Diese Dissertation befasste sich mit der Entwicklung von optischen Sensormaterialien für anionische Zielmoleküle durch die Kopplung der herausragenden Erkennungsfähigkeiten von molekular geprägten Polymere (molecularly imprinted polymers, MIPs) mit der Empfindlichkeit fluorometrischer Nachweisverfahren. In dieser Arbeit wurde dabei der direkte Einschritt-Nachweis für das Design der Sensormaterialien adaptiert. Hierbei wird eine Fluoreszenzsonde für die Signalübertragung kovalent in die Hohlräume der MIP-Matrix eingebaut. MIP-Sensormaterialien wurde in monolithischen, Dünnfilm- und Kern/Schale-Partikel-Formaten hergestellt. Die hergestellten Materialien wurden unter Verwendung unterschiedlicher Techniken charakterisiert. Die Performanz der Sensormaterialien wurde auch in Bezug auf die Sensitivität, Selektivität sowie Ansprechzeit bewertet. In dieser Arbeit wurden dabei Systeme untersucht, bei denen die Signalerzeugung sowohl auf dem „Einschalten“ als auch auf dem „Ausschalten“ der Fluoreszenz beruhte. Mit den hergestellten Materialien wurden dabei viele Ziele des Projekts erreicht. Sowohl die synthetisierten dünnen Filme als auch die Kern/Schale-Partikel zeigten eine hohe Selektivität für die geprägten Aminosäuren, auch in Bezug auf die Unterscheidung von Enantiomeren. Diese Sensormaterialien waren ebenfalls durch eine niedrige Nachweisgrenze bis 60 µM und eine schnelle Ansprechzeit von 20 Sekunden gekennzeichnet. Insbesondere die Kern/Schale-Partikel können mit verschiedenen Detektionstechniken gekoppelt werden und sind potentiell für die Entwicklung von miniaturisierten Messinstrumenten für die on-line-Detektion sowie Point-of-Care-Diagnostik (patientennahe Labordiagnostik) einsetzbar. / This dissertation derives from the DFG project aimed on preparing optical sensor material for anionic target through combing the outstanding recognition of Molecularly imprinted polymer (MIPs) and sensitive fluorescence technique. A single step direct sensing strategy is adopted to prepare the sensor material in this thesis. Here, a fluorescence probe is covalently embedded into the MIP cavity for signal transduction. MIP sensor material are prepared in forms of bulk, thin film and particles. The material is characterized using various techniques. The performance of the sensor materials is also assessed in terms of sensibility, selectivity as well response time. Both the switching on and off signaling methods are tested in this thesis. The prepared material achieves the goal of the project. Both the prepared thin film as well as core-shell particle show prominent selectivity even a strong enantioselective discrimination. These sensing materials also have low LOD to 60 µM and fast sensing response of 20 seconds. Especially the core-shell sensing particle can be coupled with various detection techniques and is potentially applicable for developing miniaturized sensing instrument for on-line detection as well as point of care diagnose.

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.

Profenofós é um organofosforado empregado como inseticida e acaricida amplamente utilizado no Brasil para o controle de pragas de cebolas, milho, soja, café, tomate, algodão, feijão, batata e outros. A irradiação é um processo empregado em todo o mundo e recomendada por diversos órgãos de saúde para a conservação de alimentos. A radiação ionizante utiliza raios gama, raios X ou aceleradores de elétrons e tem sido aplicada para eliminar ou reduzir a ação de agentes patogênicos e contribuir para aumentar o tempo de estocagem de vários alimentos. Os objetivos desse trabalho foram: (a) avaliar a degradação de soluções aquosas de profenofós submetidas à radiação ionizante, identificar e quantificar a formação de novos produtos por GC-MS; (b) analisar o efeito de feixe de elétrons em ervilhas inoculadas com soluções aquosas de profenofós; (c) sintetizar Polímeros Molecularmente Impressos (MIP) e Sílica Impressa Molecularmente (MIS), posteriormente, caracterizar os adsorventes em fase sólida e verificar sua seletividade para profenofós. O tratamento com aceleradores de elétrons com dose 31,6 kGy promoveu a formação de um novo produto de degradação e redução de 93,40 % de profenofós em soluções aquosas. Em ervilhas inoculadas com 1 μg de profenofós submetidas à radiação ionizante de 30,4 kGy promoveu uma redução na concentração de profenofós em 57,46 %. Além disso, foram realizadas sínteses de MIP e MIS para a extração em fase sólida de profenofós. Os MIS sintetizados por sol-gel mostraram-se eficazes para o reconhecimento molecular e extração seletiva de profenofós. / Profenofos is an organophosphate widely used in Brazil as insecticide and acaricide in the control of pests in onions, corn, soybeans, coffee, tomato, cotton, beans, potatoes among others. Irradiation is a process used worldwide and recommended by many health agencies for food preservation. Food irradiation preserving process uses accelerated electrons, gamma rays or X-rays. Ionizing radiation treatment is applied to eliminate or to reduce the action of pathogens and to increase the shelf life of some foods. The objective of this study were (a) to evaluate the degradation of aqueous solutions of Profenofos by ionizing radiation, identify and quantify the formation of new products by GC-MS; (b) to analyze the effects of electron beam in peas inoculated with aqueous solutions of Profenofos; (c) to synthesize Molecularly Imprinted Polymer (MIP) and Molecularly Imprinted Silica (MIS), subsequently characterize the adsorbents in solid phase and check its selectivity for profenofos. The treatment with electron accelerators with 31.6 kGy dose promoted the formation of a new by-product and 93.40 % reduction of profenofos in aqueous solutions. In peas inoculated with 1 μg of profenofos by ionizing radiation of 30.4 kGy promoted a reduction of 57.46 % in the concentration of profenofos. Furthermore, the MIP and MIS were performed for solid phase extraction of profenofos. The MIS synthesized by sol-gel proved to be effective for the recognition molecular and selective extraction of profenofos.

Profenofós é um organofosforado empregado como inseticida e acaricida amplamente utilizado no Brasil para o controle de pragas de cebolas, milho, soja, café, tomate, algodão, feijão, batata e outros. A irradiação é um processo empregado em todo o mundo e recomendada por diversos órgãos de saúde para a conservação de alimentos. A radiação ionizante utiliza raios gama, raios X ou aceleradores de elétrons e tem sido aplicada para eliminar ou reduzir a ação de agentes patogênicos e contribuir para aumentar o tempo de estocagem de vários alimentos. Os objetivos desse trabalho foram: (a) avaliar a degradação de soluções aquosas de profenofós submetidas à radiação ionizante, identificar e quantificar a formação de novos produtos por GC-MS; (b) analisar o efeito de feixe de elétrons em ervilhas inoculadas com soluções aquosas de profenofós; (c) sintetizar Polímeros Molecularmente Impressos (MIP) e Sílica Impressa Molecularmente (MIS), posteriormente, caracterizar os adsorventes em fase sólida e verificar sua seletividade para profenofós. O tratamento com aceleradores de elétrons com dose 31,6 kGy promoveu a formação de um novo produto de degradação e redução de 93,40 % de profenofós em soluções aquosas. Em ervilhas inoculadas com 1 μg de profenofós submetidas à radiação ionizante de 30,4 kGy promoveu uma redução na concentração de profenofós em 57,46 %. Além disso, foram realizadas sínteses de MIP e MIS para a extração em fase sólida de profenofós. Os MIS sintetizados por sol-gel mostraram-se eficazes para o reconhecimento molecular e extração seletiva de profenofós. / Profenofos is an organophosphate widely used in Brazil as insecticide and acaricide in the control of pests in onions, corn, soybeans, coffee, tomato, cotton, beans, potatoes among others. Irradiation is a process used worldwide and recommended by many health agencies for food preservation. Food irradiation preserving process uses accelerated electrons, gamma rays or X-rays. Ionizing radiation treatment is applied to eliminate or to reduce the action of pathogens and to increase the shelf life of some foods. The objective of this study were (a) to evaluate the degradation of aqueous solutions of Profenofos by ionizing radiation, identify and quantify the formation of new products by GC-MS; (b) to analyze the effects of electron beam in peas inoculated with aqueous solutions of Profenofos; (c) to synthesize Molecularly Imprinted Polymer (MIP) and Molecularly Imprinted Silica (MIS), subsequently characterize the adsorbents in solid phase and check its selectivity for profenofos. The treatment with electron accelerators with 31.6 kGy dose promoted the formation of a new by-product and 93.40 % reduction of profenofos in aqueous solutions. In peas inoculated with 1 μg of profenofos by ionizing radiation of 30.4 kGy promoted a reduction of 57.46 % in the concentration of profenofos. Furthermore, the MIP and MIS were performed for solid phase extraction of profenofos. The MIS synthesized by sol-gel proved to be effective for the recognition molecular and selective extraction of profenofos.

<p>The thesis describes the synthesis of chromatographic media using several different approaches, their characterizations and applications in liquid chromatography. The steps to achieve a separation column for a specific analyte are presented. The main focus of the study was the design of novel molecularly imprinted polymers.</p><p>Attachment of monolithic polymeric substrates to the walls of fused silica capillaries was studied in Paper I. With a broad literature survey, a set of common methods were tested by four techniques and ranked by their ability to improve anchoring of polymers. The best procedure was thus used for all further studies.</p><p>Synthesis of monoliths in capillary columns was studied in Paper II. With the goal of separating proteins without denaturation, various monoliths were polymerized in situ using a set of common monomers and cross-linkers mixed with poly(ethylene glycol) as porogen. The resulting network was expected to present “protein-friendly pores”. Chemometrics were used to find and describe a set of co-porogens added to the polymerization cocktails in order to get good porosity and flow-through properties.</p><p>Assessment of the macroporous structure of a monolith was described in Paper III. An alternative method to mercury intrusion porosimetry was proposed. The capillaries were embedded in a stained resin and observed under transmission electron microscope. Images were then computed to determine the pore sizes.</p><p>Synthesis of molecularly imprinted polymers grafted to a core mono-lith in a capillary was described in Paper IV. The resulting material, imprinted with local anaesthetics, was tested for its chromatographic performance. Similar imprinted polymers were characterized by microcalorimetry in Paper V. Finally, imprinted monoliths were also synthesized in a glass tube and further introduced in a NMR rotor to describe the interactions between stationary phase and template in Paper VI.</p>

The thesis describes the synthesis of chromatographic media using several different approaches, their characterizations and applications in liquid chromatography. The steps to achieve a separation column for a specific analyte are presented. The main focus of the study was the design of novel molecularly imprinted polymers. Attachment of monolithic polymeric substrates to the walls of fused silica capillaries was studied in Paper I. With a broad literature survey, a set of common methods were tested by four techniques and ranked by their ability to improve anchoring of polymers. The best procedure was thus used for all further studies. Synthesis of monoliths in capillary columns was studied in Paper II. With the goal of separating proteins without denaturation, various monoliths were polymerized in situ using a set of common monomers and cross-linkers mixed with poly(ethylene glycol) as porogen. The resulting network was expected to present “protein-friendly pores”. Chemometrics were used to find and describe a set of co-porogens added to the polymerization cocktails in order to get good porosity and flow-through properties. Assessment of the macroporous structure of a monolith was described in Paper III. An alternative method to mercury intrusion porosimetry was proposed. The capillaries were embedded in a stained resin and observed under transmission electron microscope. Images were then computed to determine the pore sizes. Synthesis of molecularly imprinted polymers grafted to a core mono-lith in a capillary was described in Paper IV. The resulting material, imprinted with local anaesthetics, was tested for its chromatographic performance. Similar imprinted polymers were characterized by microcalorimetry in Paper V. Finally, imprinted monoliths were also synthesized in a glass tube and further introduced in a NMR rotor to describe the interactions between stationary phase and template in Paper VI.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-08-20T13:36:48Z No. of bitstreams: 2 Dissertação - Lidya Cardozo da Silva - 2018.pdf: 2382834 bytes, checksum: e5a79619d923d442540c5bf0549318bd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-08-20T13:38:04Z (GMT) No. of bitstreams: 2 Dissertação - Lidya Cardozo da Silva - 2018.pdf: 2382834 bytes, checksum: e5a79619d923d442540c5bf0549318bd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-20T13:38:04Z (GMT). No. of bitstreams: 2 Dissertação - Lidya Cardozo da Silva - 2018.pdf: 2382834 bytes, checksum: e5a79619d923d442540c5bf0549318bd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-07-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Jatropha curcas L. is a euphorbiaceous oilseed plant considered toxic to humans and animals due to the presence of phorbol esters (PEs). Traditionally, the detection of these toxic compounds has been done in J. curcas seeds and derivates via chromatographic separation methods such as HPLC-UV and HPLC-MS. Although efficient, these techniques are laborious and require high time and solvent consumption, thus it would be interesting the development of new analytical methods to determine these compounds with more practicality. Probe electrospray ionization is frequently used in ambient mass spectrometry allowing analysis with minimum sample preparation. However, for complex samples analysis, this technique presents low sensitivity and ionization suppression. In this study, a molecularly imprinted polymer-coated probe electrospray ionization mass spectrometry (MIPCPESI-MS) method was developed for determination of phorbol esters in methanolic extracts of Jatropha curcas leaves with direct extraction form the ionization source. The synthesized molecularly imprinted polymer (MIP) proved to be adequate for extraction of the PEs in methanolic extracts of J. curcas leaves with better performance as extraction phase in comparison with the non-imprinted polymer (NIP). The MIPCPESI method allowed detection of phorbol 12,13-diacetate (PDA) and other three PEs metabolite ions from Jatropha leaves with minimal sample preparation, and with higher signal intensities compared to analysis with conventional PESI. For the PDA, calibration curve exhibited linearity with R2 > 0.99, LOD and LOQ in µg.mL-1 range, precision and accuracy values, respectively, between 4.06 to 13.49% and -1.60 to -15.26 %. Finally, MIPCPESI was employed for PDA quantification in methanolic extracts of six different J. curcas leaves genotypes resulting in concentrations ranging from 222.19 ± 23.55 to 528.23 ± 19.72 µg.g-1 for toxic samples. / A Jatropha curcas L. é uma oleaginosa euforbiácea considerada tóxica para humanos e animais devido à presença de ésteres de forbol (PEs). Tradicionalmente, a detecção destes compostos tóxicos tem sido feita em tortas e sementes de J. curcas por meio do uso de técnicas de separação cromatográfica como HPLC-UV e HPLC-MS que apesar de eficientes são laboriosas e requerem alto consumo de tempo e solventes. Dessa forma, seria interessante o desenvolvimento de novas técnicas analíticas para determinação desses compostos com maior praticidade. Probe electrosrpay ionization (PESI) é uma das técnicas de ionização utilizadas na espectrometria de massas ambiente que permite análises rápidas com mínimo preparo de amostras. No entanto, para análise de amostras complexas essa técnica apresenta baixa sensibilidade e supressão iônica. Neste estudo, foi desenvolvido um método de análise por espectrometria de massas por Probe electrospray revestido com polímero molecularmente impresso (MIPCPESI-MS) para determinação de ésteres de forbol em extratos metanólicos de folhas de Jatropha curcas com extração direta da fonte de ionização. O polímero molecularmente impresso (MIP) sintetizado mostrou-se adequado para extração de PEs em extratos metanólicos de folhas de J. curcas tendo melhor desempenho como fase extratora quando comparado ao polímero não molecularmente impresso (NIP). O método MIPCPESI-MS possibilitou a detecção do forbol 12,13-diacetato (PDA) e de outros três íons metabólitos presentes nas folhas de J. curcas com mínimo preparo de amostras e com maior intensidade de sinais quando comparado às análises com PESI convencional. Para o PDA, a curva de calibração apresentou linearidade com R2 > 0.99, LOD e LOQ na faixa de µg.mL-1, valores de precisão entre 4.06 e 13.49 % e exatidão entre -1.60 e -15.26 %. Posteriormente, o método MIPCPESI foi empregado na quantificação de PDA em seis extratos metanólicos de diferentes genótipos de folhas de J. curcas resultando em valores concentrações entre 222.19 ± 23.55 a 528.23 ± 19.72 µg.g-1 nas amostras tóxicas.

Au cours des dernières années, les matériaux énergétiques, tels que le 2,4,6-trinitrotoluène (TNT) et le 1,3,5-trinitro perhydro-1,3,5-triazine (RDX), ont été utilisés lors des activités d’entrainement militaire, ayant un impact potentiel sur l’environnement, les bases militaires, la population environnante, la faune et la flore. Sur les champs de tir, les sols à proximité des cibles et autour des positions de tir nécessitent une surveillance particulière, puisqu’il est possible de trouver une importante quantité de résidus d’explosif qui peuvent être transportés vers les eaux de surface et des eaux souterraines avec les précipitations. Toutefois, la procédure actuelle pour détecter des matériaux énergétiques dans l'eau naturelle est complexe, longue, nécessite un personnel hautement spécialisé et augmente le risque de contamination croisée. Par conséquent, il est difficile d'assurer un contrôle rapide et continu des contaminants. En fait, l'objectif des travaux présentés dans ce mémoire est de développer une technique pour identifier et quantifier les explosifs et leurs produits de dégradation dans les eaux naturelles. En outre, ce test doit être in situ, en continu, peu coûteux et rapide. La résonance des plasmons de surface (SPR) a été donc utilisée pour quantifier les matériaux énergétiques. Une matrice de bis-aniline réticulée avec des nanoparticules d’or (AuNPs) est utilisée en tant que polymère à empreinte moléculaire (MIP) sur le film d'or pour capturer sélectivement le composé d’intérêt. L'association de la molécule d’intérêt, tel que le TNT ou RDX, au MIP par interactions π-donneur-accepteur permet la détection d'explosifs suite aux changements d’indice de réfraction sur la sonde. Le couplage entre les plasmons des AuNPs et de la couche d'or peut également augmenter les signaux SPR. Le test optimisé a ensuite été utilisé sur une base militaire canadienne. L'utilisation de cet essai à base de MIP fournit un outil pour l'extraction et la pré-concentration de TNT ou RDX à la surface d’une sonde SPR et permet leur détection en continu de faibles concentrations dans les eaux naturelles. / Over the last few years, energetic materials such as 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) have been used and show a probable environmental impact on military bases, the surrounding population, fauna and flora, caused by military training involving munitions. On shooting ranges, soils near the firing positions and around targets require special monitoring, since the quantities of explosives residues found can be significant, and these compounds can be transported to surface water and groundwater by precipitation. However, the current procedure to detect energetic materials in natural water is complex, long and poorly adapted to. These operations require highly specialized personnel and increase the risk of cross contamination. Therefore, it is difficult to ensure a fast and continuous monitoring of the contaminants. Here, the objective is to develop a technique for identifying and quantifying explosives and their degradation products in natural water. Also, this test has to be in-situ, inexpensive and fast. Surface plasmon resonance (SPR) has been proposed to probe energetic materials. A bis-aniline-cross-linked gold nanoparticles (AuNPs) matrix is used as a molecular imprinted polymer (MIP) on gold film to selectively capture the target compound. The association of the target such as TNT or RDX to the MIP with π-donor-acceptor interactions have allow the detection of explosives by following SPR refractive changes. Plasmon coupling effects between the AuNPs and the gold film could also increase the SPR signals. The optimal sensor was then used on site to detect RDX in underground water of a Canadian military base. The utilisation of MIP based assay will provide a tool for the extraction and pre-concentration of TNT or RDX on the detector’s surface and will allow the detection of lower concentrations in natural water.

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.