Cette thèse décrit le développement de capteurs à fibre optique hautement sélectifs, utilisant des polymères à empreintes moléculaires (MIPs, de l’anglais molecularly imprinted polymers) comme éléments de reconnaissance, et se basant sur la fluorescence pour la détection. Nous avons étendu l’étude à d’autres types de capteurs et de méthodes de détection optiques, toujours basés sur les MIPs. Les MIPs sont des récepteurs synthétiques biomimétiques possédant des cavités spécifiques pour une molécule cible. Produits par un processus de moulage à l’échelle moléculaire, les MIPs sont capables de reconnaître et de se lier à leurs molécules cibles, avec des spécificités et affinités comparables aux récepteurs naturels. De plus, comparé aux récepteurs biologiques, les MIPs sont plus stables, moins chers et plus faciles à intégrer dans les procédés standard industriels de fabrication. Ainsi, les MIPs apparaissent comme une alternative intéressante aux biomolécules entant qu’éléments de reconnaissance dans les biocapteurs. Dans la première partie de la thèse (Chapitre 2), les MIPs ont été synthétisés en tant que micropointe à l’extrémité d’une fibre optique, par polymérisation in-situ induite par un laser en seulement quelques secondes. Les paramètres photoniques et physico-chimiques ont été optimisés pour moduler les propriétés des micro-objets de polymères. Des nanoparticules d’or ont été incorporées dans la micropointe de MIP afin d’induire une exaltation du signal. Afin de prouver l’efficacité de notre capteur, les études initiales ont été réalisées avec un MIP synthétisé avec le Ncarbobenzyloxy- L-phenylalanine (Z-L-Phe) comme template et le dérivé d’acide aminé fluorescentdansyl-L-phenylalanine, comme analyte. La fluorescence a été collectée de l’extérieur au niveau de la micropointe par une fibre optique connectée à un spectrofluorimètre, ou par collection du signal fluorescent ré-émis dans l’un des bras d’une fibre bifurquée en Y. L’analyse fluorescent a pu être détecté à des concentrations de l’ordre du nM. Afin de quantifier les analytes non fluorescents, un monomère fluorescent, possédant un groupe naphthalimide, a été incorporé dans le MIP ; celui-çidéployant une augmentation de la fluorescence quand l’analyte se lie. Utilisant ce système avec un MIP spécifique pour l’herbicide 2,4-D (acide 2,4-dichlorophénoxyacétique), des concentrations aussi basses que 2,5 nM en 2,4-D ont pu être mesurées. Le capteur MIP a également été appliqué à des analytes d’intérêt pour la sécurité alimentaire et le domaine nanostructurées. Cette étude pose les bases pour le développement futur de nanocapteurs et de Dans la dernière partie (Chapitre 4), une méthode d’analyse novatrice, basée sur l’utilisation des MIPs et l’analyse par polarisation de fluorescence, a été développée en vue de permettre la quantification directe et rapide d’analytes dans des échantillons alimentaires et environnementaux. Cette technique a été appliquée avec succès pour détecter des antibiotiques fluoroquinolones dans l’eau du robinet et le lait, en-dessous de leur limite maximale de résidus. En conclusion, nous pouvons dire que ce travail ouvre la voie vers l'application d'une nouvelle génération de capteurs optiques portables, robustes et miniaturisables basés sur les MIPs, pour des mesures «sur-site » et la quantification en temps réel d’analytes biologiques et environnementaux dans des milieux complexes. / This thesis describes the development of highly selective fiber optic sensors using molecularly imprinted polymers (MIPs) as recognition elements associated with fluorescence for detection. Additionally, we extended the study to the development of other MIP-based optical sensors and sensing methods. MIPs are synthetic biomimetic receptors possessing specific cavities designed for a target molecule. Produced by a templating process at the molecular level, MIPs are capable of recognizingand binding target molecules with selectivities and affinities comparable to those of natural receptors. Compared to biological recognition elements, MIPs are more stable, cheaper and easier to integrate into standard industrial fabrication processes. Hence, MIPs have become interesting alternatives to biomolecules as recognition elements for biosensing. In the first part of this thesis (Chapter 2), MIPs were synthesized by in-situ laser-induced photopolymerization in only a few seconds, as a micrometer-sized tip at the extremity of a telecommunication optical fiber. Photonic and physico-chemical parameters were optimized to tailor the properties of the polymer micro-objects. Gold nanoparticles were incorporated into the MIP microtip for signal enhancement. To prove the efficiency of the sensor, initial studies were performed with a MIP templated with N-carbobenzyloxy-L-phenylalanine (Z-L-Phe) and the fluorescent amino acid derivative dansyl-L-phenylalanine as analyte. The fluorescence was collected either externally at the tip level by an optical fiber connected to a spectrofluorimeter or by collection of the fluorescent signal re-emitted into the fiber through the second arm of a Y-shaped bifurcated fiber. The fluorescent analyte could be detected in the low nM concentrations. In order to monitor nonfluorescent analytes, a naphthalimide-based fluorescent monomer was incorporated into the MIP during its synthesis; fluorescence enhancement was observed when analyte binding occurs. Using this system, the sensor containing a MIP specific for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), could detect and quantify this analyte at concentrations as low as 2.5 nM. The signaling MIP-based sensor was also applied to analytes of interest for food safety and biomedical applications, such as the mycotoxin citrinin and the sphingolipid, D-erythro-sphingosine-1-phosphate. In the second part of the thesis (Chapter 3), a different type of fiber optic sensor: cheap, fast and made for “single-use”, was developed by using 4-cm long disposable polystyrene evanescent wave optical fiber waveguides. The coating of the MIP was either performed ex-situ, by dip-coating the fiber in a suspension of MIP particles synthesized beforehand, or in-situ by evanescent-wave photopolymerization directly on the fiber. The resulting fiber optic sensor could detect 2,4-D in the low nM range and demonstrated specific and selective recognition of the herbicide over its structural analogues and other non-related carboxyl-containing analytes. Additionally, we demonstrated the versatility of the system by applying the evanescent wave fiber optic sensor to detect citrinin, a mycotoxin, by simply coating the waveguide with a MIP specific for citrinin. This type of technology could possibly be extended to detect other carboxyl-containing analytes, as long as a specific MIP for the concerned analyte is available. In parallel, the technique of evanescent-wave photopolymerization was used for the synthesis of signaling MIP microdots on continuous and nanostructured gold films. This study lays the foundations for future development of plasmonic MIP nanosensors and microchips. In the last part of the thesis (Chapter 4), an innovative sensing method, based on the use of MIPs and analysis by fluorescence polarization, was developed in order to allow the fast and directquantification of analytes in food and environmental samples.

This thesis describes the development of highly selective fiber optic sensors using molecularly imprinted polymers (MIPs) as recognition elements associated with fluorescence for detection. Additionally, we extended the study to the development of other MIP-based optical sensors and sensing methods. MIPs are synthetic biomimetic receptors possessing specific cavities designed for a target molecule. Produced by a templating process at the molecular level, MIPs are capable of recognizingand binding target molecules with selectivities and affinities comparable to those of natural receptors. Compared to biological recognition elements, MIPs are more stable, cheaper and easier to integrate into standard industrial fabrication processes. Hence, MIPs have become interesting alternatives to biomolecules as recognition elements for biosensing. In the first part of this thesis (Chapter 2), MIPs were synthesized by in-situ laser-induced photopolymerization in only a few seconds, as a micrometer-sized tip at the extremity of a telecommunication optical fiber. Photonic and physico-chemical parameters were optimized to tailor the properties of the polymer micro-objects. Gold nanoparticles were incorporated into the MIP microtip for signal enhancement. To prove the efficiency of the sensor, initial studies were performed with a MIP templated with N-carbobenzyloxy-L-phenylalanine (Z-L-Phe) and the fluorescent amino acid derivative dansyl-L-phenylalanine as analyte. The fluorescence was collected either externally at the tip level by an optical fiber connected to a spectrofluorimeter or by collection of the fluorescent signal re-emitted into the fiber through the second arm of a Y-shaped bifurcated fiber. The fluorescent analyte could be detected in the low nM concentrations. In order to monitor nonfluorescent analytes, a naphthalimide-based fluorescent monomer was incorporated into the MIP during its synthesis; fluorescence enhancement was observed when analyte binding occurs. Using this system, the sensor containing a MIP specific for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), could detect and quantify this analyte at concentrations as low as 2.5 nM. The signaling MIP-based sensor was also applied to analytes of interest for food safety and biomedical applications, such as the mycotoxin citrinin and the sphingolipid, D-erythro-sphingosine-1-phosphate. In the second part of the thesis (Chapter 3), a different type of fiber optic sensor: cheap, fast and made for "single-use", was developed by using 4-cm long disposable polystyrene evanescent wave optical fiber waveguides. The coating of the MIP was either performed ex-situ, by dip-coating the fiber in a suspension of MIP particles synthesized beforehand, or in-situ by evanescent-wave photopolymerization directly on the fiber. The resulting fiber optic sensor could detect 2,4-D in the low nM range and demonstrated specific and selective recognition of the herbicide over its structural analogues and other non-related carboxyl-containing analytes. Additionally, we demonstrated the versatility of the system by applying the evanescent wave fiber optic sensor to detect citrinin, a mycotoxin, by simply coating the waveguide with a MIP specific for citrinin. This type of technology could possibly be extended to detect other carboxyl-containing analytes, as long as a specific MIP for the concerned analyte is available. In parallel, the technique of evanescent-wave photopolymerization was used for the synthesis of signaling MIP microdots on continuous and nanostructured gold films. This study lays the foundations for future development of plasmonic MIP nanosensors and microchips. In the last part of the thesis (Chapter 4), an innovative sensing method, based on the use of MIPs and analysis by fluorescence polarization, was developed in order to allow the fast and directquantification of analytes in food and environmental samples.

This thesis describes the development of highly selective fiber optic sensors using molecularly imprinted polymers (MIPs) as recognition elements associated with fluorescence for detection. Additionally, we extended the study to the development of other MIP-based optical sensors and sensing methods. MIPs are synthetic biomimetic receptors possessing specific cavities designed for a target molecule. Produced by a templating process at the molecular level, MIPs are capable of recognizingand binding target molecules with selectivities and affinities comparable to those of natural receptors. Compared to biological recognition elements, MIPs are more stable, cheaper and easier to integrate into standard industrial fabrication processes. Hence, MIPs have become interesting alternatives to biomolecules as recognition elements for biosensing. In the first part of this thesis (Chapter 2), MIPs were synthesized by in-situ laser-induced photopolymerization in only a few seconds, as a micrometer-sized tip at the extremity of a telecommunication optical fiber. Photonic and physico-chemical parameters were optimized to tailor the properties of the polymer micro-objects. Gold nanoparticles were incorporated into the MIP microtip for signal enhancement. To prove the efficiency of the sensor, initial studies were performed with a MIP templated with N-carbobenzyloxy-L-phenylalanine (Z-L-Phe) and the fluorescent amino acid derivative dansyl-L-phenylalanine as analyte. The fluorescence was collected either externally at the tip level by an optical fiber connected to a spectrofluorimeter or by collection of the fluorescent signal re-emitted into the fiber through the second arm of a Y-shaped bifurcated fiber. The fluorescent analyte could be detected in the low nM concentrations. In order to monitor nonfluorescent analytes, a naphthalimide-based fluorescent monomer was incorporated into the MIP during its synthesis; fluorescence enhancement was observed when analyte binding occurs. Using this system, the sensor containing a MIP specific for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), could detect and quantify this analyte at concentrations as low as 2.5 nM. The signaling MIP-based sensor was also applied to analytes of interest for food safety and biomedical applications, such as the mycotoxin citrinin and the sphingolipid, D-erythro-sphingosine-1-phosphate. In the second part of the thesis (Chapter 3), a different type of fiber optic sensor: cheap, fast and made for "single-use", was developed by using 4-cm long disposable polystyrene evanescent wave optical fiber waveguides. The coating of the MIP was either performed ex-situ, by dip-coating the fiber in a suspension of MIP particles synthesized beforehand, or in-situ by evanescent-wave photopolymerization directly on the fiber. The resulting fiber optic sensor could detect 2,4-D in the low nM range and demonstrated specific and selective recognition of the herbicide over its structural analogues and other non-related carboxyl-containing analytes. Additionally, we demonstrated the versatility of the system by applying the evanescent wave fiber optic sensor to detect citrinin, a mycotoxin, by simply coating the waveguide with a MIP specific for citrinin. This type of technology could possibly be extended to detect other carboxyl-containing analytes, as long as a specific MIP for the concerned analyte is available. In parallel, the technique of evanescent-wave photopolymerization was used for the synthesis of signaling MIP microdots on continuous and nanostructured gold films. This study lays the foundations for future development of plasmonic MIP nanosensors and microchips. In the last part of the thesis (Chapter 4), an innovative sensing method, based on the use of MIPs and analysis by fluorescence polarization, was developed in order to allow the fast and directquantification of analytes in food and environmental samples.

Dans cette thèse, nous avons démontré la faisabilité de la préparation de polymères à empreinte moléculaires (MIP) hautement sélectifs pour la reconnaissance dans des matrices aqueuses complexes avec des applications dans les cosmétiques et en technologie alimentaire. Les MIP (de l'anglais molecularly imprinted polymers) sont des récepteurs synthétiques comparables aux anticorps, qui sont synthétisés par co-polymérisation de monomères fonctionnels et réticulants en présence d'un gabarit moléculaire. Leurs propriétés de reconnaissance moléculaire, associées à leur grande stabilité, robustesse mécanique, faible coût et leur synthèse facile les rendent extrêmement intéressants comme matériaux de capture sélective, avec des applications dans les séparations analytiques, la détection et la vectorisation des médicaments. Cependant, leur reconnaissance sélective dans des milieux aqueux reste toujours problématique et c'est l'une des raisons de leur expansion commerciale restreinte. Dans une première partie, nous avons développé un MIP fonctionnant en milieu aqueux pour son application comme ingrédient actif dans un déodorant. Les odeurs corporelles sont principalement dues à des acides gras volatils générés à partir de leurs précurseurs, des conjugués de glutamine par des enzymes hydrolytiques produites à partir de bactéries présentes sur la peau. La plupart des anti-transpirants et des déodorants actuellement commercialisés contiennent des sels d'aluminium et des agents antibactériens non spécifiques, respectivement. Cependant, l'utilisation extrêmement étendue de ces produits nécessite des solutions alternatives en ce qui concerne divers problèmes (environnement, respect de l'écosystème de la peau, toxicité, etc.). Pour cette raison, un MIP a été synthétisé pour capturer les précurseurs conjugués de glutamine afin qu'ils ne soient plus disponibles aux bactéries, empêchant ainsi leur transformation en composés malodorants. Afin de générer des liaisons sélectifs dans des environnements aqueux, un monomère à base d'amidinium qui peut former une interaction électrostatique stoechiométrique forte avec les groupes carboxyle sur le gabarit moléculaire a été synthétisé. Le MIP, mélangé dans une formulation dermo-cosmétique, pourrait capter sélectivement les précurseurs conjugués de glutamine, au milieu d'une multitude d'autres molécules présentes dans la sueur humaine. En outre, le MIP n’affecte pas les bactéries de la peau, ouvrant la voie à des déodorants innovateurs de nouvelle génération, moins problématiques pour la santé. Dans une deuxième partie, nous avons développé une procédure rapide et efficace basée sur l'extraction en phase solide à empreinte moléculaire (MISPE) pour la purification sélective de la bétanine et de son stéréoisomère l’isobétanine à partir d'extraits de betterave. La bétanine est un pigment naturel ayant un fort pouvoir antioxydant et dont les propriétés pharmacologiques sont de plus en plus étudiées. Ce pigment est actuellement utilisé comme simple colorant alimentaire. Dans notre étude, l'acide dipicolinique a été utilisé comme gabarit moléculaire pour la synthèse de MIP, en raison de sa similarité structurelle avec le groupe chromophore de la bétanine. Les procédures MISPE ont été optimisées permettant l'élimination presque complète des glucides issus de la matrice végétale ainsi que la majorité des protéines, ce qui permet d'obtenir un rendement élevé d'extraction de la bétanine / isobétanine en une seule étape. De plus, toute la procédure d'extraction a été réalisée dans des solvants respectueux de l'environnement, tels que l'éthanol ou l'eau. Pour conclure, nous sommes convaincus que ce travail pave le chemin au développement d'une nouvelle génération des MIP fonctionnant en milieu aqueux avec des propriétés de reconnaissance améliorées dans des environnements complexes, qui pourra s'appliquer également à d'autres domaines biotechnologiques et biomédicaux. / In this thesis, we have demonstrated the feasibility of preparing highly selective molecularly imprinted polymers (MIPs) for recognition in complex aqueous matrices with applications in cosmetics and food technology. MIPs are synthetic tailor-made receptors, with binding affinities and specificities comparable to those of natural antibodies. Their molecular recognition properties, combined with their high stability, mechanical robustness, low cost and easy synthesis make them extremely attractive as selective capture materials with applications in analytical and preparative separations, sensing and drug delivery, among others. However, their selective recognition in aqueous samples still remains problematic and is one of the reasons for their so far lilited commercial expansion. In the first part, we developed a water compatible MIP for its application as an active ingredient in a deodorant. Body odors are mainly due to volatile fatty acids generated from their glutamine conjugate precursors by hydrolytic enzymes from bacteria present on the skin. Most currently marketed anti-perspirants and deodorants contain, respectively aluminum salts and unspecific antibacterials. However, the extremely wide use of these products requires alternative solutions with regard to various problems (environmental, respect of skin ecosystem, toxicity, etc.). For this reason, a MIP was developed to capture the glutamine conjugate precursors so that they are no longer available to the bacteria, thus preventing their transformation to malodorous compounds. In order to generate binding selectivity in aqueous environments, an amidinium-based monomer which can form a strong stoichiometric electrostatic interaction with the carboxyl groups on the template, was synthesized. The MIP, blended in a dermo-cosmetic formulation, could capture selectively the glutamine precursors, amidst a multitude of other molecules present in human sweat. Furthermore, the MIP did not affect the skin bacteria, paving the way to an innovative and 'safer ' future-generation deodorant. In the second part, we developed a fast and efficient procedure based on molecularly imprinted solid­ phase extraction (MISPE) for the selective clean-up of betanin and its stereoisomer isobetanin from red beetroot extracts. Betanin is a natural pigment with significant antioxidant and biological activities currently used as food colorant. Dipicolinic acid was used as template for the MIP synthesis, because of its structural similarity to the chromophore group of betanin The MISPE procedures were optimized allowing the almost complete removal of carbohydrates and the majority of proteins, resulting in high extraction recovery of betanin / isobetanin in a single step. Moreover, the whole extraction procedure was performed in environmentally friendly solvents with either ethanol or water. To conclude, we believe that this study paves the way towards the development of a new generation of water compatible MIPs with improved recognition properties in highly complex aqueous environments, and should be applicable to other biotechnological and biomedical areas as well.

This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated. The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction. The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude. Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support. The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.