L’épithélium intestinal est composé d’une monocouche de cellules épithéliales qui recouvrent à la fois les villi qui projettent dans le lumen et les cryptes invaginées dans le tissu conjonctif sous-jacent. Les cellules souches intestinales prolifèrent dans les cryptes et se différencient en 5 types de cellules différenciées incluant les entérocytes, les cellules de Paneth, les cellules caliciformes, les cellules entéroendocrines et les cellules Tuft. La plupart de ces cellules différenciées migrent vers le pôle apical du villus où elles meurent par apoptose exception faite des cellules de Paneth qui sont présentes uniquement dans les cryptes. Les cellules épithéliales adhèrent à la membrane basale qui sépare l’épithélium du stroma principalement constitué de collagène I et de fibroblastes. L’épithélium intestinal est renouvelé chaque semaine. Plusieurs voies de signalisation biochimiques qui gouvernent l’homéostasie intestinale ont été isolées en utilisant des modèles murins. En complément des études menées in vivo, des systèmes in vitro ont été développés de répondre à des questions difficiles à étudier in vivo, on peut notamment citer les organoides. Malgré leur indiscutable intérêt, les organoides ne reproduisent pas certaines caractéristiques majeures de l’intestin, à savoir que le nombre de total de cellules ne reste pas constant, qu’ils ne forment pas spontanément des villi et que le stroma est absent de ces modèles. Présentement, uniquement deux microsystèmes ont tenté de pallier l’absence de villi de ces modèles en reproduisant des caractéristiques dynamique (i.e le mouvement péristaltique) ou architecturale (i.e la topographie des villi) de l’intestin dans le but d’induire la formation des villi. Cependant, dans ces deux systèmes, les cellules ne reposent pas sur un substrat physiologique et sont directement ensemencées sur un support élastomérique. Quand bien même la surface de ces substrats est traitée avec des molécules constitutives de la matrice extracellulaire (ECM), ils ne reproduisent pas la micro-architecture (e.g sa structure microfibrillaire et la possibilité d’être remodelée par les cellules ensemencées) et les propriétés mécaniques spécifiques de l’ECM intestinale. Il est ainsi fort probable que ces systèmes induisent des phénotypes différents de ceux comprenant un substrat physiologique. Pour éviter ces phénomènes, nous avons développé un système innovant qui reproduit à la fois la composition et la topographie de la matrice intestinale. Le collagène I, en tant que principal composant des matrices extracellulaires, des mammifères a naturellement été choisi comme substrat cellulaire. La composition ainsi que les propriétés rhéologiques du collagène commercial ont été comparées au collagène extrait de queue de rat dans le laboratoire. Les techniques de lithographies ont été adaptées pour microstructurer les hydrogels en collagène en une sinusoïde tridimensionnelle de 400µm de période et 400µm d’amplitude en accord avec les dimensions anatomiques des intestins de souris. Les cellules épithéliales de lignées Caco2 qui sont considérées comme un modèle de cellules intestinales ont été ensemencées à la surface de la structure et ont colonisé les micro-structures en formant une monocouche cellulaire. L’utilisation du collagène I permet l’inclusion de fibroblastes primaires dans la matrice où ils évoluent in vivo. Les forces de tension développées par la monocouche épithéliale à la surface de la matrice mais également par les fibroblastes dans l’hydrogel affaissent les structures. Plus la concentration en collagène des gels est importante moins les structures sont déformées. Cependant, pour des concentrations supérieures à 6mg/mL, les fibroblastes présentent des difficultés pour s’étaler et proliférer dans la matrice probablement dues à une diffusion réduite des nutriments dans de telles matrices mais également à une réduction de la taille du maillage fibrillaire qui empêche l’étalement des cellules. / The epithelium of the small intestine is composed of a single layer of epithelial cells lining the villi that project into the lumen of the gut, and the crypts that descend into the underlying connective tissue. Dividing stem cells are contained within the crypts and give rise to five types of specialized epithelial cells including enterocytes, Goblet cells, Paneth cells, enteroendocrine cells and Tuft cells. Most of those cells travel upwards from the crypt towards the villus tip where they shed into the lumen except for Paneth cells that remain confined into the crypt. The basement membrane underlines the basal surface of epithelium and separates it from the stroma mostly composed of collagen I and fibroblasts. The whole intestinal epithelium is renewed every week. Many biochemical pathways that control intestinal homeostasis are discovered using mouse models. In contrast, in vitro models systems, such as organoids, provide a mean to investigate questions hard to be addressed in vivo. Despite their obvious interest, organoids do not fully recapitulate intestinal features: the total number of cells does not remain constant, villi-like structures are missing as well as cells and matrix constitutive of the stroma. Only two microfabricated devices have been developed to overcome this absence of villi by replicating dynamic (i.e the peristaltic motion) or structural feature (i.e the topography of the villus) of the intestine in order to induce the formation of villi. However they both do not provide the cells a physiological substrate as cells are directly seeded on an elastomeric synthetic scaffold. Even though those substrates are coated with ECM molecules, as they miss the micro-architecture specific of ECM (e.g. fibrillar structure and capacity to be remodeled by cells) as well as their mechanical properties; they might induce a different phenotype to the cells than if they were seeded on/in an ECM-like hydrogel. To address this lack, we developed an innovative device that recapitulates both the composition and topography of the intestinal lining. We chose collagen I as the constituent of our substrate since collagen I is the most abundant protein in mammals and the main constituent of all ECM in the body. We first characterized the composition and the rheological properties of commercial rat tail collagen I hydrogel and compared it to the one we extracted from rat tails. To reproduce the 3D structure of the intestine, we microstructured collagen I scaffolds as 3D sinusoids with 400µm period and 400µm height that respect the anatomic dimensions of mice intestine by adapting methods from soft lithography field. Epithelial cells from Caco2 cell line which are considered as an intestinal model were first seeded on the surface of the scaffold and successfully colonized the structure as a monolayer. Primary fibroblasts were embedded in collagen scaffolds were they actually belong in vivo. The force exterted by the epithelial monolayer at the surface of the scaffold but also by the fibroblasts inside the gels flattened the structures. The higher the concentration of collagen was the less the structures were deformed. However, for collagen concentrations higher than 6mg/mL, the fibroblasts experienced difficulties to spread and proliferate in the matrix probably related to a reduced diffusion of nutrients in such matrix or to the reduced mesh size of the fibrillar network that prevent cell spreading. Two main approaches were investigated to stiffen the collagen scaffold while maintaining a porosity suitable for fibroblasts spreading and proliferation. One consisted in the addition of a stiffer biocompatible polymer to generate a hybrid semi-interpenetrating network hydrogel with improved mechanical properties. The other resided in the addition of a cross-linker that covalently bonded the fibrils constitutive of the collagen network.

Made available in DSpace on 2015-05-14T13:21:46Z (GMT). No. of bitstreams: 1 parte1.pdf: 2204692 bytes, checksum: df26e8eadaeacfb0702b39229f465ea2 (MD5) Previous issue date: 2010-03-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This work describes the construction of a new flow-batch analyzer (FB) in micro scale (micro Flow-Batch-μFBA) using the technique of deep ultraviolet photolithography with commercial resin based in urethane acrylate oligomers (UA). The device was constructed from the union of two layers of pre-polymerized resin of 3.4 mm thick each, thus forming a single structure. The main feature of the new device is the small volume of its analysis chamber, which can be 100-200 μL, allowing the homogenization of reagent solutions in a time of 2.s. In this sense, the decrease in reagents consumption and hence generation of waste (ten times smaller than a conventional FB), wich is in line with the requirements of green chemistry and with the new position of the eco-efficiency. The system offers the following progress in relation to micro fabrication with urethane acrylate resin: (i) depth control of the channels during photolithography; (ii) axial mixer incorporated; (iii) LED - Light emitting diode (530 nm) and detector (photodiode) coupled to the device body; The depth control of the channels allows adjustment of the volume of the chamber and the LED to the mixing chamber. The LED and detector connector pins is the elements that makes the device fixed on its box. In this first assembly, system were used in photometric determination of Fe (II) in pharmaceuticals. The model for the calibration curve was validated by analysis of variance (ANOVA), and their analytical results were compared with those obtained in batch by the reference method, the application of paired t-test found no statistically significant differences at a confidence level of 95%. / No presente trabalho é descrita a construção de um novo analisador Flow- Batch (FB) em escala micro (micro Flow-Batch - μFBA) utilizando a técnica de fotolitografia profunda no ultravioleta com resina comercial à base de oligômeros uretano e acrilato (UA). O dispositivo foi construído a partir da união de duas camadas da resina pré-polimerizada de 3,4 mm de espessura cada uma, formando assim uma estrutura única. A característica principal do novo dispositivo é o pequeno volume da câmara de análise, que pode ser de 100 a 200 μL, permitindo a homogeneização das soluções reagentes em um tempo de 2.s. Nesse sentido, a diminuição do consumo de reagentes e, conseqüentemente, de resíduos gerados (dez vezes menor que um FB convencional), apontam na direção dos requisitos da química verde e se alinham com a nova postura da ecoeficiência. O sistema apresenta os seguintes avanços em relação à microfabricação com resina uretanoacrilato: (i) controle da profundidade dos canais durante a fotolitografia; (ii) agitador do tipo axial incorporado; (iii) LED emissor de luz (530nm) e detector (fotodiodo) acoplados ao corpo do dispositivo. O controle da profundidade permitiu o ajuste do volume da câmara e do LED à câmara de mistura. Os próprios pinos conectores do LED e do detector foram utilizados como elementos de fixação da peça em sua caixa. Nesta primeira montagem, o sistema foi empregado na determinação fotométrica de Fe (II) em medicamentos. O modelo para a curva de calibração foi validado através da Análise de Variância (ANOVA), e seus resultados analíticos foram comparados com aqueles obtidos em análises de referência através da aplicação do teste-t emparelhado, não apresentando diferenças estatísticas significativas a um nível de confiança de 95%.

Este trabalho demonstra o uso da fotopolimerização via absorção de dois fótons na produção de microestruturas dopadas com compostos orgânicos e nanopartículas de Au. A capacidade de produção de microestruturas com propriedades variadas é extremamente relevante, pois viabiliza o desenvolvimento de uma nova geração de dispositivos ópticos. Além disso, realizamos a conexão entre as microestruturas fabricadas e fontes de excitação, por meio de nanofibras de vidro. A integração entre essas estruturas, e destas com meios externos de excitação e detecção, é um passo essencial para o desenvolvimento de microcircuitos fotônicos, que podem representar uma nova revolução tecnológica, a exemplo do que foram os microcircuitos eletrônicos. Exploramos as possibilidades de dopagem da resina usando: (i) um composto fluorescente, (ii) um composto com birrefringência fotoinduzida e (iii) nanopartículas de ouro. Microestruturas contendo Rodamina B apresentaram boa integridade estrutural e fluorescência, tendo sido usadas para demonstrar a conexão dos microelementos com meios externos de excitação. Através de nanofibras e de micromanipuladores, comprovamos a capacidade de excitação seletiva de microestruturas através do guiamento da luz de um laser de Ar+. Estruturas birrefringentes foram obtidas pela dopagem com o azopolímero HEMA-DR13. Montamos um aparato que permite a observação da dinâmica de indução de birrefringência nas microestruturas, o qual representa um grande passo na caracterização deste tipo de microelementos. Com base nesse estudo, foi possível alcançar uma fração de birrefringência residual nas microestruturas de 35%. Por fim, propomos um método para a dopagem de microestruturas poliméricas com nanopartículas de ouro. Por se tratar de um método de dopagem indireta, ele evita interferências das nanopartículas no processo de microfabricação. Dessa forma, este trabalho abre possibilidades para a fabricação de microdispositivos funcionais com diversas propriedades especiais, bem como a integração desses microdispositivos em circuitos fotônicos. / This work demonstrates the use of two-photon photopolymerization in the fabrication of microstructures doped with organic compounds and gold nanoparticles. The ability to produce microstructures with different properties is extremely relevant, because it opens the possibility for the development of a new generation of optical devices. Besides, we have accomplished the connection between fabricated microstructures and excitation sources by means of silica nanowires. The connection among structures and with external means of detection and excitation is an essential step towards the development of new technological breakthrough in photonic microcircuits. We have explored the resin doping possibilities by using: (i) a fluorescent compound, (ii) a photoinduced birefringent compound and (iii) gold nanoparticles. Rhodamine B doped microstructures present good structural integrity and fluorescence, and were able to demonstrate the connection of microelements with external means of excitation. Through the use of nanofiber tapers and micromanipulators, we have shown the selective excitation capability of this method by guiding Ar+ laser light onto one single microstructure. Birefringent samples were obtained by doping the resin with the azopolymer HEMA-DR13. We have assembled an apparatus that allows observing the photoinduced birefringence dynamics, which represents a great step towards a better characterization of these kinds of microelements. Based on this study we were able to achieve a residual birefringence fraction of 35% in microscopic samples. Finally, we have proposed a new method for the doping of polymeric microstructures with gold nanoparticles. Because it is an indirect doping technique, it prevents gold nanoparticles from interfering with the microfabrication process. Thus, the work presented here paves the way for the fabrication of functional microdevices with a wide range of special properties, as well as for the connection of these microstructures for photonic microcircuit.

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No. of bitstreams: 2 Dissertação - Lucas da Costa Duarte - 2016.pdf: 3454791 bytes, checksum: 29ace536ee1c0aefc19dfbcdcec01bbe (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-08-05 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / This study describes the assembly and use of a RepRap 3D printer with a modeling technology fused deposition modeling (FDM) for manufacturing microfluidic devices for direct spray ionization mass spectrometry (DS-MS) assisted by paper tip as well as for generation of droplets of water/oil with integrated electrochemical detection. The low-cost 3D printer has been properly assembled and calibrated by means of pieces of kit purchased commercially. The smaller channel width obtained which allowed the uniformity 3D printing without any obstruction was 400 μm. For studies of DS-MS were used microfluidic devices consisting of a single channel (the dimensions 30 mm × 0.5 mm × 0.5 mm) connected to a sample reservoir with 3 mm diameter with low cost (R$ 0.20) and time printing (20 min). A tip paper (5 mm × 0.5 mm) was cut out and inserted at the end of the printed channel to facilitate the formation of the spray. The spraying was promoted by the application of 4 kV in the sample reservoir containing 0.1% formic acid in methanol prepared. This organic medium showed high compatibility with the polymeric material used for the printing of microfluidic chips. Using a glucose solution as a template, the spray formed by the proposed microfluidic device was extremely stable compared to the spray generated by conventional paper devices for at least 10 min. The analytical devices for viability of the printed DS-MS was successfully demonstrated by qualitative analysis of ball pens inks, caffeine, xylose and lysozyme. The DS-MS devices exhibited significant repeatability and reproducibility, making it possible to reuse. For the generation of droplets of oil/water it was built with a device channel in the T-junction configuration with 0.75 mm × 50.0 mm × 0.7 mm and containing two integrated electrodes (1.5 mm × 2.0 mm and spacing 0.5 mm) in antiparallel arrangement. These electrodes consisting of a mixture of ABS and carbon nanotubes were used for conductivity detection capacitively coupled contact (C4D). The droplets were generated by varying the flow rate of continuous phase comprised of oil + Span80/water + Triton between 10 to 60 μL/min. Through the printed electrodes it was possible to employ the system C4D system to detect the droplets generated with a satisfactory response. The intensity and width of peaks obtained exponentially decrease the extent that it increases the flow of the continuous phase. Moreover, it was possible to correlate the length of the droplet generated from the signals obtained with C4D, indicating potential as a new technique for droplet measures. Finally, the 3D printer made possible the manufacture of parts contributed to other works in cooperation to include the construction of an electrochemical cell for by batch injection analysis, a support for Snitrosothiols decomposition, a cover for conductivity detection cell, micromixers, devices for ELISA assays and mold for manufacturing zones for colorimetric analysis. / modelagem por fusão e deposição (fused deposition modeling, FDM) para fabricar dispositivos microfluídicos para ionização direta por spray em espectrometria de massas (DS-MS) assistida por ponta de papel, bem como para geração de gotículas de água/óleo com detecção eletroquímica integrada. A impressora 3D de baixo custo foi devidamente montada e calibrada por meio do kit de peças adquirido comercialmente. A menor largura de canal obtida que permitiu a uniformidade de impressão 3D sem qualquer obstrução foi de 400 μm. Para os estudos de DS-MS, foram utilizados dispositivos microfluídicos constituídos de um único canal (nas dimensões 30 mm × 0,5 mm × 0,5 mm) conectado a um reservatório de amostra com 3 mm de diâmetro com baixo custo (R$ 0,20) e tempo de impressão (20 min). Uma ponta de papel (5 mm × 0,5 mm) foi cortada e inserida na extremidade do canal impresso para facilitar a formação do spray. A pulverização foi promovida através da aplicação de 4 kV no reservatório de amostra contendo 0,1% de ácido fórmico preparada em metanol. Este meio orgânico demonstrou grande compatibilidade com o material polimérico utilizado para a impressão dos chips microfluídicos. Usando-se uma solução de glicose como modelo, o spray formado pelo dispositivo microfluídico proposto foi extremamente estável quando comparado com o spray gerado pelos dispositivos convencionais de papel por pelo menos 10 min. A viabilidade analítica dos dispositivos impressos para DS-MS foi demonstrado com sucesso por meio de análise qualitativa de tintas de canetas esferográficas, cafeína, xilose e lisozima. Os dispositivos de DS-MS exibiram repetitividade e reprodutibilidade significativa, tornando possível a sua reutilização. Para a geração de gotículas de óleo/água foi construído um dispositivo com canais na configuração junção em T com 50,0 mm × 0,75 mm × 0,7 mm contendo dois eletrodos integrados (1,5mm × 2,0 mm e espaçamento de 0,5 mm) na disposição antiparalela. Estes eletrodos, constituídos de uma mistura de ABS e nanotubos de carbono, foram utilizados para detecção condutométrica sem contato capacitivamente acoplada (C4D). As gotículas foram geradas variando a vazão de fase contínua formadas por óleo+Span80/ água+Triton de 10 a 60 μL/min. Por meio dos eletrodos impressos foi possível empregar o sistema C4D para detecção das gotas geradas com resposta satisfatória. A intensidade e largura dos picos obtidos diminuem exponencialmente a medida em que se aumenta a vazão da fase contínua. Além disso, foi possível correlacionar o comprimento da gota gerada com os sinais obtidos com C4D, indicando potencialidade como uma nova técnica para medidas de gotículas. Por fim, a impressora 3D possibilitou a fabricação de peças que contribuíram para outros trabalhos em colaboração que incluem a construção de uma célula eletroquímica para análise por injeção em batelada, um suporte para decomposição de S-nitrosotióis, uma tampa para cela de detecção condutométrica, micromisturadores, dispositivos para ensaios de ELISA, e molde para fabricação de zonas para análises colorimétrica.

O projeto de novos materiais para aplicações tecnológicas em biomateriais e bioengenharia é altamente dependente de como as células aderem à superfície de um material. A adesão e crescimento em biomateriais depende de propriedades do substrato, tais como molhabilidade da superfície, a topografia e a composição química de superfície. O objetivo deste estudo foi investigar as interações de diversos materiais com culturas celulares de células epitelial CHO (Ovário de Hamster Chinês). Os materiais utilizados foram SU-8 2005 (elétron-resiste, Microchem), PDMS (Poli (dimetil siloxano), Down Corning), DLC (Diamond-like Carbon) e vidro foi utilizado como referência. Superfícies de vidro, SU-8, PDMS e DLC lisas (planas) e isentas de modificação ou tratamento específico foram avaliadas quanto ao cultivo de células CHO. Valores médios dos fatores de forma (Ff) de 450 células foram calculados para cada uma das culturas realizadas sobre os 4 substratos. Foram obtidos Ff próximos a 0,52 para o vidro, o SU-8 liso e o DLC, demonstrando um bom espraiamento das células nessas superfícies. A superfície de PDMS apresentou valor unitário para o fator de forma (Ff), que está relacionado a um baixo espraiamento das células. A energia de superfície (ES) obtida para o PDMS é compatível com o resultado de fator de forma (Ff), uma vez que o menor valor para ES é coerente com a baixa adesão celular, o que gerou células com elevado fator de forma (Ff). O SU-8 foi modificado por implantação iônica com uma dose de 1,2x1016 átomos/cm2 e a energia de implantação foi de 8 keV, como referência foi utilizada uma superfície lisa de SU-8 sem implantação. Os resultados mostraram que o número de células vivas por unidade de área foi superior na superfície de SU-8 com prata implantada, mostrando o bom desempenho da cultura nesse substrato. As superfícies de DLC modificadas por tratamento com plasma de oxigênio (DLC-O) e com plasma de hexafluoreto de enxofre (DLC-F) foram utilizadas para cultura celular, os resultados de três experimentos independentes de contagem de número de núcleos (marcados com DAPI) por unidade de área confirmaram os resultados obtidos através do teste de viabilidade (marcados com trypan blue). A superfície de DLC-O, apresentou um maior número de núcleos por unidade de área, quando comparado à superfície DLC-F, da mesma forma que nos resultados obtidos pelo teste de viabilidade. As energias de superfície para as amostras de DLC-F e DLC-O indicaram que a superfície DLC-O é mais hidrofílica do que a superfície DLC-F, que está coerente com o que é conhecido da literatura e com os resultados obtidos em nosso trabalho. Cultura de células CHO foram realizadas em superfícies litografadas com estruturas hexagonais periódicas com o parâmetro 2R (diâmetro do círculo inscrito) sendo 12 µm, 30 µm, 80 µm, 280 µm, 560 µm e também em SU-8 liso. Estas superfícies foram caracterizadas por microscopia óptica de fluorescência com relação ao número de núcleos (marcados com o fluoróforo DAPI) por unidade de área, isto é, núcleos/mm2. Obteve-se histogramas com o número médio de núcleos por mm2 em três experimentos independentes, onde o número núcleos/mm2 foi consideravelmente maior para 80 µm. As superfícies contendo cavidades periódicas de 12 µm e 30 µm apresentaram dificuldade para as células CHO aderirem à superfície. Em uma outra etapa realizou-se culturas celulares em triplicata dos substratos com as superfícies 12 µm, 80 µm, 280 µm, 560 µm e também em SU-8 liso. As células em cada uma das superfícies foram analisadas por microscopia óptica (MO) para avaliação da viabilidade celular, utilizando marcador trypan blue. Obteve-se histogramas com os valores médios para o número de células vivas/mm2 para as culturas celulares que corrobora os resultados obtidos no histograma da cultura celular que tiveram os núcleos marcados pelo fluoróforo DAPI. Assim, fica confirmado o melhor desempenho da cultura celular no substrato 80 µm que apresentou o maior número de células vivas/mm2 As micrografias obtidas através de marcação por DAPI foram analisadas através da função de correlação com intuito de se entender como as células estavam organizadas. Isso foi feito para cada uma das superfícies litografadas, 12 µm, 30 µm, 80 µm, 280 µm, 560 µm e também em SU-8 liso. As superfícies dos substratos 80 µm apresentaram os menores valores de distâncias para primeiros e segundos vizinhos, ou seja, as células estão mais próximas umas das outras. As demais superfícies tendem a separar mais as células. Obteve-se também os valores de raio de aglomerado (rc), distância entre os aglomerados (dc) e o número de primeiros vizinhos (Np) através do ajuste da função de correlação. A análise de correlação mostrou com clareza o que não era evidenciado apenas visualizando-se as imagens. Ela mostra que as células, mesmo em SU-8 liso tem a forte tendência de formar aglomerados de células com raio de aproximadamente 45 µm. No caso de substratos lisos, células CHO apresentaram a melhor adesão na superfície do SU-8, seguido do DLC, enquanto que o PDMS foi a pior situação, devido à baixa molhabilidade do material. No caso de superfícies com microestrutura, SU-8 contendo microcavidades hexagonais de 12 e 30 µm mostraram ser as situações mais adversas para o crescimento de células CHO, provavelmente por causa da topografia das cavidades serem de menor tamanho quando comparadas ao tamanho das células CHO. Em vez disso, SU-8, contendo microcavidades hexagonais de 80 µm foi a superfície mais favorável para o crescimento de células CHO. / The design of new materials for technological applications in biomaterials and bioengineering is highly dependent on how the cells adhere to the material surface. The cells adhesion and growth on biomaterials depends on substrate properties such as surface wettability, topography and the chemical composition. The aim of this study was to investigate the interactions of various materials with cell cultures of epithelial cells CHO (Chinese Hamster Ovary). The materials used were SU-8 2005 (electron resists, Microchem), PDMS (poly (dimethyl siloxane), Dow Corning), DLC (Diamond-like Carbon) and glass was used as reference. Unmodified and flat surfaces of glass, SU-8, PDMS and DLC were evaluated for the culture of CHO cells. Form factor (Ff) values were calculated as average of 450 cells for each of the cultures performed on the four substrates. Ff close to 0.52 was obtained for flat surfaces of glass, SU-8 and DLC, showing a good cell spreading on these surfaces. The surface of PDMS presented a form factor (Ff) near unity, which is related to low spreading cell. The surface energy (ES) obtained for the PDMS is coherent with the Ff result, since the smallest value of ES is consistent with the low cell adhesion, which resulted in cells with a high Ff. The SU-8 was modified by ion implantation using a dose of 1.2x1016 atoms/cm2 and an implantation energy of 8 keV, unmodified flat SU-8 was used as a reference. The cell culture results showed that the number of live cells per unit area was greater in the SU-8 surface implanted with silver, showing a good performance in the culture substrate. The DLC surfaces modified by plasma treatment with oxygen (DLC-O) and sulfur hexafluoride (DLC-F) were used for cell culture. The results of three independent experiments, counting the number of nuclei (marked with DAPI) per unit area, confirmed the results obtained by the viability test (marked with trypan-blue). The surface of the DLC-O had higher number of nuclei per unit area when compared to the surface of the DLC-F, similarly to the results obtained for the viability test. The surface energies of the DLC-F and DLC-O samples indicated that the DLC-O surface is more hydrophilic than the DLC-F surface, which is consistent with results obtained with our work and with the literature. CHO cell culture were performed on surfaces with periodic hexagonal structures with the diameter of inscribed circle (2R) given by 12 µm, 30 µm, 80 µm, 280 µm, 560 µm and also on flat SU-8. These surfaces were characterized by fluorescence optical microscopy with respect to the number of nuclei (marked with fluorophore DAPI) per unit area, i.e. nuclei/mm2. Histograms were obtained for the average number of nuclei per mm2 in three independent experiments, where the substrate with periodic hexagonal structures with 2R = 80 µm presented considerably higher nuclei/mm2. Surfaces containing periodic cavities of 2R =12 µm and 30 µm were adverse for CHO cells adhesion. In another approach, cell culture were analyzed by light microscopy (LM) for evaluation of cell viability using trypan-blue marker. This was carried out in triplicate cell culture on substrates with surfaces 12 µm, 80 µm, 280 µm, 560 µm and also on flat SU-8. Histograms were generated for average number of living cells/mm2 for each substrate, which corroborates with the results obtained for the cell culture marked with fluorophore DAPI. Thus, it is confirmed the better performance of the cell culture on substrates with 2R = 80 µm, presenting the highest number of living cells/mm2. The micrographs obtained with cells marked with DAPI were analyzed through the correlation function with the aim of understanding how the cells were organized. This was performed for each of the lithographed surfaces 12 µm, 30 µm, 80 µm, 280 µm, 560 µm and also flat SU-8. The surfaces of the substrates with 2R = 80 µm had the lowest values for length between its neighbors, that is, the cells are closer to each other. The remaining surfaces tend to separate the cells. Also were obtained the cluster radius values (rc), the distance between the clusters (dc) and the number of nearest neighbors (Np) through the correlation function fitting. The correlation analysis clearly showed what was not possible to observe by viewing the images. It shows that the cells, even in flat SU-8, have a strong tendency to form clusters of cells within about 45 micrometers. In the case of flat substrates, CHO cells exhibited better adhesion to the surface of SU-8, followed by the DLC, while the PDMS was worse due to low wettability of the material. In the case of surfaces with microstructures, SU-8 containing hexagonal microstructures of 12 and 30 µm showed to be the most adverse conditions for the CHO cell growth, probably because of the topography of the cavities being smaller in size compared to the size of CHO cells. SU-8 with 80 µm hexagonal microstructures was more favorable surface for the growth of CHO cells.

L’utilisation de photodiodes avalanche monophotoniques (PAMP) pour une utilisation au sein d’imageur préclinique par tomographie d’émission par positrons est d’intérêt. En effet, l’utilisation de ces photodétecteurs intégrés au CMOS est poussée par leurs excellentes performances de résolution en temps ainsi que leur haute sensibilité. Cependant, l’utilisation de ces détecteurs nécessite également un circuit intégré de contrôle visant à protéger les photodiodes de courants trop élevés lors de déclenchement d’avalanches et de contrôler leurs temps mort. Ces circuits de plus en plus sophistiqués nécessitent un espace significatif diminuant ainsi la surface photosensible à la surface de la puce et diminuant leurs sensibilités. L’assemblage 3D puce-à-puce est donc nécessaire dans le but d’augmenter la surface photosensible et de ne pas limiter les fonctionnalités de contrôles électroniques individuelles à chaque PAMP. Ce document présente le développement d’un procédé d’assemblage 3D puce-à-puce visant l’intégration de matrices de PAMP. Les étapes de microfabrication nécessaires visent l’intégration d’interconnexions verticales au travers du substrat (TSV) permettant de transmettre les signaux d’une couche à l’autre et le collage 3D de ceux-ci. De plus, des mesures de caractéristiques de bruits ont été effectuées sur des puces ayant subi certaines étapes de microfabrication du procédé d’assemblage 3D. Ces mesures ont été effectuées dans le but de déterminer l’impact potentiel du procédé d’assemblage sur les performances des PAMP intégrés en 3D.

Optical polymers are a subject of research and industry implementation for many decades. Optical polymers are inexpensive, easy to process and flexible enough to meet a broad range of application-specific requirements. These advantages allow a development of cost-efficient polymer photonic integrated circuits for on-chip optical communications. However, low refractive index contrast between core and cladding limits light confinement in a core and, consequently, integrated polymer device miniaturization. Also, polymers lack active functionality like light emission, amplification, modulation, etc. In this work, we improved a performance of integrated polymer waveguides and demonstrated active waveguide devices. Also, we present novel Si QD/polymer optical materials. In the integrated device part, we demonstrate optical waveguides with enhanced performance. Decreased radiation losses in air-suspended curved waveguides allow low-loss bending with radii of only 15 µm, which is far better than &gt;100 µm for typical polymer waveguides. Another study shows a positive effect of thermal treatment on acrylate waveguides. By heating higher than polymer glass transition temperature, surface roughness is reflown, minimizing scattering losses. This treatment method enhances microring resonator Q factor more than 2 times. We also fabricated and evaluated all-optical intensity modulator based on PMMA waveguides doped with Si QDs. We developed novel hybrid optical materials. Si QDs are encapsulated into PMMA and OSTE polymers. Obtained materials show stable photoluminescence with high quantum yield. We achieved the highest up to date ~65% QY for solid-state Si QD composites. Demonstrated materials are a step towards Si light sources and active devices. Integrated devices and materials presented in this work enhance the performance and expand functionality of polymer PICs. The components described here can also serve as building blocks for on-chip sensing applications, microfluidics, etc. / <p>QC 20171207</p>

La recherche de biomarqueurs pour le dépistage, le diagnostique ou le traitement de maladie requiert le développement de dispositifs hautement sensibles alliant un faible coût d’analyse, un faible encombrement et une réponse rapide. Dans ce cadre, nous développons un biocapteur acoustique utilisant des ondes de volume pour permettre la détection d’analyte particulière dans un milieu biologique complexe. La géométrie retenue est une membrane résonante à excitation et détection piézoélectriques intégrées vibrant sur un mode de cisaillement d’épaisseur généré par un champ latéral. Le transducteur utilise les propriétés particulières de l’arséniure de gallium pour assurer une détection sensible et sélective, aussi bien grâce à ses propriétés piézoélectriques que ses possibilités de microfabrication ou de biofonctionnalisation. Dans un premier temps, nous avons dimensionné le dispositif et modélisé son comportement. Une sensibilité à un ajout de masse a pu être estimée à environ 0.1 ng.Hz-1. Nous avons ensuite envisagé la microfabrication du capteur en utilisant uniquement des techniques de microfabrication à faible coût (gravure humide et photolithogravure). Ceci a permis d’obtenir des membranes épaisses (50 μm) de géométrie et d’état de surface maitrisés. Nous avons ensuite envisagé la réalisation de la biointerface grâce au développement d’une interface chimique spécifique permettant d’immobiliser covalemment une monocouche dense de protéine à la surface du GaAs. Cette monocouche a été caractérisée par une analyse originale couplant la microscopie à force atomique (AFM) et la spectrométrie de masse MALDI-TOF. Enfin, les interfaces fluidiques et électriques ont été mises au point et ont permis de tester le dispositif par une mesure d’impédance. / The biomarkers detection for screening, diagnosis or treatment of disease requires the development of highly sensitive devices combining low cost of analysis, a small size and quick responses. In this context, we develop a biosensor using bulk acoustic wave to allow the detection of specific analyte in a complex biological medium. The geometry used is a piezoelectric resonant membrane using shear mode vibration excited by lateral field. The transducer uses the specific properties of gallium arsenide to provide a highly sensitive and selective detection thanks to its piezoelectric properties and also its microfabrication or biofonctionnalisation facilities. First, we dimensioned the device and modeled it behavior. A sensitivity to adding mass has been estimated at 0.1 ng.Hz-1. Then, we considered the sensor microfabrication using only low cost process (photolithography and wet etching). Through these processes, we obtained well formed thick membranes (50μm) with specific surface properties and microstructuration. Next, we realize the biointerface through the development of a specific chemical interface in order to immobilize a dense protein monolayer covalently attached to the GaAs surface. This monolayer was characterized by an original analysis coupling the atomic force microscopy and the mass spectroscopy MALDI-TOF. Finally, fluid and electrical interfaces have been developed and we tested the device by impedance measurements

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This dissertation describes advances in the microfabrication of thin layer chromatography (TLC) plates. These plates are prepared by the patterning of carbon nanotube (CNT) forests on substrates, followed by their infiltration with an inorganic material. This document is divided into ten sections or chapters. Chapter 1 reviews the basics of conventional TLC technology. This technology has not changed substantially in decades. This chapter also mentions some of the downsides of the conventional approach, which include unwanted interactions of the binder in the plates with the analytes, relatively slow development times, and only moderately high efficiencies. Chapter 2 focuses primarily on the tuning of the iron catalyst used to grow the CNTs, which directly influences the diameters of the CNTs grown that are produced. Chapter 3 focuses on the atomic layer deposition (ALD) of SiO2 from a silicon precursor and ozone onto carbon-nanotubes to obtain an aluminum free stationary phase. This approach allowed us to overcome the tailing issues associated with the earlier plates prepared in our laboratory. Chapter 4 is a study of the hydroxylation state of the silica in our TLC plates. A linear correlation was obtained between the SiOH+/Si+ time-of-flight secondary ion mass spectrometry (ToF-SIMS) peak ratio and the isolated silanol peak position at ca. 3740 cm-1 in the diffuse reflectance infrared spectroscopy (DRIFT) spectra. We also compared the hydroxylation efficiencies on our plates of ammonium hydroxide and HF. Chapter 5 reports a series of improvements in TLC plate preparation. The first is the low-pressure chemical vapor deposition (LPCVD) of silicon nitride onto CNTs, which can be used to make very robust TLC plates that have the necessary SiO2 surfaces. These TLC plates are the best we have prepared to date. We also describe here the ALD deposition of ZnO into these devices, which can make them fluorescent. Chapters 6 – 10 consist of contributions to Surface Science Spectra (SSS) of ToF-SIMS spectra of the materials used in our microfabrication process. SSS is a peer-reviewed database that has been useful to many in the surface community. The ToF-SIMS spectra archived include those of (i) Si/SiO2, (ii) Si/SiO2/Al2O3, (iii) Si/SiO2/Al2O3/Fe, (iv) Si/SiO2/Fe (annealed at 750 °C in H2), and (v) Si/SiO2/Al2O3/Fe(annealed)/CNTs. Both positive and negative ion spectra have been submitted. In summary, the present work is a description of advances in the development, thorough characterization, optimization, and application development of microfabricated thin layer chromatography plates that are superior to their commercial counterparts.