Stuttgart, Univ., Habil.-Schr., 2004.

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-08-14T20:32:07Z No. of bitstreams: 1 Dissertação_ICS_ Luciana Bastianelli Knop.pdf: 1536859 bytes, checksum: 7cfedb077faecccffed02fda3ab313f1 (MD5) / Made available in DSpace on 2013-08-14T20:32:07Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Luciana Bastianelli Knop.pdf: 1536859 bytes, checksum: 7cfedb077faecccffed02fda3ab313f1 (MD5) / O avanço tecnológico das últimas décadas nas técnicas dos imunodiagnósticos permitiu o desenvolvimento de métodos capazes de detectar o complexo antígeno-anticorpo com elevada eficiência e confiabilidade. Contudo, essas ténicas ainda não alcançaram um patamar de baixo custo, fácil manuseio, de resultado imediato para amostras múltiplas e de pessoal sem qualificação técnica para a aplicação dos testes. A Precision Photonic Corporation (PPC), juntamente com a mBio Inc. e em parceria com a Universidade de San Diego (UCSD), EUA, criaram um leitor de circuito integrado (Biochip Reader), baseado em arranjos multiplex para a detecção de multimarcadores biológicos a um só tempo, com a utilização de sistemas ópticos de baixo custo e fluorescência, rápido e de fácil manuseio. O objetivo deste estudo piloto, realizado na Bahia, em parceria com a Universidade Federal da Bahia (UFBA), foi o de avaliar a operacionalidade do sistema de leitor de circuito integrado (Biochip Reader), dos protocolos encaminhados e dos resultados dos testes multiplex para detecção de anticorpos contra HIV e HCV. Foram testadas 65 amostras que apresentaram uma sensibilidade e especificidade de 100% quando comparadas com os resultados realizados por ELISA para HIV e HCV. Apesar disto, a análise sobre a operacionalidade do sistema, dos protocolos e dos resultados obtidos na Bahia apresentaram instabilidade das lâminas devido à suceptibilidade excessiva à umidade, formação de cristais e resíduos de trealose, excesso de etapas e manipulação das lâminas, e levarem a alterações dos protocolos. Portanto, apesar da similaridade dos resultados encontrados nos testes deste estudo-piloto quaondo comparado com os testes realizados por ELISA nos pacientes testados, o protótipo necessita de aprimoramento tecnológico, ampliação dos biomarcadores e mais experimentos de validação, a fim de que o sistema de biochip como uma ferramenta eficaz para o diagnóstico de doenças infecciosas seja inserido no mercado. / Salvador

Du fait de leur haute stabilité et bas coût de production, les aptamères suscitent un intérêt croissant, depuis près de 20 ans, dans le design de biocapteurs en tant qu'élément de reconnaissance idéal. Le but de ce travail de thèse est de démontrer l'intérêt et la pertinence d'un outil tel qu'une biopuce à aptamères, associant les avantages des sondes aptamères à ceux d'une détection par SPRi (Surface Plasmon Resonance imaging), permettant une détection sans marquage et en temps réel d'interactions moléculaires. Dans ce but, deux aptamères anti-thrombine (APT1 = 5′- GGT-TGG-TGT-GGT-TGG -3′ et APT2 = 5′-AGT-CCG-TGG-TAG-GGG-AGG-TTG-GGG-TGA-CT-3′) ont été choisis comme objets d'étude modèles. Ce choix a permis d'orienter différents axes de recherche : utilisés indépendamment comme sondes lors de l'élaboration de notre biopuce, ils ont tout d'abord permis de réaliser une détection cinétique optimisée de la thrombine, avec des performances remarquables pour une détection de ce type, ainsi que le calcul de constantes de dissociation en solution et à la surface des biopuces. Mais au-delà d'un simple biocapteur, la biopuce a également pu être utilisée comme véritable plateforme d'étude de la thrombine et de ses interactions, au sein de structures plus complexes telles que la structure « sandwich » entre les deux aptamères, ou d'autres interactions impliquant la thrombine en tant qu'acteur de la cascade de coagulation (inhibition de la thrombine par l'antithrombine III et le cofacteur II de l'héparine, transformation de la prothrombine au sein du complexe prothrombinase). / For 20 years, aptamers have been raising an increasing interest for biosensor applications as replacements for antibodies, due to their high stability and low cost. The main objective of this Ph.D. thesis is to show the great capacities of an aptamer biochip that combines the advantages of aptamer probes associated with a SPRi (Surface Plasomn Resonance imaging) detection to monitor, in real-time and in a label-free manner, molecular interactions occurring on the surface of the biochip. Two aptamers selected against the thrombin protein (APT1 = 5′- GGT-TGG-TGT-GGT-TGG -3′ and APT2 = 5′-AGT-CCG-TGG-TAG-GGG-AGG-TTG-GGG-TGA-CT-3′) were chosen as models for our study. This choice led to the exploration of different lines of research. First, both aptamers were used independently to develop a kinetic biosensor with remarkable performances for the quantification of thrombin. This tool served to determine independently, and compare, both the solution- and surface-phase affinities of the trombin-APT2 interaction. But more than a simple and effective biosensor, this kind of biochip represents a true platform to study the protein and its interactions within complex structures, such as the sandwich-like architecture with APT1 and APT2, or its interactions with other factors of the coagulation cascade (inhibition of thrombin by antithrombin III and heparin cofactor II, conversion of prothrombin into thrombin by the prothrombinase complex).

La détection rapide et sensible des agents pathogènes est d’une très grande importance pour la biosécurité. Les biopuces sont bien adaptées à cet effet, car elles permettent la détection multiplexe des cibles. Une limitation cruciale des biopuces est leur manque de fiabilité et de sensibilité. L’objectif de cette thèse est de développer une architecture reproductible de biopuces à base de couche mince de silicium amorphe carboné (a-SiC:H) déposée sur un réflecteur en aluminium pour une détection fiable et sensible des pathogènes. Nous avons choisi comme système modèle l’interaction de la toxine alimentaire ochratoxine A (OTA) avec son aptamère AntiOTA de longueur 36mer. Les aptamères (simples brins d’ADN) sont de plus en plus utilisés comme sondes en raison de leur grande spécificité et affinité vis-à-vis d’une large gamme de cibles (i.e. protéines, bactéries…). La stratégie de fabrication consiste en un greffage de monocouches organiques d’acides carboxyliques via des liaisons Si-C robustes, suivi de l’accrochage covalent des aptamères par un couplage peptidique. Les processus de greffage ont été mis au point sur silicium cristallin permettant la quantification des couches greffées par spectroscopie infrarouge en mode ATR (Attenuated total reflexion). La quantification IR des interactions OTA – AntiOTA a été montrée pour la première fois sur des surfaces par IR-ATR. La spécificité de l’aptamère a été démontrée en utilisant une molécule chimiquement similaire (warfarin), pour laquelle l’AntiOTA ne montre aucune affinité. Ces protocoles bien contrôlés ont été transférés sur l’architecture de la biopuce a-SiC:H. Les aptamères immobilisés sont hybridés avec des brins complémentaires marqués avec des fluorophores. En présence de l’OTA une déshybridation des brins complémentaires est attendue, conduisant à une diminution du signal fluorescent. Différentes longueurs de brins complémentaires ont été comparées, montrant jusqu’à 13% de diminution due à l’interaction de l’OTA. / Rapid and sensitive detection of pathogenic targets play a crucial role in biosecurity. Biochips are ideal for this, as they allow easy and multiplex detection of targets. A crucial limitation in biochips is that they often suffer from low reliability and sensitivity. The goal of this thesis is to develop a stable and reproducible architecture for biochips based on an amorphous silicon carbon alloy (a-SiC:H) deposited on an aluminium back-reflector for reliable and sensitive detection of pathogens. On these biochips we introduced the interaction of the food and feed toxin ochratoxin A (OTA) with its 36mer aptamer AntiOTA as a model system. Aptamers (single strands of DNA) are ideal as probes for biochips as they display high specificity and affinity towards a wide range of targets (i.e. proteins, bacteria…). The well-controlled multi-step fabrication process consists of the reliable photochemical grafting of acid-terminated organic monolayers on silicon surfaces by robust Si C bonds, which in turn were functionalized with aptamers by stable peptide coupling. Carrying out this process on crystalline silicon allowed monitoring and quantification of every step by infrared spectroscopy (IR-ATR). The interaction OTA – AntiOTA was shown for the first time on surfaces by IR, and an IR in situ calibration allowed the quantification of OTA which was bound by the aptamers on the surface. The specificity of AntiOTA towards OTA was demonstrated by using a chemically similar molecule (warfarin), for which AntiOTA shows no affinity. The well-controlled protocols were transferred to the a-SiC:H biochip. The immobilized aptamers were hybridized with complementary and fluorescent-labeled DNA-strands. In presence of OTA, dehybridization of the complementary strands is expected, resulting in a decrease of fluorescent signal. Different lengths of complementary strands were compared, exhibiting up to 13% signal decrease due to OTA.

No description available.

Biochip technology is highly effective method that allows monitoring of biomaterials e.g., yeast and bacteria at a time in computerized automatic operations. Miniaturized nanostructure p-n junction test sites, which are arranged on a solid substrate, are proposed to sense and count the biomaterials. This PhD thesis reports on the impedance of p-n junction-based Si biochips with gold ring top electrodes and unstructured gold bottom electrodes, which allows for counting target biomaterial in a liquid-filled ring top electrode region. The phosphor and boron implanted biochips prepared in dissimilar annealing and doping conditions have been considered and three different types of top electrodes designed and tested to check the influence of the properties of the top electrode on the biochips to achieve more convenient samples for bio sensing technology. The systematic experiments on p-n junction-based Si biochips fabricated by two different sets of implantation parameters (i.e., biochips PS5 and BS5) are studied and the comparable significant change of impedance characteristics in the biochips in dependence on the number of bacteria suspensions, i.e., Lysinibacillus sphaericus JG-A12, in Deionized water at optical density at 600 nm from OD600 = 4–16 in the electrode ring region is demonstrated. The number of biomaterials and the microscopic images can be linked to the impedance changes of the biochip. The electrical equivalent circuit models for the devices have been proposed by using characterized frequency dependent capacitance and conductance of biochips. While the Nyquist spectrum of the biochips are not compromise on perfect semicircle, combination of constant phase elements with resistor in parallel fashion and series inductor and resistor have been utilized to model the impedance of the biochips. Corresponding parameters i.e., capacitors, resistors and inductors have been extracted from the modeling results and the changes in their values by adding the biomaterials obtained. As the result, the linear relation between the numbers of the biomaterial and the impedance of the biochips has been showed. Furthermore, Deionized water and glucose with yeast (Saccharomyces cerevisiae) at optical density OD600 ranging from 4 to 16 has been put in the ring electrode region of impedance biochips and impedance has been measured in dependence on the added volume (20, 21, 22, 23, 24, 25 µL). Modeled impedance of the biochip reveals a linear relationship between the impedance model parameters and yeast concentration. Presented biochips allow for continuous impedance measurements without interrupting the cultivation of the yeast. A multiparameter fit of the impedance model parameters allows to determine the concentration of yeast cy in the range from cy = 3.3x10^7 to cy = 17x10^7 cells/mL. This work shows that independent on the liquid, DI water or glucose, the change of the impedance model parameters with increasing added volume of the liquid is clearly distinguishable from the change of impedance model parameters with increasing concentration of added yeast in the ring electrode region of the impedance biochips. We also counted bacterial cells of E. coli strain K12 in several-microliter DI water or in several-microliter PBS at the low optical density (OD) range (OD = 0.05–1.08) in contact with the surface of Si-based impedance biochips with ring electrodes by impedance measurements. The multiparameter fit of the impedance data allowed calibration of the impedance data with the concentration cb of the E. coli cells in the range of cb = 0.06 to 1.26 × 10^9 cells/mL. The results showed that for E. coli in DI water and in PBS, the modelled impedance parameters depend linearly on the concentration of cells in the range of cb = 0.06 to 1.26 × 10^9 cells/mL, whereas the OD, which was independently measured with a spectrophotometer, was only linearly dependent on the concentration of the E. coli cells in the range of cb = 0.06 to 0.50 × 10^9 cells/mL. with the help of the newly developed ring electrode structure, the modeled capacitance and resistance parameters of the electrical equivalent circuit describing the p-n junction-based biochips depend linearly on the number of bacteria in the ring top electrode region, which successfully proves the potential performance of p-n junction-based Si biochips in observing the bacterial suspension. The proposed p-n junction-based biochips reveal perspective applications in medicine and biology for diagnosis, monitoring, management, and treatment of diseases.

Studies of biomolecular interactions are of interest for several reasons. Beside basic research, the knowledge gained from such studies is also very valuable in for example drug target identification. Medical care is another area where biomolecules may be used as biomarkers to aid physicians in making correct diagnosis. In addition, the highly specific interactions between antibodies and almost any substance opens up the possibilities to design systems for detection of trace amounts of both biological and non-biological substances within environmental restoration, law enforcement, correctional care, customs service and national security. A biochip, which contains a biologically active material, offers a means of monitoring the molecular interactions in the above applications in a sensitive and specific manner. The biochip is a key component of a biosensor, which also includes components for transforming the interaction events into a human-readable signal. This thesis describes the use of poly(ethylene glycol) (PEG) in biochip design. Two different approaches are presented, the first based on ethylene glycol (EG)-containing alkyl thiol self-assembled monolayers (SAMs) on flat gold and the second on photo-induced graft copolymerisation of PEG-containing methacrylate monomers onto various substrates. The former is a two dimensional system where EG-terminated thiols are mixed with similar thiols presenting tail groups that mimic the explosive substance 2,4,6-trinitrotoluene (TNT). In an immunoassay, the detection limit for TNT was determined to fall in the range 1-10 µg/L. In the second approach, a branched three dimensional biosensor matrix (hydrogel) is proposed. The carboxymethylated (CM) dextran matrix, which is commonly used within the biosensing community, is not always ideal for studies of biointeractions, due to the non-specific binding frequently encountered in work with complex biological solutions and various proteins. To employ PEG, which displays a low non-specific binding of such species, is therefore an interesting option worth investigating. The use of a branched graft polymerised PEG matrix in biosensor applications is novel as compared to previous reports which have focused on linear PEG chains. The latter approach provides, at maximum, one functional group, per surface anchoring point, for immobilisation of sensor elements. Thus, it has the inherited disadvantage that it limits the number of available immobilisation sites. The present PEG matrix contains a large number of functional groups, for immobilisation of sensor elements, per grafting site and offers the potential of improved response upon binding to the analyte as demonstrated in a series of successful sensor experiments. Furthermore, the nature of the process enables easy preparation of matrix patterns and gradients. In a PEG matrix gradient, protein permeability is studied and the capabilities of immobilising proteins are demonstrated. By combining the patterning technique with different monomers in a two-step process, an inert platform, lacking chemical attachment sites, is provided with arrays of spots (with immobilisation capabilities), which are conveniently addressed via microdispensing and used for biosensor purposes. The EG-terminated thiols present another means of generating such inert platforms, a route which is also investigated. To further explore the sensor quality of these spots, the concepts of patterning and gradient formation are combined and studied. / Det är intressant att studera biomolekylära interaktioner av många anledningar. För att kunna bedriva framgångsrik läkemedelsutveckling är det oerhört viktigt att känna till hur olika molekyler samverkar i människokroppen. Inom sjukvården kan biomolekyler användas som biomarkörer, då närvaro av dem eller förändringar av deras koncentrationer är kopplade till sjukdomstillstånd, och därmed hjälper läkaren att ställa rätt diagnos. Dessutom kan de mycket specifika interaktionerna mellan antikroppar och (i princip) valfri substans användas för detektion av spårämnen vid miljösaneringsarbete, gränskontroller, polisarbete, fängelser och arbete med nationell säkerhet. Den här avhandlingen beskriver hur polymeren polyetylenglykol (PEG) kan användas vid design av biochip. Ett biochip är en liten anordning, som kan användas för att detektera specifika molekyler med hjälp av en biologisk interaktion. Traditionellt har PEG använts inom biomaterialsektorn, men återfinns även i hygienartiklar som tvål och tandkräm. Ett annat användningsområde är konservering av bärgade träskepp och i en del litiumjonbatterier ingår PEG som en komponent. Dessutom pågår utveckling av PEG-innehållande skyddsvästar. I det här arbetet används PEG framför allt på grund av sin förmåga att minimera ospecifik inbindning av proteiner, som utgör en stor del av gruppen biomolekyler, till ytor på biochip. Två olika typer av ytbeläggningar, som innehåller den här polymeren, har använts. Den första typen ger mycket tunna (~0.000003 mm), tvådimensionella filmer medan den andra ger en något tjockare (~0.00005 mm), tredimensionell struktur (matris). De tvådimensionella filmerna har använts för att utveckla en sprängämnesdetektor med mycket hög känslighet (detektionsgräns mellan 1-10 ppb). En viktig beståndsdel i detta system är antikroppar riktade mot sprängämnet trinitrotoluen (TNT). Den tredimensionella matrisen är mer generell och kan användas för att studera många olika molekylära interaktioner. Tillverkningsmetoden av matrisen är baserad på belysning med ultraviolett ljus och är därmed lämpad för att skapa mönstrade ytor. Genom att blockera delar av ljusflödet begränsas tillväxten av matrisen till de belysta delarna. På så sätt har bland annat så kallade mikro-arrayer, bestående av mikrometerstora (tusendels millimeter) strukturer i ett regelbundet mönster, tillverkats. Tekniken tillåter även tillverkning av gradienter, där matrisens tjocklek varierar längs med provet, genom att belysa olika delar av provytan olika länge. Genom att undersöka dessa gradienter har information om matrisens genomsläpplighet för proteiner kunnat extraheras. Gradientkonceptet har även kombinerats med mikro-arraytillverkningen och gett möjlighet att studera interaktioner mellan flera olika modellproteiner och deras motsvarande antikroppar i olika tjocka matriser på en och samma yta. Det finns ett stort antal sätt att utnyttja interaktionerna mellan olika molekyler på ett biochip. Ett tilltalande tillvägagångssätt är exempelvis att i en mikro-array binda in olika molekyler som kan fånga kliniskt intressanta biomolekyler, i syfte att skapa en hälsoprofil. Ett sådant biochip skulle ge möjlighet att parallellt detektera eller bestämma koncentrationen av ett stort antal biomolekyler i till exempel en droppe blod. På så sätt kan en diagnos snabbt ställas, kanske till och med utan att patienten behöver uppsöka sjukvården. Den utvecklade PEG-matrisen har god potential att fungera i en sådan applikation.

La microscopie par résonance de plasmon de surface (SPR) à couplage par prisme a vu le jour à la fin des années 60. Le principal avantage de cette technique d'imagerie optique réside dans sa très grande sensibilité à de faibles variations d'indice optique ou d'épaisseurs à la surface d'un métal. De ce fait, le suivi d'interactions biologiques peut se faire en temps réel sans avoir recours à l'utilisation de marqueurs fluorescents ou enzymatiques. Depuis plus de 30 ans, la microscopie SPR s'est imposée comme la technique de référence de biodétection sans marquage. Ses champs d'application vont de la détermination de constantes d'affinité à la détection de bactéries pathogènes, en passant par la biologie cellulaire. Jusqu'à présent, on pensait la résolution spatiale limitée par la longueur de propagation des plasmons de surface. Or, de nombreux exemples ne corroborent pas cette hypothèse. Dans cette thèse, nous montrons qu'à ce phénomène de propagation se rajoute des aberrations optiques induites par l'utilisation d'un prisme pour coupler la lumière et les plasmons de surface. Nous expliquons ainsi pourquoi les résolutions expérimentales étaient souvent bien moins bonnes que celles attendues. Par l'analyse de la formation des images et la quantification des aberrations, nous aboutissons à deux nouvelles configurations optiques optimisées pour la résolution. Nous analysons ensuite quel métal offre le meilleur compromis entre longueur de propagation et sensibilité. Expérimentalement, nous obtenons une résolution comprise entre 1,5 et 4 μm suivant la direction, sur des champs de vision de plusieurs mm2, et ce, avec une sensibilité standard en biodétection. Nous sommes ainsi en mesure d'observer simultanément plusieurs milliers de cellules individuelles, eucaryotes et procaryotes. Finalement, nous développons un prototype dédié au suivi en temps réel de sécrétions de protéines par des cellules immunitaires. Les limites de la microscopie SPR et les solutions qui permettraient de faire aboutir ce type d'étude sont examinées. Des études préliminaires sont aussi menées sur l'amélioration de la détection de bactéries. / Prism-based surface plasmon resonance (SPR) microscopy is an optical imaging technique invented in the late 60s'. Its main advantage lies in its high sensitivity to optical index or thickness variations at a metal surface. Therefore, the monitoring of biological reactions can be performed in real-time without labeling agent such as fluorescence or enzymes. Over the last 30 years, SPR microscopy has become the major technique in label-free biodetection. The field of application range from the determination of affinity constant in biochemistry to the detection of pathogenic bacteria via cellular biology. Until now, the propagation length of the surface plasmons has been considered as the spatial resolution limit. However, many examples do not support this statement. In this PhD thesis, we demonstrate that the resolution is also limited by optical aberrations induced by the prism used to couple light and surface plasmons. Thus, we are able to explain why the experimental resolution was usually worse than the predicted one. The analysis of the image formation and the quantification of aberrations lead us to suggest two new optical configurations optimized for resolution. We also analyze which metal exhibits the better trade-off between propagation length and sensitivity. Experimentally, we obtain a resolution between 1.5 and 4 μm depending on the direction, on field-of-view up to several mm2, and with a standard sensitivity for biodetection (monolayer of DNA). We are then able to observe simultaneously several thousands of individual eukaryote and prokaryote cells. Finally, we develop a prototype dedicated to the real-time monitoring of protein secretion by immune cells. The limits of SPR microscopy and the solutions which could allow this kind of study are discussed. Preliminary results on the improvement of bacterial detection are also presented.

Une pléthore de processus biologiquement pertinents dépend directement de la sécrétion de biomolécules dans le milieu extracellulaire, aux fonctions régulatrices ou composants structurels. L’analyse de processus biologiques complexes nécessite ainsi la mise au point de nouveaux outils de biodétection. Par conséquent, le but de cette thèse est de fournir des stratégies polyvalentes pour la génération de biocapteurs et de biopuces innovants basés sur la Résonance des Plasmons de Surface (SPR). À l’issue de ces travaux, une méthode de photofonctionnalisation indirecte a été mise au point. Ce procédé a permis de générer des micro-réseaux de protéines dans des conditions entièrement aqueuses, et ainsi de préserver la fonctionnalité des protéines greffées. De plus, nous avons créé et évalué une nouvelle biopuce SPR microstructurée pour le suivi en temps réel des sécrétions cellulaires. Cette biopuce microstructurée présente deux phénomènes optiques différents qui peuvent être utilisés pour la détection cellulaire et le suivi de leurs sécrétions. Enfin, de multiples stratégies de fonctionnalisation ont été évaluées pour la conception d’une biopuce SPR nanostructurée à faisceau de fibres optiques. Parmi ces approches, la génération de monocouches photoréactives auto-assemblées a été la plus adaptée à ce système et est en cours d’optimisation. Une fois réalisée, cette biopuce nanostructurée pourrait ouvrir la voie à la poursuite du développement de systèmes prometteurs de biodétection in vivo. / A plethora of biologically relevant processes depends directly on the effective secretion of biomolecules, from regulatory molecules to structural components. Thus, the analysis of complex biological processes requires the development of novel biosensing tools. Therefore, the aim of this thesis is to provide versatile strategies for the generation of innovative biosensors and biochips based on Surface Plasmon Resonance (SPR). As a result from this research, an indirect photofunctionalization method was developed. This procedure allowed the generation of protein microarrays in fully aqueous conditions while preserving the functionality of the grafted proteins. Furthermore, we created and evaluated a novel microstructured SPR biochip for real-time monitoring of cellular secretions. This microstructured biochip presents two different optical phenomena which could be used for cell detection and the monitoring of their secretions. Finally, multiple functionalization strategies were evaluated for the conception of a nanostructured fiber-bundle SPR biochip. Among the approaches, the generation of photoreactive self-assembled monolayers was the most adapted to this system and currently is being optimized. Once achieved, this nanostructured biochip could pave the way for further development of promising in vivo biosensing systems.

Le domaine du diagnostic moléculaire connait un essor impressionnant depuis plusieurs dizaines d’années. Différents outils d’analyse d’interactions moléculaires sont présents sur le marché. La plupart d’entre eux sont basés sur des tests immunologiques utilisant la fluorescence comme technique de lecture. Or, l’utilisation de techniques de détection avec marquage comme la fluorescence augmente le coût d’une analyse et peut dénaturer un échantillon. Dans cette perspective, une technique de lecture optique sans marquage, qui est une alternative à la fluorescence, a été développée. Le principe de lecture est basé sur le suivi des modifications du spectre de diffraction de réseaux périodiques, composés de molécules sondes, lors d’interactions avec différentes solutions à analyser. Cette thèse CIFRE est le fruit d'une collaboration entre le LAAS CNRS et la société Innopsys, spécialisée dans la commercialisation d'outils de lecture optique. Elle porte sur le développement d’une plateforme dédiée à l’analyse biomoléculaire (ADN, protéines) au travers de l’utilisation de biopuces multiplexées et d’un instrument de lecture optique sans marquage automatisée. Nous montrons que cette technologie de biodétection sans marquage nécessite le développement d’une chimie de surface permettant l’organisation de molécules sondes en réseaux de lignes périodiques, tout en minimisant l’adsorption non-spécifique entre les lignes. Nous présentons l’optimisation d’un procédé de bi-fonctionnalisation de surface, qui met en jeu un dépôt multiplexé par microcontact printing sur des couches de polymères passivantes. Ces surfaces structurées à l’échelle moléculaire ont permis la détection d’interactions protéines/protéines sans marquage et le concept semble également transférable pour la détection d’hybridation de courtes séquences d’ADN / Development of bioassays has become the matter of intense research in the field of molecular diagnostic. Biodetection techniques have been drastically used in laboratory since the past 20 years and tend now to reach the in-vitro diagnostic industry. Most commercially available biosensing methods rely on immunoassays and use fluorescence as reading technique. However, the use of labeling methods such as fluorescence increases the cost of a single bioassay and may interfere with the biological functions of molecules. In this perspective, we have developed an optical label-free technique of microarray reading, which is an alternative to fluorescence. In this work, we use a label free biosensing method based on the diffraction of light by molecular gratings. Molecular gratings are employed as diffractive probe arrays for protein interaction analysis, as the diffraction efficiency changes in response to analyte binding. This Ph.D is supported by the French company Innopsys, which provides optical solutions for microarray reading and the Nanobiosystems group at the LAAS-CNRS. This work deals with the development of a detection platform for biomolecular interactions analysis, through the use of multiplexed biochips and the validation of an optical scanner. We present a special surface chemistry, based on blocking layers to reduce the non-specific protein adsorption and consequently decrease the limit of detection. Thanks to bi-functionalized biochips and this label free instrument, we have detected proteins interactions involving low molecular weight molecules