Neste trabalho propôs-se desenvolver eletrodos compósitos à base de grafite-poliuretana modificados com polímeros metacrilatos com impressão molecular (MIP-TC), visando a determinação do antibiótico tetraciclina em formulações farmacêuticas e urina sintética. O trabalho também visou contribuir no desenvolvimento de um sensor com melhor desempenho no que diz respeito à sensibilidade e seletividade do eletrodo compósito modificado, na quantificação do analito/molécula molde em matrizes complexas. Técnicas eletroanalíticas, tais como voltametria cíclica (CV) e voltametria de pulso diferencial (DPV) foram utilizadas para avaliar a resposta em termos de sensibilidade e seletividade do eletrodo compósito na detecção da tetraciclina (TC), proposto neste trabalho. A otimização dos parâmetros, tais como composição do MIP (5,0 %, m/m), amplitude de pulso (50 mV), velocidade de varredura (10 mV s-1), tempo (300 s) e potencial de acumulação [(+ 0,7 V (vs. SCE)] e faixa granulométrica do MIP (150 a 250 μm), usando voltametria de redissolução anódica por pulso diferencial foi realizada para maximizar a determinação da tetraciclina em tampão fosfato 0,10 mol L -1 (pH 2,5), permitindo obter limites de detecção de 0,555 μmol L-1, 2,70 μmol L-1 e 4,71 μmol L-1, respectivamente, para o EGPU-MIP-TC, EGPU e EGPU-NIP, sem necessidade de renovação da superfície do eletrodo entre as sucessivas medidas. Regiões lineares entre 4,0 e 60,0 μmol L-1, 8,0 e 60,0 μmol L-1 e 20,0 e 100,0 μmol L-1/sup> foram observadas, respectivamente, para o EGPU-MIP-TC, EGPU e EGPU-NIP, para o pico anódico em + 0,9 V (vs. SCE). Os EGPU também foram testados quanto à seletividade frente à possíveis interferentes, sendo utilizados clortetraciclina (CTC) e oxitetraciclina (OTC) para os testes de interferência. Em todos os casos, a interferência do EGPU-MIP-TC foi menor do que a obtida com o EGPU-NIP, confirmando que o EGPU-MIP-TC foi mais efetivo na capacidade de reconhecer seletivamente o analito na presença dos interferentes CTC e OTC. Após as etapas de síntese dos MIP/NIP e confecção dos eletrodos, otimização dos parâmetros, avaliações e testes de sensibilidade e seletividade dos EGPU, os eletrodos foram submetidos à aplicação em duas amostras de medicamentos comerciais, TetraMed® 500 mg e genérico de cloridrato de tetraciclina 500 mg e uma amostra sintética de urina. Os resultados foram comparados ao método oficial por HPLC, apresentando 95 % de confiança, de acordo com o teste t-Student. / In this work, graphite-polyurethane composites electrodes were modified with methacrylate molecularly imprinted polymers (MIP-TC) in order to evaluate the electrodes in the determination of tetracycline (TC) antibiotic in pharmaceutical formulations as well as in biological fluid. The work aimed to contribute in development of a sensor with better performance about the sensitivity and selectivity of the composites electrodes modified in quantification of this analyte. Electroanalytical techniques such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to evaluate the selectivity and sensitivity of the composite electrodes in the detection of tetracycline. The parameters optimization, such as MIP composition (5.0 %, m/m), pulse amplitude (50 mV), scan rate (10 mV s-1), accumulation time (300 s), accumulation potential [+ 0.7 V (vs. SCE)], and MIP granulometry (150 to 250 μm), using differential pulse anodic stripping voltammetry was done to maximize the determination of tetracycline in phosphate buffer 0.1 mol·L-1 pH 2.5 resulting in LOD of 0.555 μmol L-1, 2.70 μmol L-1 and 4.71 μmol L-1, respectively for the EGPU-MIP-TC, EGPU and EGPU-NIP, without the need for surface renovation between the measures. A linear dynamic range from 4.0 to 60.0 μmol L-1, 8.0 to 60.0 μmol L-1 and 20.0 to 100.0 μmol L-1, respectively for the EGPU-MIP-TC, EGPU and EGPU-NIP was observed for the anodic peak at + 0.9 V (vs. SCE). The EGPU were also tested regarding the selectivity compared to possible interferents, such as chlortetracycline (CTC) and oxytetracycline (OTC) for the interference study. In all cases, the interference of EGPU-MIP-TC was lower than the one that was got with EGPU-NIP, confirming that the EGPU-MIP-TC was more selective than the non-imprinted polymer in the capacity of recognizing, selectively, the analyte in presence of CTC and OTC interferents. After the steps of synthesis of MIP/NIP and electrodes confection, parameters optimization, evaluation and EGPU sensitivity and selectivity studies, the electrodes were submitted to the application in two commercial drugs containing 500 mg of tetracycline cloridrate, TetraMed® and generic; and a synthetic urine sample. The results were compared to the official method by HPLC, presenting 95 % confidence according to the t-Student test.

Philosophiae Doctor - PhD (Chemistry) / There is growing concern on endocrine disrupting compounds (EDCs). The presence of drugs in water supplies was first realized in Germany in the early 1990s when environmental scientists discovered clofibric acid. Clofibric acid has the ability to lower cholesterol in ground water below a water treatment plant. Endocrine disrupting compounds can be defined as those chemicals with the ability to alter daily functioning of the endocrine system in living organisms. There are numerous molecules that are regarded or referred to as EDCs such as but not limited to organochlorinated pesticides, industrial chemicals, plastics and plasticizers, fuels, estrogens and many other chemicals that are found in the environment or are in widespread use. 17?- estradiol is the principal estrogen found in mammals during reproductive years. Estriol is produced in large quantities during pregnancy. 17?-estradiol is the strongest, estriol the weakest. Estriol is water soluble, estrone and estradiol are not. Although estrogen is produced in women they are also at risk of over exposure to estrogen. Pesticides are extensively used today in agricultural settings to prevent and control pests. Various pesticides, including banned organochlorines (OCs) and modern non-persistent pesticides, have shown the ability to disrupt thyroid activity, disturbing the homeostasis of the thyroid system. Because these EDCs have adverse effects on health of both human and wildlife, it is imperative to develop viable costeffective analytical methods for the detection of these EDCs in complicated samples and at very low concentrations. Very high selectivity towards particular compounds is a very important property for the suitability of a detection method. This is because these compounds mostly coexist in complex matrices which makes the detection of a specific compound very challenging. It is paramount to develop highly sensitive and selective methods for the detection of these estrogens and phosphoric acid-based pesticides at trace levels. / 2021-08-31

The research described in this thesis contributes to the development of new strategies facilitating advanced understanding of the fundamental principles governing selective recognition of molecularly imprinted polymers (MIPs) at a molecular level for the rational optimization of biomimetic materials. The nature of non-covalent interactions involved in the templating process of molecularly imprinted polymers based on the self-assembly approach were investigated with a variety of analytical techniques addressing molecular level interactions. For this purpose, the concerted application of IR and 1H-NMR spectroscopy enabled studying the complexation of the template molecules 2,4-dichlorophenoxyacetic acid, quercetin, and o-, m-, and p-nitrophenol with a variety of functional monomers in the pre-polymerization solution by systematically varying the ratio of the involved components. In aqueous and non protic porogenic solvents, information on the interaction types, thermodynamics, and complex stoichiometry was applied toward predicting the optimum imprinting building blocks and ratios. Molecular dynamics simulations of 2,4-dichlorophenoxyacetic acid and its interactions with the functional monomer 4-vinylpyridine in aqueous and aprotic explicit solvent allowed demonstrating the fundamental potential of computer MD simulations for predicting optimized pre-polymerization ratios and the involved interaction types. The obtained results clearly demonstrate that the application of rapid IR/NMR pre-screening methods in combination with molecular modeling strategies is a promising strategy towards optimized imprinting protocols in lieu of the conventionally applied labor intensive and time-consuming trial-and-error approach. Furthermore, HPLC characterization of the produced MIPs compared to control polymers enabled a systematic approach to imprinting based on advanced understanding of the factors governing the formation of high-affinity binding sites during the polymerization. In addition, the importance of the combination of size, shape, and molecular functionalities for the selective recognition properties of MIPs was investigated. MIPs for the mycotoxins deoxynivalenol and zearalenone and for the antioxidant quercetin were applied as separation materials for advanced sample preparation in beverage analysis. The obtained results demonstrated the potential of MIPs for rapid one-step sample clean-up and pre-concentration from beverages such as wine and beer.

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.

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 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 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.