Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequences SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized; one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system. / Anaplasmose, uma infecção intraeritrocitária obrigatória de bovinos, causada pela Anaplasma marginale, causa severa anemia e alta taxa de aborto, resultando em perda significativa para a indústria de laticínios e carne. O diagnóstico clínico é baseado nos sintomas e exames laboratorias confirmatórios são necessários. Atualmente, todos os ensaios de diagnósticos têm sido desenvolvidos com ELISA indireto de antígenos totais baseado em epítopos múltiplos. Em uma investigação pioneira demonstramos o uso de motivos críticos de um epítopo como biomarcador para a aplicação em imunossensores. Mimotopos do epítopo funcional da proteína MSP1a foram obtidos por meio de Phage Display, após três ciclos de seleção, de uma biblioteca randômica de peptídeos 12-mer contra o anticorpo monoclonal 15D2. Trinta e nove clones foram selecionados aleatoriamente, sequenciados, traduzidos e alinhados com a sequência nativa. Foi obtida a sequência consenso SxSSQSEASTSSQLGA, que está localizada na extremidade C-terminal do motivo repetitivo de 28-aa da proteína MSP1a, mas o alinhamento e a variação das sequências entre os mimotopos nos permitiu mapear o motivo crítico STSSxL dentro da sequência consenso. Com base nesses resultados, dois peptídeos foram quimicamente sintetizados; um baseado no motivo crítico (STSSQL, Am1) e o outro baseado na sequência consenso alinhada com o epítopo nativo (SEASTSSQLGA, Am2). Soro de 24 animais infectados e 52 animais saudáveis foram testados por ELISA quanto à reatividade contra Am1 e Am2, que apresentou sensibilidades de 96% e 100%, respectivamente. O peptide Am1 foi incorporado a um bioeletrodo (grafite modificado com ácido poli-3-hidroxyfenilacético) e a detecção direta de soro foi demonstrada por impedância, voltametria de pulso diferencial e microscopia de força atômica. O sistema de sensor eletroquímico provou ser altamente eficaz em soro de animais positivos de negativos. Este imunossensor foi altamente sensível e seletivo para IgG positiva, contaminantes não afetaram as medições, e foram baseados e um sistema simples, rápido e reprodutível. / Doutor em Genética e Bioquímica

L’objectif général de cette thèse est de développer une plateforme d’immobilisation d’enzymes efficace pour application en biopile. Grâce à la microencapsulation ainsi qu’au choix judicieux des matériaux polymériques pour la fabrication de la plateforme d’immobilisation, l’efficacité du transfert électronique entre l’enzyme encapsulée et l’électrode serait amélioré. Du même coup, les biopiles employant cette plateforme d’immobilisation d’enzymes pourrait voir leur puissance délivrée être grandement augmentée et atteindre les niveaux nécessaires à l’alimentation d’implants artificiels pouvant remplacer des organes telque le pancréas, les reins, le sphincter urinaire et le coeur. Dans un premier temps, le p-phénylènediamine a été employé comme substrat pour la caractérisation de la laccase encapsulée dans des microcapsules de poly(éthylèneimine). La diffusion de ce substrat à travers les microcapsules a été étudiée sous diverses conditions par l’entremise de son oxidation électrochimique et enzymatique afin d’en évaluer sa réversibilité et sa stabilité. La voltampérométrie cyclique, l’électrode à disque tournante (rotating disk electrode - RDE) et l’électrode à O2 ont été les techniques employées pour cette étude. Par la suite, la famille des poly(aminocarbazoles) et leurs dérivés a été identifée pour remplacer le poly(éthylèneimine) dans la conception de microcapsules. Ces polymères possèdent sur leurs unités de répétition (mono- ou diamino) des amines primaires qui seraient disponibles lors de la polymérisation interfaciale avec un agent réticulant tel qu’un chlorure de diacide. De plus, le 1,8-diaminocarbazole (unité de répétition) possède, une fois polymérisé, les propriétés électrochimiques recherchées pour un transfert d’électrons efficace entre l’enzyme et l’électrode. Il a toutefois été nécessaire de développer une route de synthèse afin d’obtenir le 1,8-diaminocarbazole puisque le protocole de synthèse disponible dans la littérature a été jugé non viable pour être utilisé à grande échelle. De plus, aucun protocole de synthèse pour obtenir du poly(1,8-diaminocarbazole) directement n’a été trouvé. Ainsi, deux isomères de structure (1,6 et 1,8-diaminocarbazole) ont pu être synthétisés en deux étapes. La première étape consistait en une substitution électrophile du 3,6-dibromocarbazole en positions 1,8 et/ou 1,6 par des groupements nitro. Par la suite, une réaction de déhalogénation réductive à été réalisée en utilisant le Et3N et 10% Pd/C comme catalyseur dans le méthanol sous atmosphère d’hydrogène. De plus, lors de la première étape de synthèse, le composé 3,6-dibromo-1-nitro-carbazole a été obtenu; un monomère clé pour la synthèse du copolymère conducteur employé. Finalement, la fabrication de microcapsules conductrices a été réalisée en incorporant le copolymère poly[(9H-octylcarbazol-3,6-diyl)-alt-co-(2-amino-9H-carbazol-3,6-diyl)] au PEI. Ce copolymère a pu être synthétisé en grande quantité pour en permettre son utilisation lors de la fabrication de microcapsules. Son comportement électrochimique s’apparentait à celui du poly(1,8-diaminocarbazole). Ces microcapsules, avec laccase encapsulée, sont suffisamment perméables au PPD pour permettre une activité enzymatique détectable par électrode à O2. Par la suite, la modification de la surface d’une électrode de platine a pu être réalisée en utilisant ces microcapsules pour l’obtention d’une bioélectrode. Ainsi, la validité de cette plateforme d’immobilisation d’enzymes développée, au cours de cette thèse, a été démontrée par le biais de l’augmentation de l’efficacité du transfert électronique entre l’enzyme encapsulée et l’électrode. / The main objective of this thesis is the development of a conductive enzyme immobilisation template for laccase through microencapsulation allowing an efficient electron transfer between the enzyme and the electrode for application in biofuel cells. First, p-phenylenediamine was used as substrate for the characterisation of the microencapsulated laccase. The diffusion of this substrate through the microcapsules was studied under various conditions by means of its electrochemical and enzymatic oxidation processes in order to assess its reversibility and stability. Cyclic voltammetry, rotating disk electrode and Clark electrode were the techniques used in this study. Moreover, poly(aminocarbazole) compounds and their derivatives were identified to replace poly(ethyleneimine) in the fabrication of the microcapsules. These polymers exhibit primary amines (mono- or di-amino) that could be available for an interfacial polymerisation using a cross-linker agent. Also, the monomer 1,8-diaminocarbazole presents the desired electrochemical propreties for an efficient electron transfer between the enzyme and the electrode. Therefore, a synthetic pathway was developed in order to synthesise this monomer since the available literature protocol was considered inappropriate for large scale synthesis. As for the direct synthesis of the poly(1,8-diaminocarbazole), to our knowledge, there is no protocol currently available. Two structural isomers (1,6 and 1,8-diaminocarbazole) were thus synthesised in two steps. The first step consists in the electrophilic substitution of 3,6-dibromocarbazole in 1,8 and/or 1,6 positions by nitro groups. This step was followed by a dehydrodehalogenation reaction that comes along with reduction of nitro to amino functions using Et3N and 10% Pd/C as the catalyst in methanol under H2 flux. During the first step, the 3,6-dibromo-1-nitro-carbazole was also obtained and appeared to be an efficient monomer in the synthesis of the desired conductive copolymer. Finally, the fabrication of the conductive microcapsules was realised by adding the copolymer poly[(9H-octylcarbazol-3,6-diyl)-alt-co-(2-amino-9H-carbazol-3,6-diyl)] to the PEI. This copolymer was synthesised in large quantities, which allowed its use in the design of microcapsules. Its electrochemical behaviour was similar in many ways to the one of poly(1,8-diaminocarbazole). These conductive microcapsules were then used to modify the surface of a platinum electrode to fabricate the bioelectrode. The main objective of this project was achieved through this final step.

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