoire des cellules dans une puce fluidique en temps réel nécessite de reconcevoir la puce, la modéliser et développer des lois de commande dédiées au contrôle en temps réel. Concernant la conception, cette thèse s’intéresse au compromis existant entre la vitesse de tri et les problématiques de suivi des cellules en temps réel. Une architecture originale basée sur deux plans d’électrodes, sur les faces supérieures et inférieures des canaux, est proposée. Des procédés de fabrication dédiés à cette architecture sont développés. En particulier, la fabrication d'électrodes transparentes et l'assemblage des deux réseaux d'électrodes parallèles sont étudiés. Concernant la modélisation, une formulation analytique du champ électrique découplant variables de commande, termes dépendant de la position de l’objet et termes dépendant uniquement de la géométrie de la puce est proposée afin de calculer rapidement et précisément la force de diélectrophorèse. Une analyse de l'anisotropie des forces de frottement présentes à proximité des électrodes vient compléter la modélisation dynamique du comportement des microparticules, et donne lieu à un modèle compatible avec la commande temps réel, validé expérimentalement sur des objets artificiels. Enfin, un contrôleur basé sur des techniques d’optimisation ainsi qu’un planificateur de trajectoires sont proposés pour le tri de cellules. Un simulateur est développé et met en avant les bonnes performances de tri d’un tel système. L’ensemble de ces méthodes permettront de contrôler la trajectoire de cellules biologiques dans des puces de tri afin d’en améliorer la sélectivité et la rapidité. / This thesis proposes to integrate the principles of micro-robotics in a lab-on-a-chip in order to improve the performance of cell sorting by dielectrophoresis. Controlling the trajectory of cells in a fluidic chip in real time requires redesigning the chip, modeling it and developing control laws dedicated to real-time control. Concerning the design, this thesis is interested in the compromise existing between the speed of sorting and the problems of cell tracking in real time. An original architecture based on two electrode planes, on the upper and lower faces of the channels, is proposed. Manufacturing processes dedicated to this architecture are developed. In particular, the manufacture of transparent electrodes and the assembly of the two parallel electrode arrays are studied. Concerning the modeling, an analytical formulation of the electric field uncoupling control variables, terms depending on the position of the object and terms depending solely on the geometry of the chip is proposed in order to quickly and accurately calculate the dielectrophoresis force. An analysis of the anisotropy of the friction forces present near the electrodes completes the dynamic modeling of the behavior of the microparticles, and gives rise to a model compatible with real-time control, validated experimentally on artificial objects. Finally, a controller based on optimization techniques and a trajectory planner are proposed for cell sorting. A simulator is developed and highlights the good sorting performance of such a system. All of these methods will control the trajectory of biological cells in sorting chips to improve selectivity and speed.

Este estudo tem por objetivo o desenvolvimento de uma metodologia de fabricação de sistemas Lab On Chip, úteis no estudo de processos celulares, a partir da adaptação de tecnologias próprias da microeletrônica. Foram exploradas todas as etapas envolvidas na fabricação de sistemas Lab On Chip em Poli-Di-Metil-Siloxano e desenvolvidos protocolos de fabricação de moldes, técnicas de moldagem e processos de ativação de PDMS com plasma de oxigênio para sua solda química sobre diferentes materiais, obtendo uniões irreversíveis que permitem a integração com outras tecnologias como a microeletrônica em silício e o encapsulamento com cerâmica verde, completando uma metodologia que permite a prototipagem de dispositivos micro-fluídicos de multicamadas com um nível de sofisticação comparável ao estado da arte. Foi desenvolvido o protótipo de um equipamento ótico para litografia por projeção que permite a fabricação de máscaras óticas com resolução de 5 m e oferece a possibilidade de litografia em escala de cinzas para gerar canais e estruturas com relevos arbitrários. Foram adicionalmente abordados três problemas de biofísica celular, para os quais foram propostos novos dispositivos para separação de células móveis de acordo às suas velocidades lineares, dispositivos para crescimento confinado de bactérias e dispositivos para manipulação da curvatura de membranas celulares. / The objective of this study is the development of a methodology for the fabrication of Lab On Chip systems, useful for the analysis of cellular processes, through the adaptation of technologies from microelectronics. All the steps involved with the fabrication of Lab on Chip system in Poly-Di-Methil-Siloxane (PDMS) were explored, developing protocols for mold fabrication, molding techniques and processes for oxygen plasma activation of PDMS for its bonding to different materials, achieving irreversible bonds that enable the integration with other technologies such as silicon microelectronics and green tape packaging. All this techniques constitute a methodology that allows the prototyping of multilayer microfluidic devices comparable with state of the art devices. It was developed the prototype of optical equipment for projection lithography capable of mask fabrication with 5 m resolution, and which offers also the capability of gray scale lithography for the generation of free form microchannels. Additionally three different problems in cellular biophysics where boarded, proposing new devices for the separation of motile cells according to their linear speeds in liquids, new devices for constrained bacterial growth and for curvature manipulation of cell membranes.

La microfluidique, domaine de recherche qui a émergé il y a juste 20 ans, a permis de réduire les dimensions des dispositifs d’analyse biologique ouvrant la porte au concept de « laboratoire-sur-puce » (lab-on-chip). Les succès de cette approche sont déjà nombreux, depuis l’analyse du génome en passant par la réduction du coût des analyses médicales. L’utilisation de gouttes comme enceinte réactionnelle au sein de ces dispositifs est une évolution récente qui permet de réduire encore le volume des échantillons biologiques et d’augmenter la vitesse d’analyse en parallélisant les mesures.Notre équipe développe des capteurs acoustiques dédiés à la détection d’analytes biologiques en milieu liquide. Ce type de capteur a pour principal défaut de ne permettre qu’une mesure contraignant au remplacement de l’interface de biodétection pour une réutilisation éventuelle du capteur. Dès lors, ils utilisent majoritairement une chambre de détection tout ou partie jetable, même si quelques travaux de recherche ont pu montrer la régénération d’un capteur par traitement chimique.Nous proposons ici de s’affranchir des étapes lourdes de remplacement ou de traitement de l’interface de détection qui conduisent entre chaque mesure au démontage du dispositif de détection. Nous employons dans ce cas les gouttes non plus comme enceinte réactionnelle mais comme interface de détection mobile. Elles ont ainsi le potentiel d’être générées et fonctionnalisées directement dans le dispositif pour détecter un analyte spécifique et peuvent être simplement évacuées afin de régénérer l’interface pour effectuer une mesure différente.Les travaux présentés dans cette thèse visent plus particulièrement la capture sur gouttes fonctionnalisées dans ce type de capteur innovant. Ils exposent le développement, incluant la fabrication et la caractérisation, de ces dispositifs microfluidiques ainsi que le montage d’un banc de test expérimental dédié. Ce sujet est suivi de deux projets ancillaires de développement de dispositifs microfluidiques liés aux capteurs acoustiques et à l’utilisation de gouttes. Le premier vise à homogénéiser les vitesses d’écoulements dans une chambre réactionnelle. Le second exploite les propriétés de génération de gouttes pour réaliser un condensateur à capacité variable. / Since two decades the research on microfluidics systems allowed creating devices for biological detection with regular improvement in compactness, functionality integration and quantity of biological sample, leading to the concept of lab-on-chip. This approach has resulted in dramatic changes in the biomedical field, for example, opening the possibility to perform genomic analysis or improving the medical analysis cost. Using droplet as reaction chamber is a recent evolution that leads to a decrease in biological sample volume and an increase in analysis speed by multiplexing.Our team develops acoustical sensors dedicated to detect biomarker of interest in liquids. The principal weakness of theses sensors lies in their need for replacement of the biodetection interface for performing a new measurement. Accordingly, they use a detection chamber partially or totally disposable. However, few research works showed reusability of sensor by regenerating the bioreceptor layer on the detection interface by chemical treatment.We are proposing to avoid the replacement or the chemical treatment of the detection interface that requires dismounting the device between measurements. We are using here droplets, not as reaction chambers but as movable detection interface. They can be generated and configured directly inside the device to detect a specific biomarker. Then, droplets can be easily evacuated and replaced through the device, which allows to chain measurement of various configurations without dismounting it.The research work conducted in this thesis focuses on the fluidic aspects of this innovative sensor. They show development, including realization and characterization, of theses microfluidic devices and its dedicated characterization setup. This project is followed by two ancillary works about development of microfluidic devices for acoustical sensors and droplets systems. The first one is aiming at the homogenization of the flow velocity inside a reaction chamber. The second one is exploiting property of droplet generation for the realization of a variable capacitance capacitor.

This thesis presents technological advancements in microfluidics. The overall goals of the work are to develop new miniaturized tests for point-of-care diagnostics and robust super-lubricating surfaces for friction reduction. To achieve these goals, novel materials, surfaces and manufacturing methods in microfluidics have been developed. Point-of-care diagnostic tests are portable miniaturized instruments that downscale and automate medical tests previously performed in the central laboratories of hospitals. The instruments are used in the doctor’s office, in the emergency room or at home as self-tests. By bringing the analysis closer to the patient, the likelihood of an accurate diagnosis, or a quick therapy adjustment is increased. Already today, there are point-of-care tests available on the market, for example blood glucose tests, rapid streptococcus tests and pregnancy tests. However, for more advanced diagnostic tests, such as DNA-tests or antibody analysis, integration of microfluidic functions for mass transport and sample preparation is required. The problem is that the polymer materials used in academic development are not always suited for prototyping microfluidic components for sensitive biosensors. Despite the enormous work that has gone into the field, very few technical solutions have been implemented commercially. The first part of the work deals with the development of prototype point of-care tests. The research has focused on two major areas: developing new manufacturing methods to leverage the performance of existing materials and developing a novel polymer material platform, adapted for the extreme demands on surfaces and materials in miniaturized laboratories. The novel manufacturing methods allow complex 3D channel networks and the integration of materials with different surface properties. The novel material platform is based on a novel off-stoichiometry formulation of thiol-enes (OSTE) and has very attractive material and manufacturing properties from a lab-on-chip perspective, such as, chemically stable surfaces, low absorption of small molecules, facile and inexpensive manufacturing process and a biocompatible bonding method. As the OSTE-platform can mirror many of the properties of commercially used polymers, while at the same time having an inexpensive and facile manufacturing method, it has potential to bridge the gap between research and commercial production. Friction in liquid flows is a critical limiting factor in microfluidics, where friction is the dominant force, but also in marine applications where frictional losses are responsible for a large part of the total energy consumption of sea vessels. Microstructured surfaces can drastically reduce the frictional losses by trapping a layer of air bubbles on the surface that can act as an air bearing for the liquid flow. The problem is that these trapped air bubbles collapse at the liquid pressures encountered in practical applications. The last part of the thesis is devoted to the development of novel low fluidfriction surfaces with increased robustness but also with active control of the surface friction. The results show that the novel surfaces can resist up to three times higher liquid pressure than previous designs, while keeping the same friction reducing capacity. The novel designs represent the first step towards practical implementation of micro-structured surfaces for friction reduction. / <p>QC 20110907</p>