Résumé : Cette thèse porte sur l’élaboration d’un outil de capture d’ADN permettant d’identifier une mutation génétique (SNP) grâce à la formation de sandwichs avec des particules de carboxylatex biofonctionnalisées avec des oligonucléotides couplée à une détection de la fluorescence. Le modèle biologique choisi pour ce projet est le génotypage plaquettaire et plus particulièrement la recherche du gène biallélique HPA-1. Le principal objectif de ce travail a été d’optimiser un outil de capture préalablement développé dans l’équipe (Trévisan, 2011) afin de réduire le nombre d’étapes et de simplifier la mise en œuvre globale du test en modifiant les interactions moléculaires utilisée pour capturer l’ADN cible et en utilisant des particules fluorescentes comme élément de détection. En présence d’ADN cible, des sandwichs sont formés entre les particules fluorescentes et les particules magnétiques biofonctionnalisées. Ces sandwichs sont purifiés par séparation magnétique et la fluorescence est détectée par trois méthodes : la cytométrie en flux, l’imagerie de fluorescence et l’Evareader (détection par ondes évanescentes). Dans un premier temps, les paramètres de fonctionnalisation chimique et biologique des différentes particules (magnétiques et fluorescentes) ont été déterminés et optimisés ainsi que les conditions d’hybridation pour la capture de l’ADN cible. Ensuite, la formation des sandwichs et leur détection ont été suivies par des mesures de fluorescence en utilisant trois méthodes différentes : la cytométrie en flux, l’imagerie de fluorescence et l’Evareader (capteur à ondes évanescentes). Les résultats obtenus avec les différentes méthodes de détection sont concordants et montrent que l’outil de capture d’ADN développé permet de capturer la cible synthétique (oligonucléotide) HPA-1 en réduisant le temps d’analyse de 45 min. Dans nos conditions, le test permet de discriminer l’allèle a de l’allèle b du gène HPA-1 qui ne diffère que d’un nucléotide. Le rapport des signaux de fluorescence issus du sandwich spécifique et du sandwich non spécifique est d’environ 2,5 à 3. Ce rapport devra être amélioré par la suite, en optimisant les conditions de formation des sandwichs. La prochaine étape consistera à optimiser le système de capture d’ADN développé pour gagner en spécificité et déterminer la limite de détection du test. Ce test devra également être validé avec des échantillons biologiques. A plus long terme, la fluorescence pourra être détectée par un photodétecteur miniaturisé actuellement développé à l’Université de Sherbrooke. Des études préliminaires présentées dans ce manuscrit montrent les potentialités de ce nouveau transducteur. // Abstract : This thesis is about the development of a new assay to capture DNA. This assay is based on the formation of sandwiches between biofunctionnalized with oligonucleotides carboxylatex microparticles combined with fluorescence detection. It should be able to discriminate single nucleotide polymorphism (SNP). This assay is designed to be applied to platelet genotyping for the research of the gene HPA-1. The main goal of this work was to improve an assay previously developed (Trévisan, 2011) by INL and EFS Rhône-Alpes. The objectives are to reduce the number of steps and to simplify the test. To do so, the molecular interactions used in order to capture target DNA are modified and fluorescent microparticles are used for the detection. In the presence of target DNA, sandwiches are formed between both biofunctionnalized fluorescent and magnetic particles. Those sandwiches are purified through magnetic separation. Then, fluorescence is detected by three methods: flow cytometry, fluorescence imaging and Evareader (detection with an evanescent wave). First, chemical and biological parameters for the functionalization of the different particles (magnetic and fluorescent) are determined. The conditions for the capture of target DNA were optimized. Then, the formation and the detection of the sandwiches were estimated by measuring the fluorescence using three different methods: flow cytometry, fluorescence imaging and Evareader. The results obtained with the three methods are consistent. They show that the new system enables to capture synthetic target (oligonucleotide) HPA-1 with a reduction of total time analysis of 45 min. In our conditions, SNP can be discriminated for HPA-1 gene. For this discrimination, the fluorescence signal ratio about 2.5 to 3. This ratio should be improved by optimizing the conditions of sandwiches formation. Next step will consist in the optimization of the system developed to capture DNA in order to gain specificity and to determine the limit of detection. This test should also be validated with biological samples. In the long term, fluorescence could be detected by a miniaturized photodetector developed in the University of Sherbrook. Preliminary studies presented in this manuscript show the potentialities of this new transducer.

Este trabalho teve por objetivo desenvolver e caracterizar um sensor de força utilizando uma fibra óptica modificada pelo processo conhecido como Fiber tapering. A fibra quando modificada deixa exposto o campo evanescente, o que a torna sensível a influências externas, e a luz guiada na fibra pode vir a sofrer reflexão interna total frustrada ao entrar em contato com materiais. Ao envolver a região modificada por um material elastomérico, a área de contato e consequentemente a atenuação torna-se uma função da intensidade da força aplicada, possibilitando então relacionar a força a atenuação da luz. Baseando-se nesse efeito, foi criado um sensor de dimensões reduzidas, de rápida resposta, linear, altamente sensível e de boa repetibilidade. Foi criado também um circuito eletrônico utilizando amplificadores operacionais para a aquisição e processamento do sinal proveniente da fibra e selecionado um sensor comercial comum para a realização de experimentos e comparações. Ambos os sensores foram posicionados sobre uma balança de precisão e submetidos a diversos esforços obtendo-se dados sobre a resposta estática. Em seguida utilizando um shaker eletrodinâmico foram medidos os tempos de resposta a uma entrada degrau, e realizando esforços repetitivos foram analisados os desvios das medidas lidas pelos sensores. / The aim of this research was to develop and characterize a force sensor using a modified optical fiber by a process known as Fiber tapering. The modified fiber leaves the evanescent field exposed and prone to external influences and the guided light may suffer frustration of total internal reflection upon contact with materials. When covering the modified fiber section with an elastomeric material, the contact area and therefore the attenuation becomes a function of the applied pressure, making it possible to relate force to attenuation in light intensity. Based on this effect, a small sensor was created, having a quick response time, with high linearity, high sensitivity and good repeatability. Along with the sensor, an electronic circuit using operational amplifiers was designed for acquisition and processing of the signal obtained from the optical fiber. In addition, in order to perform experiments and comparisons, a standard force sensor was chosen. Both sensors were placed over a precision weighing scale and had different intensities of force applied on them, and after that, data regarding static measurements was gathered. The response time was obtained using an electrodynamic shaker and applying a step input. Furthermore, data was gathered about the deviations on the measurements by performing a repetitive set of compressions.

Este trabalho teve por objetivo desenvolver e caracterizar um sensor de força utilizando uma fibra óptica modificada pelo processo conhecido como Fiber tapering. A fibra quando modificada deixa exposto o campo evanescente, o que a torna sensível a influências externas, e a luz guiada na fibra pode vir a sofrer reflexão interna total frustrada ao entrar em contato com materiais. Ao envolver a região modificada por um material elastomérico, a área de contato e consequentemente a atenuação torna-se uma função da intensidade da força aplicada, possibilitando então relacionar a força a atenuação da luz. Baseando-se nesse efeito, foi criado um sensor de dimensões reduzidas, de rápida resposta, linear, altamente sensível e de boa repetibilidade. Foi criado também um circuito eletrônico utilizando amplificadores operacionais para a aquisição e processamento do sinal proveniente da fibra e selecionado um sensor comercial comum para a realização de experimentos e comparações. Ambos os sensores foram posicionados sobre uma balança de precisão e submetidos a diversos esforços obtendo-se dados sobre a resposta estática. Em seguida utilizando um shaker eletrodinâmico foram medidos os tempos de resposta a uma entrada degrau, e realizando esforços repetitivos foram analisados os desvios das medidas lidas pelos sensores. / The aim of this research was to develop and characterize a force sensor using a modified optical fiber by a process known as Fiber tapering. The modified fiber leaves the evanescent field exposed and prone to external influences and the guided light may suffer frustration of total internal reflection upon contact with materials. When covering the modified fiber section with an elastomeric material, the contact area and therefore the attenuation becomes a function of the applied pressure, making it possible to relate force to attenuation in light intensity. Based on this effect, a small sensor was created, having a quick response time, with high linearity, high sensitivity and good repeatability. Along with the sensor, an electronic circuit using operational amplifiers was designed for acquisition and processing of the signal obtained from the optical fiber. In addition, in order to perform experiments and comparisons, a standard force sensor was chosen. Both sensors were placed over a precision weighing scale and had different intensities of force applied on them, and after that, data regarding static measurements was gathered. The response time was obtained using an electrodynamic shaker and applying a step input. Furthermore, data was gathered about the deviations on the measurements by performing a repetitive set of compressions.