This thesis aimed at elaborating an optical sensor to detect molecules in a biological fluid. Two steroids and a xenobiotic were identified as biomarkers released in some body fluids: cyproterone acetate, cortisol and 2,4-dichlorophenoxyacetic acid respectively. On one hand, detection was performed by Molecularly Imprinted Polymers (MIPs). These tailor-made synthetic receptors display numerous qualities that foster their integration in sensors. MIPs were therefore developed against the targeted analytes. Formulation optimization was led thanks to experimental designs. On the other hand, optical transduction was made possible thanks to the structuring of a polymer into a photonic crystal. Opals were manufactured with a new process suitable for large scales and were used to mold MIPs in inverse opals. Thus, submicron structures of the polymer are responsible for the color of the sensor. A change of color is triggered by the recognition of the analyte by the polymer (upon swelling). Polymers studied displayed sufficient swelling observed by spectrophotometry. Finally, the work of this thesis consisted in elaborating polymer formulations and their integration in a sensor so as to detect an analyte with direct, rapid and unobtrusive means.

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 le domaine des capteurs chimiques et biologiques, les besoins toujours croissants en termes de sensibilité, de rapidité et de sélectivité de l’analyse nécessitent le développement de couches sensibles transductrices de plus en plus performantes. Dans ce contexte et dans l’optique de pouvoir détecter de petites molécules non électroactives, telles que l’atrazine (ATZ), nous avons conçu, caractérisé et développé des couches sensibles constituées de polymères conducteurs fonctionnalisés à empreintes moléculaires (MICP) et les avons intégrées au sein de capteurs électrochimiques et gravimétriques. A partir de solutions d’acétonitrile contenant de l’ATZ, molécule empreinte en interaction avec des monomères fonctionnels dérivés du thiophène (FM = TMA, TAA, EDOT, TMeOH ou Th), différents polythiophènes FM-MICP de structures et de fonctionnalités différentes ont été électrosynthétisés sur substrats d’or et utilisés pour la détection de l’ATZ. Nous avons montré que les propriétés de reconnaissance des FM-MICP résultaient de la présence en leur sein d’empreintes moléculaires, fonctionnalisées par les résidus FM, qui conservaient la mémoire géométrique et fonctionnelle des molécules cibles. Néanmoins, une adsorption non-spécifique se produit systématiquement à la surface des couches sensibles et affecte par conséquent la sélectivité de la reconnaissance. Les techniques de caractérisation de surface employées nous ont permis de mettre en évidence l’influence de l’épaisseur et des propriétés structurales des couches sensibles sur l’efficacité de la détection. En outre, nous avons montré que du fait de la porosité de la couche polymère, le processus de reconnaissance se produisait en volume. Par ailleurs, des mesures électrochimiques corrélées à des calculs semi-empiriques ont permis de démontrer l’influence de la nature de FM d’abord sur la force de l’interaction ATZ-FM au sein de la solution de pré-polymérisation, ensuite sur le nombre d’empreintes moléculaires et enfin sur la sensibilité des FM-MICP vis-à-vis de l’ATZ. La couche TAA-MICP, qui présente un faible seuil de détection (10-9 mol L-1) ainsi qu’une large gamme dynamique (10-8 à 10-4 mol L-1), est la plus performante des couches sensibles puisqu’elle offre le meilleur compromis entre une détection spécifique de l’ATZ relativement élevée et une adsorption non-spécifique relativement faible. Enfin, le TAA-MICP a été utilisé comme couche sensible au sein d’un capteur électrochimique original à ondes acoustiques de surface (ESAW) dans l’optique de réaliser des mesures gravimétriques et électrochimiques couplées et simultanées. / In the field of chemical and biological sensors, the increased need for better sensitivity, faster response and higher selectivity during an analysis process, requires the development of more and more efficient transducing sensing layers. In this context, and with the aim to detect small non-electroactive molecules, such as atrazine (ATZ), we designed, characterized and developed sensing layers constituted by functionalized Molecularly Imprinted Conducting Polymers (MICP) and we integrated them into electrochemical and gravimetrical sensors. Starting from acetonitrile pre-polymerization media containing ATZ as template molecules in the presence of thiophene-based functional monomers (FM, namely TMA, TAA, EDOT, TMeOH or Th), differently functionalized and structurally different polythiophene-based FM-MICP films were electrosynthesized onto gold substrates and used for ATZ detection. The sensing properties of FM-MICP layers were shown to result from the presence in their backbones of pre-shaped FM-functionalized imprinted cavities which keep the memory of the targets. Nevertheless, non-specific adsorption onto the surface of the sensing layers takes place systematically, which affects the selectivity of the recognition process. Thanks to surface characterization techniques, we highlighted the influence of the thickness and of the structural properties of the layers on the efficiency of the recognition process. Besides, this latter was shown to operate in the bulk of the polymer matrixes thanks to layers porosity. On another hand, electrochemical measurements correlated with semi-empirical calculations demonstrated the influence of the nature of FM on the strength of the ATZ-FM interaction in the pre-polymerization medium, and then on the number of ATZ molecular imprints and on the sensitivity towards ATZ of the FM-MICP layers. We showed that TAA-MICP, which presents a low limit of detection (10-9 mol L-1) and a large dynamic range (10-8 to 10-4 mol L-1), is the best sensing layer since it offers the best compromise between high level of specific detection of ATZ and low level of non-specific adsorption. Finally, TAA-MICP was used as sensitive layer in an original Electrochemical Surface Acoustic Wave sensor (ESAW) which enabled simultaneous coupled gravimetric and electrochemical measurements.

L’utilisation croissante de pesticides dans l'agriculture peut entraîner de graves risques pour la santé humaine. En effet, des résidus de pesticides peuvent être retrouvés à l’état de trace dans de nombreux produits de grande consommation. Leur identification et leur analyse quantitative à l’état de trace dans des échantillons complexes, tel que les huiles végétales, constituent un défi analytique majeur. Malgré le potentiel élevé de méthodes analytiques comme la chromatographie en phase liquide couplée à la spectrométrie de masse (LC-MS/MS), l’introduction d’une étape d’extraction et de purification des extraits avant l’analyse chromatographique s’avère nécessaire. Afin d'augmenter la sélectivité de cette étape de traitement de l'échantillon, la synthèse des supports à empreintes moléculaires générant un mécanisme de reconnaissance moléculaire a été envisagé. Cette étude se concentre sur une famille de pesticides, les organophosphorés (OP), qui présentent des disparités structurales importantes et une gamme de polarité assez large (log P compris entre 0,7 et 4,7). Deux approches ont été envisagées pour la synthèse de ces supports imprimés. La première approche consiste à réaliser une polymérisation par voie radicalaire en utilisant des monomères organiques dans des solvants organiques peu polaires pour obtenir des polymères à empreintes moléculaires (MIP). La seconde approche consiste à produire les supports par voie sol-gel via l’hydrolyse puis la condensation d'organosilanes dans un milieu polaire pour produire des silices imprimées (MIS). Pour les deux approches, différentes conditions de synthèse ont été criblées en utilisant différentes molécules empreintes, monomères et solvants. La sélectivité des polymères imprimés résultants a d'abord été évaluée en étudiant les profils de rétention des OP en milieu pur. Les interactions non-spécifiques ont été évaluées en étudiant parallèlement la rétention des OP sur des supports non-imprimés (NIP/NIS) qui ont été synthétisés dans les mêmes conditions que les MIP/MIS mais sans introduire la molécule empreinte. Il est apparu que les supports MIP/MIS présentaient une complémentarité en termes d'extraction sélective des OP visés : les OP les plus polaires ont été extraits sélectivement par le MIS alors que les OP modérément polaires ont été extraits sélectivement par le MIP. La capacité de ces supports a été évaluée et se révèle adaptée à l'analyse des OP à l’état de traces dans des huiles végétales. Après avoir vérifié la répétabilité de la procédure d'extraction et des synthèses, les performances de ces supports ont été étudiées dans des milieux réels. / The increasing use of pesticides in agriculture causes serious health risks to humans. These pesticides may possibly be found in vegetable oils used as cosmetic ingredients. Their identification and reliable quantitative analysis at trace levels constitute a challenge for the safe use of such oils despite the high potential of analytical methods such as liquid chromatography coupled to mass spectrometry (LC-MS/MS). Their determination at low concentration levels in complex oil samples requires an extraction and a purification step. In order to increase the selectivity of the sample treatment step, the synthesis of imprinted sorbents can be considered. This study focusses on a group of pesticides, the organophosphorus (OPs) that present some structural disparity and belong to a wide range of polarity (log P values between 0.7 and 4.7). To produce imprinted sorbents, a first approach of synthesis consists in the radical polymerization of organic monomers in moderately polar organic solvents to obtain molecularly imprinted polymers (MIPs). The second one, the Sol-Gel approach, consists in the hydrolysis and then condensation of organosilanes in a polar medium to produce molecularly imprinted silicas (MIS). For both approaches, different conditions of synthesis were screened using different template molecules, monomers and solvents. The selectivity of the resulting imprinting polymers was first evaluated by studying the extraction profiles of OPs in pure media on MIP and MIS. The non-specific interactions were estimated by studying in parallel the retention of OPs on non-imprinted polymers synthesized in the same conditions as imprinted sorbents but in the absence of the template molecule. Both sorbents MIP/MIS present a complementarity in terms of selective extraction of the target OPs: polar OPs were extracted selectively using the MIS while moderately polar OPs were selectively extracted by the MIP. The capacity of these supports was evaluated and was consistent with the analysis of OPs at trace levels in real oil samples. After studying the repeatability of the extraction procedure and of the reliability of the syntheses, the performances of these supports were studied in real media. For this, MIP/MIS were applied to the selective extraction of OPs from different vegetable oils (almond, olive and sunflower oil) and similar results were obtained for the three different oils. Their potential in terms of ability to remove matrix interfering compounds were higher than those of the conventional method based on the use of C18 silica. The estimated limits of quantifications were lower than the Maximum Residue Levels (MRLs) established by EU Regulation 396/2005 for these compounds in oils.

Submitted by JEFFERSON HONORIO FRANCO null (jeffersonhfranco@gmail.com) on 2016-12-01T15:16:17Z No. of bitstreams: 1 TESE FINAL-IMPRESSÃO CD.pdf: 4568825 bytes, checksum: 666b30b163102c69eb93110502678419 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-05T12:50:59Z (GMT) No. of bitstreams: 1 franco_jh_dr_araiq_par.pdf: 1149871 bytes, checksum: 4c942fc016c94499d690f6474dda7078 (MD5) / Made available in DSpace on 2016-12-05T12:50:59Z (GMT). No. of bitstreams: 1 franco_jh_dr_araiq_par.pdf: 1149871 bytes, checksum: 4c942fc016c94499d690f6474dda7078 (MD5) Previous issue date: 2016-11-21 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Corantes sintéticos do tipo azo têm sido um assunto de grande preocupação ambiental devido ao potencial genotóxico e mutagênico dos produtos de biotransformação. Deste modo, nos últimos anos a consequência da ingestão destes corantes presentes na agua potável servida à população é discutida por diversos autores. Este estudo avalia a ação de microssomas de fígado de rato, enzimas redutoras produzidas pela bactéria Escherichia coli (E. coli) e nitroredutase imobilizada na biotransformação de três corantes dispersos que possuem grupos azo, Disperse Red 73 (DR 73), Disperse Red 78 (DR 78) e Disperse Red 167 (DR 167). A técnica de Espectrofotometria de absorção molecular na região do Uv- visível, Cromatografia Líquida de Alta Eficiência com detector de arranjo de diodos (CLAE-DAD) e Cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS) foram técnicas usadas para identificar os principais produtos gerados após os processos de degradação dos corantes. Polímeros de impressão molecular magnéticos (MMIPs) foram investigados usando reações de polimerização por precipitação para pré-concentração do corante DR 73, juntamente com a degradação por fotoeletrocatálise e subsequente análise dos produtos por LC-MS/MS. Os estudos in vitro do metabolismo de biotransformação dos corantes têxteis com microssoma de fígado de rato mostraram que as reações ocorreram preferencialmente no grupo azo e nitro dos corantes, indicando a redução destes grupos pelas enzimas do citocromo P-450. Foram obtidos dois produtos de degradação para cada corante após reação com a bactéria E. coli; o corante DR 73 originou os produtos 3-((4-aminofenil)(etil)amino)propanitrila e 4-nitroanilina, os produtos 3-((4-aminofenil)(etil)amino)propanitrila e 2-cloro-4-nitroanilina foram obtidos após reação com o corante DR78 e o DR 167 originou dimetil 3,3`-((3-acetamido-4aminofenil)azanediyl)dipropanoato e 2-cloro-4-nitroanilina, indicando a clivagem do grupo azo, possivelmente, pela enzima azoredutase, produzida pela bacteria. A enzima nitroredutase, imobilizada em partículas magnéticas modificadas com tosil, mostrou que a redução dos corantes ocorreu preferencialmente no grupo nitro, enquanto que a enzima livre no meio reacional resultou em mais de um produto de biotransformação para cada corante, atuando em mais de um sítio da molécula, comprovando a eficácia da imobilização enzimática para estudos de biotransformação e formação de produtos majoritários. A mutagenicidade dos corantes foi avaliado pelo ensaio de Salmonella/microssoma realizado nas estirpes TA 98 e TA 100, com e sem S9. De acordo com este ensaio, DR 73 foi o mais mutagênico. O MMIP para o corante DR 73 apresentou excelentes valores de religação (16 mg g−1 e 6 mg g−1, para MMIP e MNIP, respectivamente) indicando que o polímero molecularmente impresso formou cavidades específicas para retenção do corante. Através dos resultados obtidos por LCMS/MS, observou-se 100% de degradação do corante em apenas 60 min de tratamento via fotoeletrocatálise para soluções mais diluidas do mesmo, comprovando a eficiência da técnica na degradação de poluentes. Sendo assim, estes resultados sugerem que o MMIP mostrou uma excelente especificidade e seletividade para o corante DR 73 e uma técnica promissora na captação de corantes mutagênicos de águas superficiais, com grande potencial de aplicação e exploração na pré-concentração antes do tratamento. Além disso, a redução destes corantes por sistemas biológicos representa uma grande preocupação ambiental devido ao aumento da genotoxicidade para os seres vivos, em especial a seres humanos, produzindo compostos nocivos, tais como aminas condenadas pela Agência Internacional de Pesquisa sobre o Câncer. / Synthetic azo dyes have been a matter of great concern due to the genotoxic and mutagenic potential of the products originating from azo dye biotransformation. Thus, in recent years the result of the intake of these dyes present in drinking water supplied to a population is discussed by several authors. This work evaluates the action of rat liver microsomes, reducing enzymes produced by the Escherichia coli (E. coli) and nitroreductase immobilized on biotransformation of three disperse dyes bearing azo groups, namely Disperse Red 73 (DR 73), Disperse Red 78 (DR 78), and Disperse Red 167 (DR 167). UV-Vis spectrophotometry, high-performance liquid chromatography with diode array detector (HPLC-DAD), and liquid chromatography coupled to mass spectrometry (LC-MS/MS) were techniques used to identify the main products generated after the process degradation of dyes. Magnetic molecularly imprinted polymers (MMIPs) were investigated using precipitation polymerization reactions for preconcentration of the dye DR 73, together with the photoelectrocatalysis degradation and subsequent analysis of the products by LC-MS/MS. In vitro studies of biotransformation metabolism of textile dyes with rat liver microsome showed that the reactions occur preferentially in the group of azo and nitro dyes, indicating the reduction of these groups by enzymes of the cytochrome P-450. There were obtained two degradation products for each dye after reaction with E. coli; the dye DR 73 gave the product 3 - ((4-aminophenyl) (ethyl) amino) propanitrila and 4-nitroaniline, the product 3 - ((4-aminophenyl) (ethyl) amino) propanitrila and 2-chloro-4-nitroaniline were obtained after reaction with the dye DR78 and DR 167 gave 3,3`-dimethyl-((3-acetamido-4-aminophenyl) azanediyl) dipropanoato and 2chloro-4-nitroaniline; indicating cleavage of the azo group, possibly by azoredutase enzyme produced by bacteria. The nitroreductase enzyme immobilized on modified magnetic particles Tosyl showed that the reduction of dyes occurred preferentially in the nitro group, while the free enzyme in the reaction medium resulted in more than a product of biotransformation for each dye, acting in more than one site of the molecule, proving the efficacy of enzyme immobilization for biotransformation studies and formation of major products. The mutagenicity of the dyes was evaluated by the Salmonella/microsome assay performed on strains TA 98 and TA 100, with and without S9. According to this assay, DR 73 was the most mutagenic. The MMIP to the dye DR 73 showed excellent rebinding values (16 mg g−1 and 6 mg g−1, for MMIP and MNIP, respectively) indicating that the molecularly imprinted polymer formed cavities for specific dye retention. Through the results obtained by LC-MS/MS, it was observed 100% dye degradation in 60 min treatment for more dilute solutions thereof, proving the efficiency of technique in pollutant degradation. Thus, these results suggest that MMIP showed excellent specificity and selectivity for the dye DR 73 and a promising technique in capturing mutagenic dyes of surface water, with great potential for application and operation in the pre-concentration before treatment. Moreover, the reduction of these dyes by biological systems is a major environmental concern due to increased of genotoxicity for living beings, especially humans, producing harmful compounds, such as condemned amines by the International Agency for Research on Cancer.

Submitted by Caroline Periotto (carol@ufscar.br) on 2016-09-19T14:54:59Z No. of bitstreams: 1 TeseACL.pdf: 2614987 bytes, checksum: b5377ebbef88bc31b06fbcdabbe6a724 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-20T17:59:42Z (GMT) No. of bitstreams: 1 TeseACL.pdf: 2614987 bytes, checksum: b5377ebbef88bc31b06fbcdabbe6a724 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-20T17:59:49Z (GMT) No. of bitstreams: 1 TeseACL.pdf: 2614987 bytes, checksum: b5377ebbef88bc31b06fbcdabbe6a724 (MD5) / Made available in DSpace on 2016-09-20T17:59:58Z (GMT). No. of bitstreams: 1 TeseACL.pdf: 2614987 bytes, checksum: b5377ebbef88bc31b06fbcdabbe6a724 (MD5) Previous issue date: 2015-06-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / This thesis describes the development of imprinted polymers for preconcentration of Se, Cd and U. The relevant factors to the FIA system and preconcentration were optimized by Doehlert matrix. Chapter I describes an atomically imprinted polymer synthesis for preconcentration of Se (0) and determination by HG-AAS. The method presents the following analytical features: linear range from 0.17 to 6.0 μg L-1 , limit of detection and quantification of 53 and 170 ng L-1 , respectively, of preconcentration factor of 232 and reading frequency analysis of 12 h-1 ; which were all applied in food samples. The accuracy was checked with certified reference material. Chapter II describes the synthesis of a hybrid imprinted polymer for uranium concentration and determination by UV-visible spectrophotometry. The polymer had adsorptive capacity of 1.55 mg g-1 , linear analytical range from 2.0 to 100.0 μg L -1 detection limit of 0.40 μg L-1 quantification limit of 1.25 μg L-1 , preconcentration factor of 97, concentration efficiency of 19.40 min-1 . The method was applied to water samples from rivers and lakes, with accuracy confirmed by alpha spectrometry. Finally, in Chapter III, a method for cadmium determination by TS-FF-AAS after preconcentration in imprinted polymer synthesized via bulk is proposed. The figures of merit were linear range 0.081 to 10 μg L-1 , detection and quantification limit of 24 and 81 ng L-1 , respectively. Preconcentration factor of 165 and frequency measures analyzes 25 h-1 . The method was applied in tea, hair, cigarette and jewelry samples. The accuracy was checked with certified reference material. / A presente tese descreve o desenvolvimento de polímeros impressos para pré- concentração de Se, U e Cd. Os fatores pertinentes ao sistema FIA e ao sistema de pré-concentração foram otimizados por matriz de Doehlert. O capítulo I descreve a síntese de um polímero atomicamente impresso para pré- concentração de Se(0) e detecção e determinação por HG-AAS. O método apresentou as seguintes características analíticas: faixa linear de 0,17 a 6,0 µg L -1 , limite de detecção e quantificação de 53 e 170 ng L-1 , respectivamente, fator de pré-concentração de 232 e frequência de leitura de 12 análises h-1 , tendo sido aplicado em amostras alimentícias. A exatidão foi checada com material de referência certificado. O Capítulo II descreve a síntese de um polímero impresso híbrido para pré-concentração de urânio e detecção por espectrofometria de UV-visível. O polímero apresentou capacidade adsortiva de 1,55 mg g-1 , faixa analítica linear de 2,0 a 100,0 µg L-1 , limite de detecção 0,40 µg L-1 , limite de quantificação de 1,25 µg L-1 , fator de pré-concentração igual a 97 e eficiência de pré-concentração de 19,40 min-1 . O método foi aplicado em amostras de água de rios e lagos, sendo a exatidão confirmada por espectrometria alfa. Finalmente no Capítulo III é proposto um método para a detecção de cádmio por TS-FF-AAS após pré-concentração em polímero impresso sintetizado via bulk. As figuras de mérito foram: faixa linear de 0,081 a 10 µg L-1 , limite de detecção e quantificação de 24 e 81 ng L-1 , respectivamente. Fator de pré-concentração igual 165 e frequência de 25 analíses h-1 . O método foi aplicado em amostras de chás, cabelo, cigarro e bijuteria. A exatidão foi checada com material de referência certificado.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-23T19:00:13Z No. of bitstreams: 1 Peçanha, Bruna Rachel de Britto [Dissertação, 2012].pdf: 3907379 bytes, checksum: f2acabc3c1c39363b86f0d651d6b5936 (MD5) / Made available in DSpace on 2017-03-23T19:00:13Z (GMT). No. of bitstreams: 1 Peçanha, Bruna Rachel de Britto [Dissertação, 2012].pdf: 3907379 bytes, checksum: f2acabc3c1c39363b86f0d651d6b5936 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Polímeros de impressão molecular (MIPs) foram sintetizados e aplicados como adsorventes na técnica de extração em fase sólida (EFS). O método de polimerização por precipitação foi utilizado para a síntese dos polímeros, devido à simplicidade de preparo, altos rendimentos e obtenção de partículas mais uniformes, devido a não trituração do polímero. O MIP foi sintetizado com ácido metacrílico (MAA) como monômero funcional, trimetacrilato de trimetilolpropano (TRIM) e dimetacrilato de etilenoglicol (EDMA) como agentes de reticulação e o cloridrato de amilorida (AMI) foi escolhido como molécula-molde. Diferentes proporções de MAA, TRIM, EDMA, volume e tipo de solvente foram utilizadas para ajuste das condições ideais de síntese. Os MIP foram avaliados quanto à capacidade de adsorção comparando-se a polímeros sintetizados na ausência da molécula-molde (NIP, polímeros não impressos). O solvente de elevada polaridade empregado na síntese (THF:MeOH:H2O) permitiu o emprego da técnica para moléculas polares como AMI. O controle no volume de solvente permitiu a obtenção de partículas maiores, de modo que a EFS foi realizada em condições usuais, o que confere um potencial para aplicação dessa técnica de polimerização na preparação de adsorventes para EFS. O polímero que apresentou maior capacidade adsortiva no ensaio realizado em tampão citrato-acetato pH 6,5 foi o MIP/NIP 12 (AMI:MAA:TRIM 1:8:10), com uma taxa média de adsorção de 83 e 88% para NIP e MIP, respectivamente. A adsorção foi elevada devido a interação iônica entre MAA e AMI promovida pelo controle de pH, porém foi não específica. O polímero MIP/NIP 12 foi aplicado como adsorvente na EFS, onde a recuperação de AMI foi avaliada nos resíduos de carregamento e eluição com solventes. O carregamento com tampão citrato-acetato pH 6,5 foi o ideal, favorecendo a interação iônica do polímero com o analito. A eluição total de AMI do cartucho somente ocorre após lavagem com o solvente na presença de ácido, que protona os grupos carboxila do polímero, rompendo assim a interação iônica com o analito / Molecularly imprinted polymers (MIPs) were synthesized and applied as adsorbents in solid-phase extraction technique (SPE). The polymers have been synthesized by precipitation polymerization method because of its simplicity, high yields and good control of final size and shape of particles. MIP was synthesized using methacrylic acid (MAA) as functional monomer, trimethylolpropane trimethacrylate (TRIM) and ethyleneglycol dimethacrylate (EDMA) as cross-linker and amiloride hydrochloride (AMI) was chosen as template. Different ratios of MAA, TRIM and EDMA, volume and type of solvent were used to adjust the optimal synthesis conditions. The MIP were tested for adsorption capacity compared to the polymers synthesized in the absence of template molecule (NIP, non-imprinted polymers). The polar solvent mixture used (THF:MeOH:H2O) allowed the synthesis of MIP of polar molecules as AMI. The solvent volume control afforded the larger particles so the SPE was performed in the usual conditions, giving a potential application for this polymerization technique in the preparation of adsorbents for SPE. The polymers with higher adsorption capacity at the test performed in citrateacetate buffer pH 6,5 was MIP/NIP 12 (AMI:MAA:TRIM 1:8:10) with adsorption rate of 83 and 88% for NIP and MIP, respectively. The recognition of MIP was due to ionic interaction between MAA and AMI promoted by pH control, but was not specific. The polymer MIP/NIP 12 was used as a solid-phase extraction sorbent and the recoveries of AMI was evaluated using different loading and elution conditions. The loading with buffer citrate-acetate pH 6,5 was optimal, due to ionic interaction of the polymer with the analyte. Total elution of AMI bound to the polymers only occurs after washing with a acid-containing solvent, because of protonation of the carboxyl groups of the polymer and disrupting the ionic interaction with the analyte

Les biocapteurs sont des moyens d'analyse en plein essor à la fois rapides, sélectifs et peu coûteux, applicables à des domaines très variés (environnement, santé, agroalimentaire…). La capacité de reconnaissance moléculaire extraordinaire de biomolécules telles que les enzymes ou les anticorps a été exploitée avec succès pour la réalisation de nombreux biocapteurs. Cependant, l'inconvénient majeur de ces récepteurs biologiques est qu'ils sont difficiles à produire et fragiles. Une manière de surmonter ces inconvénients consiste à les remplacer par des récepteurs artificiels présentant des propriétés de reconnaissance similaires. Parmi les matériaux biomimétiques prometteurs figurent les polymères à empreintes moléculaires (MIPs). Dans ce travail, nous nous sommes intéressés au développement de deux capteurs biomimétiques impédimétriques, le premier basé sur l'utilisation de poly(éthylène co-alcool vinylique) imprimé pour la détection de la créatinine et le deuxième sur des MIPs de polyméthacrylate pour la détection de la testostérone. Dans le premier cas, le polymère imprimé a été produit et déposé à la surface d'électrodes en or, soit par drop-coating, soit sous forme de nanofibres par la technique d'électrofilage. Dans le deuxième, le MIP a été synthétisé par polymérisation radicalaire de l'acide méthacrylique en présence d'éthylèneglycol diméthacrylate (réticulant), d'initiateur et de testostérone en utilisant la méthode du « grafting from » qui consiste à greffer d'abord l'initiateur sur la surface du transducteur mais pour la polymérisation on a utilisé deux approches (spin-coating d'une solution de prépolymérisation sur la surface du transducteur ou l'immersion de ce dernier dans la solution de monomère plus testostérone) suivie de l'exposition à une source d'energie pour effectuer la polymérisation. Les performances des capteurs (limite de détection, sélectivité, reproductibilité) ont ensuite été évaluées / Biosensors are rapid, selective and low-cost analytical devices of growing interest for a wide range of application fields (e.g. environment, food, health). The extraordinary molecular recognition capabilities of sensing biomolecules such as enzymes and antibodies have been successfully exploited in the elaboration of a number of biosensors. However, these biorecognition elements are often produced via complex and costful protocols and require specific handling conditions because of their poor stability. To circumvent these limitations, artificial receptors of similar recognition properties are now proposed as alternatives to natural receptors in sensor technology. Molecular imprinted polymers are among the most promising biomimetic materials reported. In this work, we developed two impedimetric biomimetic sensors. The first one is based on imprinted poly(ethylene co-vinyl alcohol) for creatinine detection and the second on polymethacrylate MIPs for testosterone analysis. In the first case, MIP was produced and deposited onto gold microelectrodes, either by spin-coating of a pre-polymerization solution, or by electrospinning. In the second case, MIPs were synthetized by photopolymerization of methacrylic acid in presence of ethyleneglycoldimethacrylate (cross-linker), an azo-initiator and testosterone as template using the “grafting from” method in which the initiator is first attached to the transducer surface but to effect polymerization we used two different approaches (dip-coating of a prepolymerization solution on the transducer surface functionalized with the initiator or immersing it in the solution of monomers and testosterone) followed by exposure to an energy source to effect polymerization. Then, analytical performances (linear range, detection limit, selectivity and reproducibility) of both creatinine and testosterone sensors were determined and compared

Les hydrocarbures aromatiques polycycliques (HAP) sont des contaminants de notre environnement et de notre alimentation. En raison de leur toxicité, la Commission européenne a réglementé leur teneur dans les denrées alimentaires et notamment dans les huiles. Les industriels des corps gras ont donc pour obligation de vérifier la conformité de leurs produits. Dans ce contexte, le groupe Lesieur souhaiterait développer un nouvel outil analytique rapide et portable. Ainsi, ce vaste projet de recherche vise à concevoir un microsystème électrophorétique capable d'analyser les HAP dans les huiles alimentaires. Première étude à s'inscrire dans ce projet, ce travail de thèse a donc consisté à développer de nouveaux protocoles analytiques. Dans une première partie, des méthodes de séparation des HAP ont été développées en électrophorèse capillaire (CE) modifiée par des cyclodextrines couplée à un détecteur de fluorescence induite par laser. En suivant des stratégies multivariées basées sur les plans d'expériences, deux méthodes de séparation ont été optimisées. Les huit HAP communs aux listes établies par l'agence de protection de l'environnement des Etats-Unis et l'agence européenne de sécurité sanitaire des aliments ont été séparés en moins de 7 min et dix-neuf HAP, également classés par ces deux organismes, ont été analysés en moins de 18 min. Ces méthodes de séparation ont été appliquées avec succès à des extraits d'huile dopés. Dans une deuxième partie, il a été question de transférer la méthode d'analyse des huit HAP au format microsystèmes. La principale difficulté rencontrée a été le manque de sensibilité du système de détection couplé aux puces. Le premier objectif a donc été d'optimiser les quantités d'échantillon injectées et les paramètres de la détection avec un composé modèle dans un tampon borate. Cependant, seulement quatre HAP sur les dix-neuf étudiés précédemment en CE ont pu être détectés. Toutefois, dans les conditions optimisées par le plan d'expériences, ils étaient séparés en moins de 4 min. Enfin, différents polymères à empreintes moléculaires (MIP) ont été synthétisés en vue d'extraire sélectivement les HAP des huiles. Après un criblage des conditions de synthèse, la sélectivité de chaque MIP a été évaluée en milieu pur en comparant sa capacité de rétention avec celle d'un polymère non-imprimé. Les huit HAP communs aux deux listes ont finalement pu être extraits sélectivement à partir d'huiles de tournesol, mais avec des rendements d'extraction encore insuffisants et qui nécessitent une amélioration de la procédure d'extraction. / Polycyclic aromatic hydrocarbons (PAHs) are widespread contaminants in the environment and food products. Given their toxicity, their concentrations are regulated by the European Commission in food products and especially in edible oils. Consequently, manufacturers of fat products have to verify the conformity of their products. In this context, Lesieur wants to develop a rapid and portable new analytical tool. Hence, this ambitious research project aims at developing a capillary electrophoresis microchip for PAH analysis in edible oils. This PhD study, first one involved in this project, consisted in developing new analytical protocols.In a first part, PAH separation methods were developed by cyclodextrin-modified capillary electrophoresis (CE) with laser-induced fluorescence detection. Using experimental design-based strategies, two separation methods were optimized. The 8 PAHs in common between the United States Environmental Protection Agency and the European Food Safety Authority lists of priority pollutants were separated in less than 7 min, while 19 PAHs, also targeted by these institutions, were separated in less than 18 min. These CE separation methods were successfully applied to the analysis of spiked edible oil extracts.The second part aimed at transferring the electrophoretic separation method of the 8 PAHs from the capillary to the microsystem format. The lack of sensitivity of the detection system hyphenated with chips was the main difficulty encountered. Thus, injected sample amounts and detection parameters were first optimized with a model compound in a borate buffer. However, only 4 out of the 19 PAHs, previously studied in CE, could be detected. Nevertheless, under conditions predicted by the design of experiments, they were baseline resolved in less than 4 min.Finally, several molecularly imprinted polymers (MIPs) were synthesized for the selective extraction of PAHs from oils. After a screening of synthesis conditions, the selectivity of each MIP was evaluated in pure media by comparing the extraction recoveries obtained on MIP with those obtained on a non-imprinted polymer. Finally, the 8 PAHs in common between the two lists were selectively extracted from sunflower oils, but with low recoveries. Improvements in extraction procedures are still required.