Le développement des technologies de recombinaison en biologie moléculaire fut un point tournant pour les sciences biologiques. Depuis cette découverte, diverses avancées extraordinaires qui ont un impact direct sur les humains ont pu être accomplies dans les domaines de recherches qui découlent de cette technologie. L’étude des enzymes produites en utilisant cette technique est le fondement de leurs applications éventuellement accessibles. À cet effet, la biocatalyse est un sous-domaine de l’enzymologie en développement continuel. Les chimistes et ingénieurs utilisent les composantes de systèmes biologiques ou même des systèmes complets afin de complémenter ou remplacer des méthodologies existantes. Cette thèse étudie la famille d’enzymes transglutaminase (TGase) comme biocatalyseur afin d’explorer et d’étendre l’ubiquité et les innovations rendues possibles grâce aux enzymes. Les TGases sont des enzymes versatiles. Leur homologue bactérien, la transglutaminase bactérienne (MTG), est couramment utilisé à l’échelle industrielle pour la transformation alimentaire. Depuis une dizaines d’années, de nombreux efforts ont été faits afin de trouver de nouvelles applications des TGases. En premier lieu, une revue des accomplissements, progrès et défis reliés au développement des TGases sera décrite. Les TGases sont intrinsèquement des catalyseurs de la formation de lien isopeptidiques entre une glutamine et une lysine. Par ce fait, elles ont été initialement testées dans cette thèse pour la synthèse de peptides. Une forme de l’enzyme TGase de mammifères fut en mesure de générer les composés dipeptidiques Gly-Xaa et D-Ala-Gly avec une faible conversion. La MTG possède plusieurs caractéristiques qui font de cette enzyme un candidat intéressant pour le développement de biotechnologies. Elle est stable, non dépendante d’un cofacteur et connait peu de compétition pour sa réaction catalytique inverse. La majeure partie de cette thèse porte exclusivement sur l’utilisation de la MTG. Nous avons développé et caractérisé une réaction chimio-enzymatique en un seul pot pour la conjugaison de peptides et protéines. La présence de glutathion en quantité suffisante permet de contourner l’incompatibilité de la MTG avec le cuivre et ouvre la porte à l’utilisation de la réaction de cycloaddition entre un alcyne et un azoture catalysée par le cuivre, afin d’effectuer le marquage fluorescent de protéines. L’utilisation d’autres méthodes de chimie « click » sans métaux fut aussi étudiée afin d’incorporer divers substrats protéiques. Le marquage de protéines avec la MTG fut investigué de manière combinatoire. Précisément, la ligation de Staudinger, la cycloaddition azoture-alcyne promue par la tension de cycle, ainsi que la ligation de tetrazine (TL) ont été testées. Différents niveaux de conversion ont été atteints, le plus prometteur étant celui obtenu avec la TL. Une étude par cristallographie a été effectuée afin d’élucider comment les substrats contenant une glutamine interagissent avec la MTG. Une méthode de purification alternative de la MTG a été développée afin d’atteindre ce but. Une discussion sur les stratégies et défis est présentée. Finalement, la conjugaison entre un système contenant la MTG comme biocatalyseur de marquage, le domaine B1 de la protéine G (GB1) comme substrat et d’un fluorophore contenant une amine comme sonde fut étudié. Comme deux des constituants de ce système sont des protéines, l’ingénierie d’enzyme peut être entreprise afin d’améliorer leurs propriétés. Une banque de 24 variantes de GB1 fut construite grâce à une approche semi-rationnelle afin d’investiguer quels facteurs sont déterminants pour la sélectivité de la MTG envers la glutamine. Chaque variante étudiée comportait une seule glutamine à une position variable afin d’évaluer l’impact des éléments de structure secondaire où se retrouve la glutamine. L’efficacité pour le marquage a pu être améliorée d’au moins un ordre de grandeur pour huit des substitutions étudiées. Comme chacune des structures secondaires fut marquée, il fut démontré que la MTG n’en préfère pas une en particulier. De plus, la réactivité de la MTG envers la variante I6Q-GB1 fut augmentée en créant des mutations dans son site actif. Ces résultats permettent de comprendre d’avantage la sélectivité de la MTG envers la glutamine, tout en démontrant le potentiel de cette enzyme à être modifiée afin d’être améliorée. / The development of recombinant molecular biology technologies was a turning point for the biological sciences, which has since evolved into dozens upon dozens of different subfields and contributed to extraordinary advances for humans. At the core of many of these advances are the enzymes produced by these techniques, with efforts to understand their form and function laying the groundwork for their application. One of these continuously advancing subfields rooted in enzymology is biocatalysis, in which chemists and engineers embrace biological components and systems to complement, or even replace, existing methodologies. This thesis seeks to further contribute to the advancement and ubiquity of enzymes to be incorporated into future innovations. To this end, transglutaminase (TGase) is the biocatalyst selected for study. TGases are versatile enzymes, with the bacterial homolog, microbial transglutaminase (MTG) being readily used in industrial processes for years, particularly for food processing. An abundance of efforts seeking to apply TGases to other processes have been made within the last decade. We commence by reviewing the accomplishments, progress, and challenges to developing TGase towards new goals. TGase naturally catalyzes the formation of isopeptide bonds utilizing a glutamine and lysine substrates, and one of its first unconventional applications we investigated was for peptide synthesis. We determined the ability and specificity of one form of TGase for various amino acid-derived substrates, observing the formation of Gly-Xaa and D-Ala-Gly dipeptide products, albeit at a low conversion. MTG exhibits several characteristics that make it an appealing candidate for biotechnological development, such as its independence from a cofactor, little competition for its reverse catalytic reaction, and increased stability relative to mammalian TGases. Therefore, the remainder of this thesis pertains exclusively to MTG. We developed and extensively characterized a one-pot chemoenzymatic peptide and protein conjugation scheme. The presence of sufficient glutathione circumvents the incompatibility of the copper-catalyzed azide-alkyne cycloaddition with MTG owing to the presence of copper. We ultimately utilized this chemoenzymatic conjugation scheme for fluorescent protein labeling. We continue to expand upon combinatorial methods to undertake protein labeling by investigating to what extent metal-free click chemistries can be utilized in combination with MTG. Specifically, the Staudinger ligation, strain-promoted azide-alkyne cycloaddition, and tetrazine ligation (TL) were assayed on protein substrates to reveal varying levels of effective conjugation, with the TL being the most promising of the three. The details surrounding the manner in which MTG interacts with its glutamine-containing substrate remains unclear. To address this knowledge gap, we sought to pursue crystallography studies, which required the development a modified purification strategy. We discuss the strategies we investigated and the challenges surrounding such efforts. Finally, we present a conjugation system consisting of MTG as the labeling biocatalyst, the B1 domain of Protein G (GB1) as a substrate, and a small-molecule amine belonging to a recently developed class of fluorophores as a probe. As two components of this system are proteins, enzyme engineering can be applied to further improve their properties. A semi-rational approach was used to generate a 24-member GB1 library to probe the structural determinants of MTG’s glutamine selectivity. Each variant contained a single glutamine at varying positions covering all secondary structure elements, and assayed for reactivity. Eight substitutions resulting in an increased labeling efficiency of at least an order of magnitude were distributed throughout all secondary structure elements, indicating that MTG does not favor one preferentially. In addition, introducing point mutations within MTG’s active site also resulted in increased reactivity towards variant I6Q-GB1. Our results contribute further to understanding the nature of MTG’s glutamine selectivity, while simultaneously demonstrating the potential enzyme engineering has to improve and adjust this system.

Tato práce navrhuje techniku separace a detekce na čipu pro kvantové tečky (QD, „quantum dots“) konjugované s různými proteiny, za účelem sledování vlivu vazebného činidla na potlačení intenzity uorescence QD způsobené konjugací s proteinem a za účelem provedení multianalytické imunoanalýzy k identifikaci malých množství daného proteinu. Za optimálních podmínek byly biokonjugované QD úspěšně odděleny od těch nezkonjugovaných během 10 minut. Částice a cílové roztoky byly smíchány a detekce na čipu byla provedena za pomoci zařízení vyvinutého v naší laboratoři. Byl použit pouze jeden zdroj excitačního světla v kombinaci s několika filtry pro různé emisní vlnové délky. Fluorescence emitovaná dvěma typy konjugovaných QD mohla být poté zaznamenána současně, protože QD emitovaly světlo na různých vlnových délkách, ačkoliv byly excitovány při stejné vlnové délce. Smícháním dvou typů QD biokonjugovaných se dvěma druhy proteinů a protilátek jsme dokázali detekovat imunokomplexní píky s různými plochami. Plocha pod píkem závisela na koncentraci QD a antigenů, na postupu reakcí protilátka–antigen a ukázalo se, že je lineárně korelována s koncentrací antigenu. Ukázali jsme, že kapilární elektroforéza QD na čipu může být použita jako citlivá technika pro detekci biologických molekul. Hlavními výhodami této metody jsou jednoduchost, malé požadavky na objem vzorku i činidla a také vysoká účinnost separace.

Les protéines modifiées ont suscité un grand intérêt en raison de leur taille extrêmement petite par rapport à l'anticorps entier. Ces petites protéines de liaison ont démontré de nombreux avantages tels qu'une bio distribution rapide, une bonne pénétration dans le tissu tumoral et une élimination rapide du sérum et des tissus non-infectés. Ainsi, ces protéines devraient être d'excellentes alternatives aux anticorps pour les applications cliniques. Cette thèse présente le développement de biocapteurs basés sur des anticorps synthétiques et le transfert d'énergie par résonance de type Förster (FRET) résolu en temps par la détection de biomarqueurs. Les tests immunologiques à base de FRET sont établis en utilisant des complexes de terbium (Tb) comme donneurs de FRET et des boîtes quantiques semi-conducteurs (QDs) comme accepteurs de FRET. Les propriétés photophysiques exceptionnelles de ce couple de FRET Tb-QD permettent une détection quantitative ultrasensible. Des anticorps monocaténaires (single-domain antibody, sdAb) et des petites protéines d’affinité synthétiques (albumin-binding domain-derived affinity protein, ADAPT) sont utilisés pour étudier différentes stratégies de conjugaison d'anticorps, et quantifier des biomarqueurs cliniques (EGFR, HER2). Ce travail peut être considéré comme une condition préalable à l’utilisation des QDs en diagnostic clinique. / Engineered affinity proteins have raised great interest due to their extremely small size compared to full length antibodies. Such small binding proteins have demonstrated many advantages such as quick biodistribution, good penetration into tumor tissue, and fast elimination from serum and nondiseased tissues. Thus, they are expected to be excellent alternatives to antibodies for clinical applications. This thesis focuses on the development of biosensors based on engineered antibodies and time-resolved Förster resonance energy transfer (FRET) through biological recognition of biomarkers. FRET-based immunoassays are established using terbium complexes (Tb) as FRET donors and semiconductor quantum dots (QDs) as FRET acceptors. The exceptional photophysical properties of the Tb-QD FRET pair allow for ultrasensitive quantitative biosensing. Single-domain antibodies (sdAb) and small engineered scaffold antibodies (ADAPT) are used to investigate different antibody-conjugation strategies for quantifying human epidermal growth factor receptors (EGFR, HER2) as clinical biomarkers. This work can be considered as a prerequisite to implementing QDs into applied clinical diagnostics.

No description available.

A range of microgels with two different functionalities were synthesized, and their utility in novel bioapplications was examined. Cationic microgels with varying properties were developed by tuning synthesis conditions. Their size and primary amine content was analyzed, and one microgel system was selected as a model construct. Its primary amine groups were conjugated to two dyes with properties favorable for utilization as contrast agents in photoacoustic imaging. The concentration of contrast agent in single particles was determined. The implications of a high local dye concentration in the generation of high intensity photoacoustic signals, are discussed. The second bioapplication involved the targeted delivery of fibrinolytics to fibrin clots, in order to bring about dissolution of abnormal thrombi. For this purpose, core/shell microgels with carboxylic acid groups in their shells were synthesized in three size ranges. Following this, their dimension based differential localization in and around porous fibrin clots was examined. Fibrin-specific peptides were then conjugated onto the shells of these particles and the conjugates were shown to demonstrate strong interactions with the fibrin clots. The microgels conjugated to the peptide with the highest binding affinity to fibrin, were observed to bring about disruption of fibrin clots, merely through interference in the dynamic interactions among clot fibers, due to the equilibrium nature of the fibrin polymer. The implications of these novel results and future studies required to facilitate a better understanding of the phenomena involved, are discussed.

Ce travail de thèse porte sur la synthèse de (co)polymères thermosensibles présentant une fonctionnalité azlactone par polymérisation radicalaire contrôlée RAFT pour l’ancrage de biomolécules. Trois stratégies différentes ont été étudiées. La première stratégie a consisté en la synthèse d’un nouvel agent de transfert permettant d’obtenir des polymères thermosensibles à fonctionnalité azlactone en position . La seconde approche a permis d’introduire la fonctionnalité azlactone en position ω de copolymères thermosensibles via la combinaison de la polymérisation RAFT et de l’addition de Michaël « thiol-ène ». La dernière stratégie a conduit à des copolymères thermosensibles à fonctionnalité azlactone en position latérale par copolymérisation RAFT de la 2-vinyl-4,4-diméthylazlactone avec d’autres monomères. Enfin, la réactivité de ces copolymères thermosensibles pour l’ancrage d’une protéine modèle (lysozyme) a été mise en évidence. / The synthesis of well-defined azlactone-functionalized thermoresponsive copolymers was performed using the RAFT polymerization and their interaction with primary amines and proteins was studied. Three different strategies have been developed. The first strategy was based on the synthesis of a novel azlactone-functionalized chain transfer agent which was used to target well defined  azlactone-functionalized thermoresponsive polymers. In the second approach, ω-azlactone-terminated thermoresponsive copolymers were prepared by a combination of RAFT polymerization and “thiol ene” Michaël’s addition. In the last strategy, RAFT copolymerization of 2-vinyl-4,4 dimethylazlactone with other monomers has been performed to target well-defined azlactone functionalized copolymers. Finally, the reactivity of such reactive thermoresponsive copolymers was successfully demonstrated by bioconjugation with a model protein (lysozyme).

Esta tese descreve o desenvolvimento e aplicação de sensores particulados (Parte A) e nanomecânicos (Parte B). Nanopartículas de poliestireno (PS) decoradas com Poli (etileno glicol), PEG, foram sintetizadas e caracterizadas antes e após adsorção do corante vermelho do Congo (VC). Colesterol oxidase (Chox), a principal enzima na oxidação do colesterol, foi imobilizada sobre PS/PEG e PS/PEG/VC para gerar sensores de colesterol. A bioconjugação entre VC e Chox permitiu respostas lineares para dosagem de colesterol HDL presente em amostras de sangue artificial. A lipase, uma hidrolase com larga aplicação científica e industrial, também foi imobilizada sobre PS/PEG e PS/PEG/VC. Os parâmetros cinéticos da hidrólise de p-nitrofenil butirato determinados para lipase livre e lipase imobilizada mostraram que sobre PS/PEG/VC a velocidade máxima de reação (Vmax) e número de renovação (kcat) aumentaram em comparação com os valores determinados para enzima livre. Lipase imobilizada sobre PS/PEG/VC pôde ser reutilizado por até sete vezes, perdendo um máximo de 10% ou 30% da atividade enzimática original a 40 °C ou 25 °C, respectivamente. Estes efeitos foram atribuídos à bioconjugação entre lipase e VC. Os comportamentos catalíticos da lipase e da Chox na ausência e na presença de grafeno (G) ou grafeno oxidado (GO) foram sistematicamente investigados. Na presença de G, ambas enzimas apresentaram valores de Vmax e de kcat superiores aos das enzimas livres. Já na presença de GO, Chox não apresenteou atividade e lipase apresentou Vmax e de kcat superiores aos da enzima livre. Estes resultados foram atribuídos à bioconjugação com as partículas de G e GO e grupos hidrofílicos presentes no plano basal de GO. Sensores nanomecânicos foram desenvolvidos a partir de microcantileveres (MC). Respostas nanomecânicas frente à variação de umidade relativa do meio foram detectadas utilizando filmes finos de Poli (hidroxietil metacrilato), PHEMA, com três diferentes massas molares médias depositados sobre MC de silício puros, um substrato hidrofílico, e sobre MC revestidos com PS, um substrato hidrofóbico. Os resultados demonstraram que as respostas nanomecânicas dependem não só do tamanho de cadeia de PHEMA, mas também da camada de água interfacial entre PHEMA e MC. Transdução nanomecânica foi usada para detectar e dosar a presença do biomarcador antígeno carcinoembrionário (CEA) presente em amostras de soro. CEA é um biomarcador de interesse clínico-diagnóstico para acompanhamento e prognóstico de câncer de cólon. O biomarcador é primeiro reconhecido pelo anticorpo (MAb 3C1) ancorado a uma nanopartícula de ouro, e posteriormente reconhecido pelo anticorpo (MAb 3C6) ancorado em um MC de silício, o qual serve como um ressonador mecânico de massa de nanopartículas de ouro capturadas. O biosensor desenvolvido é capaz de dosar CEA sérico em concentrações traços, correspondente a 1,0 10-16 g ml-1. / This thesis reports the development and application of particulate (Part A) and nanomechanics sensors (Part B). Poly (ethylene glycol), PEG, decorated polystyrene (PS) nanoparticles were synthesized and characterized before and after adsorption of the dye Congo red (CR). Cholesterol oxidase (Chox), the key enzyme in the oxidation of cholesterol, was immobilized onto PS/PEG and PS/PEG/CR particles for generating cholesterol sensors. The bioconjugation between CR and Chox allowed linear responses for HDL cholesterol content in artificial blood samples. Lipase, a hydrolase with large scientific and industrial applications, was also immobilized onto PS/PEG and PS/PEG/VC nanoparticles. The kinetic parameters of the hydrolysis of p-nitrophenyl butyrate were determined for free lipase and immobilized onto PS/PEG/CR particles. The results showed that maximum reaction velocity (Vmax) and catalytic efficiency (kcat) increased compared to the values determined for the free enzyme. Lipase immobilized onto PS/PEG/CR particles could be recycled seven times, losing maximum 10% or 30% of the original enzymatic activity at 40 °C or 25 °C, respectively. These effects were attributed to bioconjugation between lipase and CR. Catalytic behavior of lipase and Chox in the absence and in the presence of graphene (G) or graphene oxide (GO) was systematically investigated. In the presence of G, both enzymes showed Vmax and kcat values higher than free enzymes. On the other hand, ChOx was inactive and the interactions between GO and lipase showed Vmax and kcat values higher than those of the free enzyme. These results were attributed to bioconjugation of G and GO particles and hydrophilic groups present in the basal plane of GO. Nanomechanical sensors were developed from microcantilevers (MC). Nanomechanical answers against the relative humidity variation of the medium were detected using thin films of poly (hydroxyethyl methacrylate), PHEMA, with three different average molecular masses deposid onto bare silicon microcantilevers, a hydrophilic substrate, and onto polystyrene (PS) coated microcantilevers, which is a hydrophobic substrate. The results found in the present study demonstrate that the micromechanical responses observed are related not only to the polymer molecular weight, but also to the polymer-interface phenomena and environment-polymer interface. Nanomechanics transductions were used to dose and detect the presence of carcinoembryonic antigen in serum samples. CEA is a biomarker of clinical diagnostic interest for monitoring and prognose of colon cancer. Biomarker is first recognized by a surface-anchored antibody to a gold nanoparticle (MAB 3C1) and later recognized by a surface-anchored antibody to a silicon MC (MAB 3C6), that acts as a mechanical resonator for \'weighing\' the mass of the captured nanoparticles. The biosensor developed is able to dose serum CEA in trace concentrations, corresponding to 1.0 10-16 g ml-1.

Esta tese descreve o desenvolvimento e aplicação de sensores particulados (Parte A) e nanomecânicos (Parte B). Nanopartículas de poliestireno (PS) decoradas com Poli (etileno glicol), PEG, foram sintetizadas e caracterizadas antes e após adsorção do corante vermelho do Congo (VC). Colesterol oxidase (Chox), a principal enzima na oxidação do colesterol, foi imobilizada sobre PS/PEG e PS/PEG/VC para gerar sensores de colesterol. A bioconjugação entre VC e Chox permitiu respostas lineares para dosagem de colesterol HDL presente em amostras de sangue artificial. A lipase, uma hidrolase com larga aplicação científica e industrial, também foi imobilizada sobre PS/PEG e PS/PEG/VC. Os parâmetros cinéticos da hidrólise de p-nitrofenil butirato determinados para lipase livre e lipase imobilizada mostraram que sobre PS/PEG/VC a velocidade máxima de reação (Vmax) e número de renovação (kcat) aumentaram em comparação com os valores determinados para enzima livre. Lipase imobilizada sobre PS/PEG/VC pôde ser reutilizado por até sete vezes, perdendo um máximo de 10% ou 30% da atividade enzimática original a 40 °C ou 25 °C, respectivamente. Estes efeitos foram atribuídos à bioconjugação entre lipase e VC. Os comportamentos catalíticos da lipase e da Chox na ausência e na presença de grafeno (G) ou grafeno oxidado (GO) foram sistematicamente investigados. Na presença de G, ambas enzimas apresentaram valores de Vmax e de kcat superiores aos das enzimas livres. Já na presença de GO, Chox não apresenteou atividade e lipase apresentou Vmax e de kcat superiores aos da enzima livre. Estes resultados foram atribuídos à bioconjugação com as partículas de G e GO e grupos hidrofílicos presentes no plano basal de GO. Sensores nanomecânicos foram desenvolvidos a partir de microcantileveres (MC). Respostas nanomecânicas frente à variação de umidade relativa do meio foram detectadas utilizando filmes finos de Poli (hidroxietil metacrilato), PHEMA, com três diferentes massas molares médias depositados sobre MC de silício puros, um substrato hidrofílico, e sobre MC revestidos com PS, um substrato hidrofóbico. Os resultados demonstraram que as respostas nanomecânicas dependem não só do tamanho de cadeia de PHEMA, mas também da camada de água interfacial entre PHEMA e MC. Transdução nanomecânica foi usada para detectar e dosar a presença do biomarcador antígeno carcinoembrionário (CEA) presente em amostras de soro. CEA é um biomarcador de interesse clínico-diagnóstico para acompanhamento e prognóstico de câncer de cólon. O biomarcador é primeiro reconhecido pelo anticorpo (MAb 3C1) ancorado a uma nanopartícula de ouro, e posteriormente reconhecido pelo anticorpo (MAb 3C6) ancorado em um MC de silício, o qual serve como um ressonador mecânico de massa de nanopartículas de ouro capturadas. O biosensor desenvolvido é capaz de dosar CEA sérico em concentrações traços, correspondente a 1,0 10-16 g ml-1. / This thesis reports the development and application of particulate (Part A) and nanomechanics sensors (Part B). Poly (ethylene glycol), PEG, decorated polystyrene (PS) nanoparticles were synthesized and characterized before and after adsorption of the dye Congo red (CR). Cholesterol oxidase (Chox), the key enzyme in the oxidation of cholesterol, was immobilized onto PS/PEG and PS/PEG/CR particles for generating cholesterol sensors. The bioconjugation between CR and Chox allowed linear responses for HDL cholesterol content in artificial blood samples. Lipase, a hydrolase with large scientific and industrial applications, was also immobilized onto PS/PEG and PS/PEG/VC nanoparticles. The kinetic parameters of the hydrolysis of p-nitrophenyl butyrate were determined for free lipase and immobilized onto PS/PEG/CR particles. The results showed that maximum reaction velocity (Vmax) and catalytic efficiency (kcat) increased compared to the values determined for the free enzyme. Lipase immobilized onto PS/PEG/CR particles could be recycled seven times, losing maximum 10% or 30% of the original enzymatic activity at 40 °C or 25 °C, respectively. These effects were attributed to bioconjugation between lipase and CR. Catalytic behavior of lipase and Chox in the absence and in the presence of graphene (G) or graphene oxide (GO) was systematically investigated. In the presence of G, both enzymes showed Vmax and kcat values higher than free enzymes. On the other hand, ChOx was inactive and the interactions between GO and lipase showed Vmax and kcat values higher than those of the free enzyme. These results were attributed to bioconjugation of G and GO particles and hydrophilic groups present in the basal plane of GO. Nanomechanical sensors were developed from microcantilevers (MC). Nanomechanical answers against the relative humidity variation of the medium were detected using thin films of poly (hydroxyethyl methacrylate), PHEMA, with three different average molecular masses deposid onto bare silicon microcantilevers, a hydrophilic substrate, and onto polystyrene (PS) coated microcantilevers, which is a hydrophobic substrate. The results found in the present study demonstrate that the micromechanical responses observed are related not only to the polymer molecular weight, but also to the polymer-interface phenomena and environment-polymer interface. Nanomechanics transductions were used to dose and detect the presence of carcinoembryonic antigen in serum samples. CEA is a biomarker of clinical diagnostic interest for monitoring and prognose of colon cancer. Biomarker is first recognized by a surface-anchored antibody to a gold nanoparticle (MAB 3C1) and later recognized by a surface-anchored antibody to a silicon MC (MAB 3C6), that acts as a mechanical resonator for \'weighing\' the mass of the captured nanoparticles. The biosensor developed is able to dose serum CEA in trace concentrations, corresponding to 1.0 10-16 g ml-1.

In this thesis will be described research towards the development of bioorthogonal bond-cleavage reactions, and their applications in targeted drug delivery (Figure 1). The first project relates to the development of a palladium mediated bond-cleavage or "decaging" reaction which can cause a propargyl carbamate to decompose and release an amine. This was further developed by the incorporation of a protein modification handle which allowed an amine-bearing drug to be covalently ligated to a protein by a palladium-cleavable linker. This chemistry was demonstrated by the conjugation of the anticancer drug doxorubicin to a tumour targeted anti-HER2 nanobody. The drug could then be delivered to cancer cells upon addition of a palladium complex. The second project relates to the development of a platinum mediated bond-cleavage reaction. This was developed with the aim of using platinum-containing anticancer drugs - such as cisplatin - as a catalyst to cause drug release reactions in tumours. In this reaction an alkyne-containing amide can decompose to release an amine upon addition of platinum complexes, and was applied to the release of prodrugs of the cytotoxins monomethylauristatin E and 5-fluorouracil in cancer cells. A cisplatin-cleavable antibody-drug conjugate was designed and synthesised, and progress towards its biological evaluation will be discussed.

A cerebral aneurysm is a weakened area of an artery in the brain, creating an abnormal expansion. Recent research for treatment is utilizing a photopolymerizable hydrogel as a possible operation for injection in situ. This paper aimed to achieve bioconjugation of peptides on a PEGDMA polymer network (using the photoinitiator PEG-BAPO) to form a biocompatible photopolymerizable hydrogel, without compromise to any of its mechanical attributes. Achieving cell adhesion to the hydrogel surface is a critical requirement as that could drive the growth of endothelium between aneurysm and artery, to considerably enhance its sustainability and decrease the risk of inflammation. The hydrogel was synthesized by functionalizing RGD with a PEG-spacer and co-polymerize it with PEGDMA using UV-radiation to create an intertwined cross-linking network. Samples of various peptide concentrations were studied in cell culture to analyze cell adhesion, followed by mechanical tests to identify possible deviations. A subsequent study was established to create a dynamic prototype as a quantifiable replication of a hydrogel inside an aneurysm in vivo. The model was designed in SolidWorks and connected with an Ibidi sticky-Slide to roughly replicate a cerebral aneurysm connected to an artery with space to introduce a hydrogel sample.