A ocorrência de N-nitrosaminas em produtos cosméticos e de higiene pessoal está relacionada com os ingredientes utilizados na formulação de tais artefatos. A N-nitrosodietanolamina (NDELA) é formada na presença da dietanolamina associada a outros componentes, tais como íons nitrito empregados como conservantes em matrizes cosméticas. São cancerígenas e, portanto, as condições em que tais itens de consumo são fabricados é um fator que deve ser mensurado. Assim, é aconselhável a realização de estudos que mostrem a ocorrência de NDELA em cosméticos, seus principais métodos de determinação, bem como os aspectos legais que indicam os níveis de concentração deste contaminante presente em diversas formulações. Foi avaliado neste trabalho o potencial de algumas técnicas de preparo de amostras na concentração de NDELA, tais como microextração líquido-líquido dispersiva em fase reversa (DLLME-RP), extração líquido-líquido com salting out e clean up no modo on-line. Os resultados obtidos foram confrontados com os dados presentes na literatura. Adicionalmente, utilizando os conceitos de polimerização via sol-gel e via precipitação para a síntese de polímeros molecularmente impressos (MIP), propõem-se uma possível rota sintética para a confecção de materiais seletivos para a pré-concentração da nitrosamina. / The occurrence of N-nitrosamines in cosmetic and personal hygiene are related to the ingredients used in the formulation of such artefacts. N-nitrosodiethanolamine (NDELA) is formed in the presence of diethanolamine associated with other components such as nitrite ions employed as preservatives in cosmetic matrices. They are carcinogenic and, therefore, the conditions under which such consumer items are produced is a factor that must be measured. It is therefore advisable to carry out studies that show the occurrence of NDELA in cosmetics, their main methods of determination as well as the legal aspects that indicate the levels of concentration of this contaminant present in various formulations. It was evaluated in this study the potential for some sample preparation techniques in the concentration of NDELA such as liquid-liquid microextraction dispersive reverse phase (DLLME-RP) liquid-liquid extraction with salting out and clean up online. The results were compared with data in the literature. Additionally, using the concepts of polymerization via sol-gel and precipitation route for the synthesis of molecularly imprinted polymers (MIP) are proposed as a possible synthetic route for the preparation of materials for selective pre-concentration of nitrosamine.

A ocorrência de N-nitrosaminas em produtos cosméticos e de higiene pessoal está relacionada com os ingredientes utilizados na formulação de tais artefatos. A N-nitrosodietanolamina (NDELA) é formada na presença da dietanolamina associada a outros componentes, tais como íons nitrito empregados como conservantes em matrizes cosméticas. São cancerígenas e, portanto, as condições em que tais itens de consumo são fabricados é um fator que deve ser mensurado. Assim, é aconselhável a realização de estudos que mostrem a ocorrência de NDELA em cosméticos, seus principais métodos de determinação, bem como os aspectos legais que indicam os níveis de concentração deste contaminante presente em diversas formulações. Foi avaliado neste trabalho o potencial de algumas técnicas de preparo de amostras na concentração de NDELA, tais como microextração líquido-líquido dispersiva em fase reversa (DLLME-RP), extração líquido-líquido com salting out e clean up no modo on-line. Os resultados obtidos foram confrontados com os dados presentes na literatura. Adicionalmente, utilizando os conceitos de polimerização via sol-gel e via precipitação para a síntese de polímeros molecularmente impressos (MIP), propõem-se uma possível rota sintética para a confecção de materiais seletivos para a pré-concentração da nitrosamina. / The occurrence of N-nitrosamines in cosmetic and personal hygiene are related to the ingredients used in the formulation of such artefacts. N-nitrosodiethanolamine (NDELA) is formed in the presence of diethanolamine associated with other components such as nitrite ions employed as preservatives in cosmetic matrices. They are carcinogenic and, therefore, the conditions under which such consumer items are produced is a factor that must be measured. It is therefore advisable to carry out studies that show the occurrence of NDELA in cosmetics, their main methods of determination as well as the legal aspects that indicate the levels of concentration of this contaminant present in various formulations. It was evaluated in this study the potential for some sample preparation techniques in the concentration of NDELA such as liquid-liquid microextraction dispersive reverse phase (DLLME-RP) liquid-liquid extraction with salting out and clean up online. The results were compared with data in the literature. Additionally, using the concepts of polymerization via sol-gel and precipitation route for the synthesis of molecularly imprinted polymers (MIP) are proposed as a possible synthetic route for the preparation of materials for selective pre-concentration of nitrosamine.

Orientadores: Adriana Vitorino Rossi, Regina Buffon / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T19:48:13Z (GMT). No. of bitstreams: 1 Manzoor_Suryyia_D.pdf: 4523839 bytes, checksum: 9aa2a7bc8376699193b0d5e83f0ec0f5 (MD5) Previous issue date: 2013 / Resumo: Este trabalho envolveu um estudo elaborado da técnica de impressão molecular para síntese, caracterização e avaliação de materiais impressos molecularmente (MIMs) para fluconazol (FLU), cafeína (CAF) e antocianinas (ACYs). O polímero de impressão molecular para FLU (FLUMIP) foi sintetizado utilizando-se ácido metacrílico (monômero funcional), etilenoglicoldimetacrilato (EGDMA) (agente reticulador) e acetonitrila em polimerização térmica. O FLUMIP foi caracterizado e aplicado como sorvente em cartuchos de extração em fase sólida (SPE). Sua capacidade de interação seletiva com o analito foi avaliada, obtendo-se alta afinidade para FLU, em comparação com análogos estruturais, com limite de detecção menor que 1,63X10 mmol/L com cromatografia de ultra alta eficiência acoplada com espectrometria de massas. Este MIP foi usado em cartuchos de SPE para extrair o analito de medicamento em cápsula, com recuperação de 91±10 % (n=9). Outro MIM obtido foi uma sílica organicamente modificada (ORMOSIL) para extração de CAF, a partir da reação de metacrilato de 3- (trimetoxisilil) propila e acetato de vinila, seguindo-se condensação e hidrólise com tetraetilortosilicato usando CAF como molécula modelo. Este ORMOLSIL foi caracterizado e testado quanto à sua eficácia de extrair CAF de amostras de café, com recuperação de 88±5 % (n=9); ele atuou como grupo seletivo com alta porcentagem de recuperação para teofilina (77 %) e teobromina (82 %). Limites de detecção e quantificação 5,14x10 e 1,71x10 mmol/L respectivamente foram obtidos com cromatografia líquida de alta eficiência. Também foi sintetizado um MIP usando rutina molécula modelo (RUTMIP), acrilamida (monômero funcional), EGDMA (agente reticulador) e tetraidrofurano por polimerização em bulk. Embora tenha sido alcançada impressão bem sucedida de rutina, confirmada pela comparação de afinidade de RUTMIP em aplicação de SPE (12 vezes maior que afinidade do polímero não impresso), não se alcançou a seletividade esperada para ACYs utilizando o RUTMIP / Abstract: This work involves an elaborative study of molecularly imprinting technique. Keeping in view its robustness and selectivity, this technique was applied for the synthesis of molecularly imprinted materials for the extraction of fluconazole (FLU), caffeine (CAF) and anthocyanins (ACYs). Molecularly imprinted polymer (MIP) for FLU (FLUMIP) was synthesized using methacrylic acid (functional monomer), ethyleneglycoldimethacrylate (crosslinker) and acetonitrile through thermal polymerization. The FLUMIP was characterized and applied as sorbent in solid phase extraction (SPE) cartridges. It was then evaluated for its ability to selectively interact with the analyte and presented an apparent affinity for FLU, which was confirmed by comparing it with structural analogues. The application of ultra high performance liquid chromatography with spectrometer mass detection, allowed a limit of detection 1.63x10 mmol/L. Furthermore, the SPE procedure was applied to extract FLU from medicine samples with recovery of 91±10 % (n=9). An organically modified silica (ORMOSIL) for CAF was also synthesized by reacting vinyl acetate and 3- (trimethoxysilyl) propyl methacrylate, followed by the condensation and hydrolysis with tetraethyl orthosilicate, using CAF as template molecule. The ORMOSIL was characterized and tested for its efficiency to extract the analyte from coffee samples and the percentage recovery of 88±5 % (n=9) was obtained. The cross reactivity studies for theophylline and theobromine showed high recovery (77 % and 82% respectively). The limit of detection and quantification, 5.14x10 and 1.71x10 mmol/L respectively, were achieved using high performance liquid chromatography. Also, a MIP for ACYs (RUTMIP) was synthesized using rutin (template molecule), EGDMA (cross linker) and tetrahydrofuran by the bulk polymerization method. A successful imprinting of rutin was attained. This can be confirmed by the high affinity of rutin for MIP (12 times greater than non imprinted polymer) during SPE procedure; however, the RUTMIP was not efficient enough to selectively extract ACYs from vegetal extracts / Doutorado / Doutora em Ciências

Cette thèse décrit la synthèse de nanoparticules de polymères à empreintes moléculaires (MIP, de l’anglais molecularly imprinted polymer) pour la reconnaissance de protéines, par une approche de synthèse en phase solide. Les polymères à empreintes moléculaires sont des récepteurs biomimétiques synthétisés sur mesure par un processus de nanomoulage du polymère autour de la molécule unique. Ils possèdent ainsi des cavités de reconnaissance spécifiques pour leur molécule cible. La technique de l'impression moléculaire pour les petites molécules cibles est bien établie, alors que l'impression de protéines reste encore un défi en raison de la flexibilité et complexité de leur structure native et de leurs nombreux sites fonctionnels, mais aussi en raison de leur faible stabilité dans des conditions inhabituelles. Par conséquent, une approche de synthèse en phase solide a été développée ici où la protéine est immobilisée sur un support avant la synthèse de nanoparticules hydrosolubles de MIP par polymérisation radicalaire. Les MIPs obtenus ont des affinités comparables à celles des anticorps, et des réactivités croisées faibles. Ils possèdent des avantages tels qu'une stabilité meilleure, un coût plus faible et peuvent potentiellement être régénérés et réutilisés, devenant ainsi des alternatives prometteuses aux anticorps naturels. Nous avons fabriqué des MIPs contre des protéases à sérine, telles la trypsine et la kallikréine, mais aussi contre un épitope peptidique de la protéine gp41 du VIH. Des nanogels de MIP thermosensibles ont été synthétisés dans un réacteur sous la forme d’une colonne thermostatée ou une boîte de Pétri, par polymérisation radicalaire initiée par voie thermique ou photochimique. Un simple changement de la température permet de libérer les MIPs de la protéine immobilisée. Ces MIPs sont hydrosolubles en fonction de la température et ont un diamètre inférieur à 100 nm. Leur affinité pour leur cible est élevée, avec un Kd du nano ou picomolaire. Ces 'anticorps synthétiques' ont été appliqués dans des tests d'adsorption sur microbalance à cristal de quartz, mais également comme 'chaperons synthétiques'. Des études préliminaires de la protection des protéines d'une dénaturation thermique ou par un pH défavorable ont été effectuées. L'utilisation d'un iniferter pour initier la photopolymérisation vivante du MIP a permis de synthétiser des nanogels de type core-shell. En introduisant des marqueurs fluorescents dans les MIPs, les tests d’immunoessai dans des fluides biologiques ont été démontrés, ce qui indique le grand potentiel de ces MIPs dans le diagnostic clinique. En conclusion, nous avons développé une nouvelle approche de synthèse de nanoparticules de MIP hydrosoluble ayant une haute affinité pour une protéine, utilisables à la place des anticorps dans des applications dans le monde réel tel que la détection de protéines biomarqueurs dans des échantillons complexes, et potentiellement comme principe actif in vivo. / This thesis describes the synthesis, by a solid-phase synthesis approach, of nanoparticles of molecularly imprinted polymers (MIPs) for the recognition of proteins. Molecularly imprinted polymers are biomimetic receptors synthesized by a nanomolding process of the polymer around single molecules. They therefore possess specific recognition cavities for their target molecule. The technique of molecular imprinting for small target molecules is well established, while protein imprinting remains a challenge due to the flexibility and complexity of their native structure and functional sites, but also because of their low stability under unusual conditions. Therefore, a solid-phase synthesis approach has been developed where the protein is immobilized on a support before the synthesis of water-soluble MIP nanogel particles by radical polymerization. The MIPs obtained have affinities comparable to those of antibodies, and low cross-reactivities. They have advantages such as better stability, lower cost, and can potentially be regenerated and reused, thus becoming promising alternatives to real antibodies. We have synthesized MIPs against serine proteases such as trypsin, and kallikrein, but also against a peptide epitope of the HIV gp41 protein. Thermosensitive MIP nanogels were synthesized in a thermostated column-type reactor or a petri dish, by thermally or photo-initiated radical polymerization. Their thermosensitivity allows the MIPs to be released from the immobilized protein by a simple temperature change. They are water-soluble as a function of temperature and have a diameter of less than 100 nm. Their affinity for their target is strong, with a Kd in the nano or picomolar range. These 'synthetic antibodies' have been applied in binding assays with quartz crystal microbalance, but also as 'synthetic chaperones'. Preliminary studies of the protection of proteins from thermal denaturation or from denaturation by an unfavorable pH have been carried out. The use of an iniferter to initiate the living photopolymerization of MIP made it possible to synthesize nanogels of core-shell type. By introducing fluorescent markers into MIPs, immunoassay applications in biological fluids have been demonstrated, indicating the great potential of these MIPs in clinical diagnostics. In conclusion, we have developed a novel approach to the synthesis of soluble MIP nanoparticles having high affinity for a protein, usable in place of antibodies in real world applications such as the detection of biomarker proteins in complex samples, and potentially as an active principle in vivo.

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.

L’objectif de cette thèse a consisté en la synthèse d’un polymère à empreinte moléculaire (MIP) permettant l’extraction du phosmet, un pesticide organophosphoré largement utilisé en oléiculture. La recherche du monomère fonctionnel (MF) disposant de la meilleure capacité à interagir de manière non-covalente avec le phosmet en présence du solvant porogène le plus approprié a été réalisée pour la toute première fois au moyen d’un capteur à acétylcholinestérase. Cette stratégie innovante a permis une meilleure compréhension des mécanismes cinétiques à l’œuvre lors de l’interaction MF-molécule cible. De par l’importance de son rôle dans la détermination de la structure d’un MIP, le choix d’un agent réticulant aux caractéristiques physico-chimiques adéquates a permis de sélectionner le meilleur MIP en s’appuyant sur l’étude des isothermes d’adsorption selon les modèles de Freundlich et Langmuir. La procédure d’extraction du phosmet selon la procédure MISPE (Molecularly Imprinted Solid Phase Extraction) a été effectuée par le biais d’une cartouche SPE dont la capacité a été évaluée à partir d’une solution standard. La validation du choix des réactifs de MIP sélectionnés a été confortée par la réalisation d’une expérience de réactivité croisée appliquée à une molécule analogue au phosmet. L’extraction du phosmet de l’huile d’olive a pu être effectuée avec succès selon un protocole d’extraction en flux inverse optimisé. Cette étude ouvre ainsi la voie à la recherche de nouvelles interactions MFs-molécules cibles au moyen de biocapteurs enzymatiques inhibant des composés toxiques tels que les herbicides, fongicides et autres pesticides. / The objective of this thesis has been the synthesis of a molecularly imprinted polymer (MIP) for the extraction of phosmet, an organophosphorus pesticide widely used in olive growing. The search for the functional monomer (FM) having the best ability to interact non-covalently with phosmet in the presence of the most suitable pore-forming solvent was carried out for the first time by means of an acetylcholinesterase sensor. This innovative strategy allowed us to better understand the kinetic mechanisms of FM-template interaction. Because of the importance of its role in determining the structure of a MIP, the selection of a crosslinking agent with adequate physicochemical characteristics made it possible to select the best MIP, whose adsorption isotherms were studied according to Freundlich and Langmuir models. Extraction of phosmet using a Molecularly Imprinted Solid Phase Extraction (MISPE) procedure was carried out via an SPE cartridge, whose capacity was evaluated from a standard solution. The choice of reagents and experimental conditions were validated by carrying out selectivity assays using another organophosphorus insecticide. Extraction of phosmet from olive oil was successfully carried out according to an optimized reverse flow extraction protocol. This work opens new opportunities for studying new FM-template interactions by means of enzymatic biosensors capable of detecting other inhibitors such as herbicides, fungicides and other pesticides.

O preparo da amostra é uma das etapas mais importantes em toda a análise química. O isolamento e a concentração dos componentes da amostra são cruciais e busca-se sempre que essas etapas sejam as mais simples e consumam o mínimo possível de tempo e reagentes. Nos últimos anos, um tipo de material tem se mostrado bastante útil para análises químicas a partir de fluidos biológicos, os polímeros molecularmente impressos (MIPs). Os MIPs são sintetizados por reações de polimerização, na presença de uma molécula molde (template). A molécula molde se liga aos monômeros funcionais do polímero durante a reação de polimerização e permanece ligada à superfície das cadeias poliméricas quando a reação se completa. Terminada a polimerização, realiza-se a completa lavagem das moléculas molde, assim, restam na superfície polimérica cavidades tridimensionais complementares à molécula empregada como molde. Essas cavidades permitem a ligação reversível e preferencial da molécula molde ou outras com estrutura química semelhante. A Cannabis sativa é a droga ilícita mais consumida em todo o mundo e nos últimos anos muita atenção tem se voltado a seus efeitos toxicológicos no corpo humano e a aplicações medicinais. Nesta dissertação, foi sintetizado um MIP com a molécula molde catequina para a extração e posterior análise por LC-MS/MS dos canabinóides Δ9-tetrahidrocanabinol (THC), 11-hidroxi-Δ9-tetrahidrocannabinol (THC-OH) e 11-nor-Δ9-tetrahidrocannabinol-9-ácido carboxílico (THC-COOH) em amostras de urina. O MIP produzido foi empacotado em microdispositivo e empregado no preparo das amostras de urina por microextração por sorvente empacotado (MEPS). O método desenvolvido apresentou boa linearidade (valores de r de 0,977 para o THC e 0,994 para THC-OH e THC-COOH). Os limites de detecção e de quantificação foram respectivamente de 5 ng mL-1 e 20 ng mL-1, para os compostos THC e THC-OH, na faixa linear de 25 a 250 ng mL-1. Para o composto THC-COOH os limites de detecção e quantificação alcançados foram de 1 ng mL-1 e 5 ng mL-1, respectivamente, na faixa linear de 5 a 170 ng mL-1. O método apresentou valores razoáveis de precisão entre 3,2% (THC-COOH) e 25,1% (THC) e de exatidão, que variou entre -18,4 e 17,4 (ambos para o THC). O MIP empregado no preparo da amostra mostrou-se mais seletivo e específico do que materiais normalmente empregados para a extração dos canabinoides das amostras de urina, além de a técnica de extração por MEPS apresentar baixo consumo de solventes e amostra para a extração dos analitos e posterior análise por LC-MS/MS. / The sample preparation is one of the most important steps in every chemical analysis. The isolation and concentration of the sample components are crucial and it is always sought that these steps are simple and consume the lowest amount of time and reagents. In the recent years, a type of material has proved to be very useful for chemical analyzes of biological fluids, the molecularly imprinted polymers (MIPs). MIPs are synthesized by polymerization reactions in the presence of a template molecule. The template molecule binds to the functional monomers of the polymer during the polymerization reaction and remains bonded to the surface of the polymeric chains after the reaction is complete. After the polymerization is finished, the complete washing of the template molecules is carried out, thus, three-dimensional cavities, complementary to the molecule used as a template, remain on the polymer surface. These cavities allow the reversible and preferential bonding of the template molecule or others with similar chemical structure. Cannabis sativa is the most commonly consumed illicit drug in the world and in recent years much attention has focused on its toxicological effects on human body and for medical applications. In this master dissertation, a MIP was synthesized with the catechin molecule as template, for extraction and subsequent analysis by LC-MS/MS of the cannabinoids Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. The MIP produced was packed in a microdevice and used in the preparation of the urine samples by microextraction by packed sorbent (MEPS). The developed method showed good linearity (r values of 0.977 for THC and 0.994 for THC-OH and THC-COOH). The detection and quantification limits were respectively 5 ng mL-1 and 20 ng mL-1 for THC and THC-OH in the linear range from 25 to 250 ng mL-1. For the compound THC-COOH the limits of detection and quantification achieved were 1 ng mL-1 and 5 ng mL-1, respectively, in the linear range from 5 to 170 ng mL-1. The method presented reasonable values of precision, between 3.2% (for THC-COOH) and 25.1% (for THC) and displayed accuracy ranging from -18.4 to 17.4 (both for THC). The MIP used in the sample preparation was more selective and specific than other materials usually employed for the extraction of the cannabinoids from the urine samples. The MEPS technique also showed low consumption of solvents and sample for sample preparation, extraction of analytes and subsequent analysis by LC-MS/MS.

O preparo da amostra é uma das etapas mais importantes em toda a análise química. O isolamento e a concentração dos componentes da amostra são cruciais e busca-se sempre que essas etapas sejam as mais simples e consumam o mínimo possível de tempo e reagentes. Nos últimos anos, um tipo de material tem se mostrado bastante útil para análises químicas a partir de fluidos biológicos, os polímeros molecularmente impressos (MIPs). Os MIPs são sintetizados por reações de polimerização, na presença de uma molécula molde (template). A molécula molde se liga aos monômeros funcionais do polímero durante a reação de polimerização e permanece ligada à superfície das cadeias poliméricas quando a reação se completa. Terminada a polimerização, realiza-se a completa lavagem das moléculas molde, assim, restam na superfície polimérica cavidades tridimensionais complementares à molécula empregada como molde. Essas cavidades permitem a ligação reversível e preferencial da molécula molde ou outras com estrutura química semelhante. A Cannabis sativa é a droga ilícita mais consumida em todo o mundo e nos últimos anos muita atenção tem se voltado a seus efeitos toxicológicos no corpo humano e a aplicações medicinais. Nesta dissertação, foi sintetizado um MIP com a molécula molde catequina para a extração e posterior análise por LC-MS/MS dos canabinóides Δ9-tetrahidrocanabinol (THC), 11-hidroxi-Δ9-tetrahidrocannabinol (THC-OH) e 11-nor-Δ9-tetrahidrocannabinol-9-ácido carboxílico (THC-COOH) em amostras de urina. O MIP produzido foi empacotado em microdispositivo e empregado no preparo das amostras de urina por microextração por sorvente empacotado (MEPS). O método desenvolvido apresentou boa linearidade (valores de r de 0,977 para o THC e 0,994 para THC-OH e THC-COOH). Os limites de detecção e de quantificação foram respectivamente de 5 ng mL-1 e 20 ng mL-1, para os compostos THC e THC-OH, na faixa linear de 25 a 250 ng mL-1. Para o composto THC-COOH os limites de detecção e quantificação alcançados foram de 1 ng mL-1 e 5 ng mL-1, respectivamente, na faixa linear de 5 a 170 ng mL-1. O método apresentou valores razoáveis de precisão entre 3,2% (THC-COOH) e 25,1% (THC) e de exatidão, que variou entre -18,4 e 17,4 (ambos para o THC). O MIP empregado no preparo da amostra mostrou-se mais seletivo e específico do que materiais normalmente empregados para a extração dos canabinoides das amostras de urina, além de a técnica de extração por MEPS apresentar baixo consumo de solventes e amostra para a extração dos analitos e posterior análise por LC-MS/MS. / The sample preparation is one of the most important steps in every chemical analysis. The isolation and concentration of the sample components are crucial and it is always sought that these steps are simple and consume the lowest amount of time and reagents. In the recent years, a type of material has proved to be very useful for chemical analyzes of biological fluids, the molecularly imprinted polymers (MIPs). MIPs are synthesized by polymerization reactions in the presence of a template molecule. The template molecule binds to the functional monomers of the polymer during the polymerization reaction and remains bonded to the surface of the polymeric chains after the reaction is complete. After the polymerization is finished, the complete washing of the template molecules is carried out, thus, three-dimensional cavities, complementary to the molecule used as a template, remain on the polymer surface. These cavities allow the reversible and preferential bonding of the template molecule or others with similar chemical structure. Cannabis sativa is the most commonly consumed illicit drug in the world and in recent years much attention has focused on its toxicological effects on human body and for medical applications. In this master dissertation, a MIP was synthesized with the catechin molecule as template, for extraction and subsequent analysis by LC-MS/MS of the cannabinoids Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. The MIP produced was packed in a microdevice and used in the preparation of the urine samples by microextraction by packed sorbent (MEPS). The developed method showed good linearity (r values of 0.977 for THC and 0.994 for THC-OH and THC-COOH). The detection and quantification limits were respectively 5 ng mL-1 and 20 ng mL-1 for THC and THC-OH in the linear range from 25 to 250 ng mL-1. For the compound THC-COOH the limits of detection and quantification achieved were 1 ng mL-1 and 5 ng mL-1, respectively, in the linear range from 5 to 170 ng mL-1. The method presented reasonable values of precision, between 3.2% (for THC-COOH) and 25.1% (for THC) and displayed accuracy ranging from -18.4 to 17.4 (both for THC). The MIP used in the sample preparation was more selective and specific than other materials usually employed for the extraction of the cannabinoids from the urine samples. The MEPS technique also showed low consumption of solvents and sample for sample preparation, extraction of analytes and subsequent analysis by LC-MS/MS.

Cette thèse décrit l'état de l'art des sondes et nanoparticules fluorescents traditionnels utilisés en imagerie de fluorescence ainsi que le développement de nouveaux nanomatériaux à base de polymère à empreinte moléculaire, aussi dénommé ‘anticorps plastique’, pour le ciblage et la bioimagerie. En biologie et en médecine, il y a un besoin constant de diagnostiquer diverses maladies pour leur éventuel traitement et prévention. Une distribution anormale et un taux élévé de glycosylation (e.g. acides hyaluronique et sialique) à la surface ou dans les cellules sont indicateurs d’une infection ou d’un cancer. Généralement, l’imagerie par fluorescence permet de visualiser, localiser et quantifier les biomarqueurs de pathologie mais à l’heure actuelle, il n’existe pas d’outil analytique fiable pour cibler spécifiquement les molécules de glycosylation car les anticorps et les lectines vendus dans le commerce ont une faible affinité et sélectivité vis-à-vis de ces cibles. Dans ce contexte, les polymères à empreintes moléculaires (MIPs) pourraient apporter une solution. Les MIPs sont des récepteurs synthétiques possédant des affinités et sélectivités comparables à ceux des anticorps, mais exhibant une stabilité physique, thermique et chimique bien plus accrue. De plus, leur fabrication est peu coûteuse et ne nécessite pas de tuer des animaux comme pour l’obtention des anticorps biologiques. Dans cette thèse, nous avons optimisé et synthétisé des MIPs biocompatibles pour leur utilisation en bioimagerie afin de détecter et quantifier l’acide hyaluronique et l’acide sialique sur les cellules et les tissus de peau humaine. L’acide glucuronique, une composante de l’acide hyaluronique et l’acide N-acétylneuraminique, l’acide sialique le plus commun, ont été utilisés comme molécules ‘patron’, générant des MIPs très sélectifs envers leur cible en milieu aqueux. Deux types de nanoparticules de MIPs fluorescents ont été synthétisés: (1) en incorporant un colorant rhodamine polymérisable dans la solution de pré-polymérisation et (2) en encapsulant des boîtes quantiques InP/ZnS générant ainsi des MIPs de type cœur-coquille. Pour cela, nous avons adopté une stratégie innovante qui consiste à synthétiser les coquilles de MIPs directement autour des boîtes quantiques en utilisant l’énergie de l’onde fluorescente émise par l’excitation des points quantiques, pour initier la polymérisation. Un protocole d'immunocoloration standard a ensuite été optimisé afin d’imager des kératinocytes humains fixés et vivants ainsi que des tissus de peau, par microscopie à épifluorescence et confocale. Les résultats étaient similaires à ceux obtenus par la méthode de référence utilisant une protéine biotinylée reconnaissant l'acide hyaluronique. L'imagerie multiplex en combinant deux MIPs couplés à deux couleurs de boîtes quantiques et l’imagerie des cellules cancéreuses ont également été démontrées. Bien que les MIPs n’étaient pas cytotoxiques aux concentrations utilisées pour la bioimagerie, la toxicité des différentes composantes du MIP pourrait être un frein à leur utilisation dans le domaine biomédical. Afin de rendre ces MIPs plus ‘inoffensifs’, nous avons supprimé l’amorceur de polymérisation, une molécule considérée comme toxique. Les MIPs ont été synthétisés en employant des monomères qui s’auto-initient sous l’effet de l’UV ou de la chaleur. La spécificité et la sélectivité des MIPs obtenus étaient similaires à ceux préparés avec des amorceurs. En conclusion, cette thèse décrit la première utilisation des MIPs comme anticorps synthétique pour la bioimagerie de fluorescence. Ce travail ouvre la voie à de nouvelles applications en détection, diagnostique et thérapie par des MIPs. / This thesis describes the state of the art in nanomaterials-based targeted bioimaging and introduces molecularly imprinted polymers, also termed ‘plastic antibodies’ as novel biorecognition agents for labeling and imaging of cells and tissues. In fundamental biology and medical diagnostics, there is a constant need to localize and quantify specific molecular targets. Abnormal glycosylation levels or distributions of hyaluronan or sialic acids on cells are indicators of infection or malignancy. In general, bioimaging with fluorescent probes enables the localization and qualitative or quantitative determination of these pathological biomarkers. However, no reliable tools for the recognition of glycosylation sites on proteins exist, because the commercially available antibodies or lectins have poor affinity and selectivity for these targets. In this context, tailor-made molecularly imprinted polymers (MIPs) are promising synthetic receptor materials since they present a series of advantages over their natural counterparts such as the ease and low cost of preparation and their physical and chemical stability. Thus, MIPs could provide a robust and specific imaging tool for revealing the location/distribution, time of appearance and structure of glycosylation sites on/in cells, which would lead to a better insight of the tremendously diverse biological processes in which these molecules are involved. Herein, we describe the synthesis of water-compatible MIPs for the molecular imaging of hyaluronan and sialylation sites on cells and tissues. Since molecular imprinting of entire biomacromolecules like oligosaccharides is challenging, we opted for what is commonly called the ‘epitope approach’, which was inspired by nature. The monosaccharides, glucuronic acid and N-acetylneuraminic acid were imprinted, and the resulting MIPs were able to bind these molecules when present and accessible on the terminal unit of hyaluronan and sialylation sites. Fluorescent MIPs were synthesized as rhodamine-labeled nanoparticles and as MIP-coated InP/ZnS core-shell quantum dot (QD) particles. For the coating of the QDs, a novel versatile solubilization and functionalization strategy was proposed, which consists of creating polymer shells directly on QDs by photopolymerization using the particles as individual internal light sources. A standard immunostaining protocol was then successfully adapted for the application of the fluorescently labeled MIPs to image fixed and living human keratinocytes and skin tissues, by epifluorescence and confocal fluorescence microscopy. The results were comparable to those obtained with a reference method where staining was done with a biotinylated hyaluronic acid binding protein. Multiplexed and cancer cell imaging were also performed, demonstrating the potential of molecularly imprinted polymers as a versatile biolabeling and bioimaging tool. Although the MIPs were not cytotoxic at the concentrations used for bioimaging, in order to render them generally applicable in biomedicine, where toxicity of the polymerization precursors is a matter of concern, we suppressed the initiator, a toxic chemical. Initiator-free MIPs were thus synthesized by using monomers that can self-initiate under UV irradiation or heat. The specificity and selectivity of the obtained MIPs were as good as the ones prepared with initiators. In conclusion, we have demonstrated for the first time the great potential of MIPs as synthetic antibody mimics for bioimaging. The possibility to associate other functionalities such as QDs and additionally attach drugs to the same material appears rather straightforward due to the synthetic polymeric nature of MIPs, which paves the way to new potential applications in theranostics.

The synthesis of materials with molecular recognition properties has become a topic of great technological and scientific interest. Molecular imprinting is one of the most effective strategies in preparing highly selective synthetic receptors. The technique of molecular imprinting involves the copolymerization of functional and cross-linking monomers in the presence of a molecular template. Following polymerization and subsequent removal of the template, the molecularly imprinted polymer (MIP) retains a “molecular memory” of the template. During rebinding, the resultant polymer shows higher affinity and selectivity towards the molecular template when compared to other structural analogs. Ease of preparation and high thermal and chemical stability of this class of materials offers a broad range of potential applications. Promising areas of application include separation, chromatography, catalysis, sensors, antibody mimics, and drug delivery etc. The thesis entitled “Molecularly Imprinted Polymers based on Fluorescent and Template binding Cross-linker” deals with the design and synthesis of several molecularly imprinted polymers (MIPs) using different functional and cross-linking monomers, the main focus being use of preformed template-monomer complex, use of fluorescent cross-linker and development of functional group containing cross-linker. Chapter 1: An Introduction to Molecularly Imprinted Polymers. The first chapter provides an introduction to the field of molecularly imprinted polymers. It presents an overview of molecular imprinting process including a brief history of its discovery and its evolution to the present form. This chapter further elaborates on the principle of molecular imprinting with an emphasis on different parameters that directly affect their performance. It also provides a brief review of the applications of molecularly imprinted polymers. Chapter 2: Highly Cross-linked Metal Ion Imprinted Polymers. The second chapter deals with the synthesis of series of highly cross-linked metal-ion imprinted polymers. The process of metal ion-imprinting usually involves carrying out the polymerization and cross-linking directly in presence of the appropriate metal ion. In the present study, chemical-immobilization method was adopted which involves the use of preformed metal complexes with polymerizable group for the imprinting. Acrylate complexes of various metal-ions, such as Cu2+, Zn2+, Co2+, Ni2+, Pb2+ and Cr3+, were synthesized prior to polymerization. These pre-assembled complexes were then used to prepare MIPs, in the anticipation that this would lead to enhanced selectivity. Ethyleneglycol dimethacrylate (EGDMA) was used as the cross-linking monomer. As a control, the respective non-imprinted polymers (NIPs) were also made in absence of the template metal ion. Following polymerization, the template metal ion was extracted from the resultant metal ion-imprinted polymer. The selectivity of the metal ion-imprinted polymers was examined by a batch process using analytical tools, such as, Atomic Absorption Spectroscopy (AAS) and Inductively Coupled Plasma Spectroscopy (ICP). The spectroscopic studies revealed significant selectivity of all the MIPs towards the template metal ion. Among all six metal ion-imprinted polymers, Pb2+ and Cr3+ ion-imprinted polymer showed remarkable selectivity, followed by Cu2+ and Zn2+ ion-imprinted polymers. The Co2+ and Ni2+ ion-imprinted polymers exhibited comparatively poor selectivity. Representative plots depicting the selectivity exhibited by Pb2+ and Cr3+ ion-imprinted polymers are shown in Figure 1. These observations were rationalized based on the size and geometric preferences imposed by the imprinted site on the ion that binds to it. Figure 1. Selectivity study for (a) Pb2+ ion-imprinted polymer, (b) Cr3+ ion-imprinted polymer. Chapter 3. Molecularly Imprinted Fluorescent Chemosensor for Copper (II). Cu(II) is a source of important pollutant and therefore, the development of sensors that can detect Cu(II) selectively as well as remove Cu(II) from contaminated samples is an important objective. The use of molecular imprinting technique is an appealing approach in this regard. For this, a fluorophore containing cross-linker, namely 9,10-bis-(acryloyloxymethyl)anthracene (BAMA) was synthesized. This fluorescent cross-linker was used along with the standard cross-linker, EGDMA, for preparing Cu2+ ion-imprinted polymer. The complex of copper methacrylate (Cu-MAA) was prepared prior to polymerization used for the preparation of MIP. The resultant imprinted polymer exhibited quenching of the fluorescence in presence of Cu2+ ion, both in organic and aqueous medium. The efficiency of quenching of NIP (prepared in absence of Cu2+ ion) was significantly lower than that of MIP. A typical stack spectra showing the quenching process, along with a comparison of the quenching efficiency of MIP and NIP is shown in Figure 2. The imprinted polymers showed significant selectivity over other non-template metal ions, thereby reaffirming the importance of the imprinting process. The sensitivity of the fluorescence detection could be enhanced by increasing the level of the fluorophore incorporation. The increased sensitivity in detecting Cu2+ ion, demonstrated by the MIP suggests that a statistically random incorporation of the fluorophore into MIP matrices could be a useful approach for imparting a sensing element to MIPs. Figure 2. Fluorescence spectra of the (a) imprinted (MIP-1) and (b) non-imprinted (NIP-1) polymers in the presence of various concentration of Cu(OAc)2 in methanol. (c) Comparison of quenching efficiency of MIP-1 and NIP-1. Data were collected 3 h after addition of copper solution. I0 and I are the fluorescence intensities at 399 nm of the polymers in the absence presence of copper respectively. Two individual runs are presented in (c). Chapter 4. Molecularly Imprinted Turn-Off-On Sensor. This chapter describes the design and synthesis of molecularly imprinted fluorescent turn-off-on sensor utilizing the same fluorescent cross-linker, BAMA. Combining the process of fluorescence resonance energy transfer (FRET) with molecular imprinting technique, a novel turn-off-on sensor was developed. A molecularly imprinted polymer was prepared using a fluorescent template Coumarin-30 (C-30). C-30 was chosen as the template to ensure a significant overlap of the emission spectra of BAMA and the absorption spectra of C-30, thereby optimizing for FRET. Figure 3. Structures of relevant molecules. The C-30 imprinted polymer exhibited simultaneous quenching in fluorescence (turn-off) of BAMA and enhancement in fluorescence (turn-on) of C-30 (Figure 4). The imprinted polymer showed significantly better performance over the non-imprinted polymer (NIP). Figure 4. Fluorescence spectra of the (a) imprinted (MIP) and (b) non-imprinted (NIP) polymers with increasing concentration of the template Coumarine-30 in methanol. The UV-vis studies revealed that the more effective quenching is indeed due to the affinity for C-30 exhibited by the higher binding imprinted polymer. The imprinted polymer also showed significant selectivity over structurally analogous molecules. Therefore, both high sensitivity and selectivity were realized in such novel off-on sensor. Extension of this concept to other biologically relevant fluorescent templates could lead to potentially useful applications. Chapter 5. Design of New Template Binding Cross-linker. In molecularly imprinted polymers (MIP), high cross-linking density (~80 to 90 mole percent) is essential to ensure high selectivity, which limits the functional (binding) monomer to about 10-20 mole percent. Methacrylic acid (MAA) and ethyleneglycol dimethacrylate (EGDMA) are the most common combination of functional monomer and cross-linker, respectively, used in molecular imprinting. Generally a molecularly imprinted polymer made with this combination, contains only 10-20% binding sites. This limitation of binding site density is an aspect that has largely been overlooked. In order to improve the efficiency of MIP materials by enhancing the number of binding sites, a new cross-linking monomer (CYDI, 1) with two carboxylic acid groups was designed and synthesized by coupling itaconic anhydride with cyclohexane dimethanol (Figure 5). Figure 5. Structures of relevant molecules. The new functional group bearing cross-linking monomer (1) Itaconate ester of cyclohexanedimethanol (CYDI), the template (2) theophylline (Theop) and the structural analogue of template (3) caffeine (Caff). This new cross-linking monomer was then employed for preparing molecularly imprinted polymer using a drug molecule, theophylline (Theop 2, a bronchodilator) as the template. Seven molecularly imprinted polymers were synthesized with different ratios of CYDI and EGDMA, keeping the cross-linking density constant. The binding efficiency and the selectivity of these imprinted polymers were thoroughly investigated. It was seen that while saturation binding values for theophylline increased continuously with functional cross-linker (CYDI) content, the optimum selectivity with respect to analogous substrate, caffeine, was attained at 40 mol% CYDI. These studies suggest that the approach of using functional group containing cross-linkers could lead to improved MIP performance.