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

[pt] Neste trabalho, polímeros de impressão molecular (MIP) foram desenvolvidos para aplicação em procedimentos de extração em fase sólida (SPE) visando separar e concentrar quercetina (flavonóide), piraclostrobina (pesticida) e kanamicina (antibiótico) em amostras contendo substâncias interferentes na determinação dos analitos-alvo. A seletividade em relação aos analitos de interesse foi conseguida pela interação específica dessas espécies químicas com os sítios de reconhecimento dos polímeros. A produção desses MIPs foi baseada na polimerização em bulk e, de modo a se comprovar a efetividade dos mesmos, o desempenho destes foram comparados com polímeros não-impressos (NIP) correspondentes, que foram produzidos sem o uso da molécula-molde. Os procedimentos para síntese são simples e o material produzido é quimicamente resistente nas condições de uso. A caracterização do material produzido foi feita por meio de microscopia de varredura eletrônica (MEV) e espectrometria de absorção na região do infravermelho (IV). O MIP preparado com quercetina foi empregado na extração seletiva deste flavonóide em amostras de urina e de suplemento alimentar e permitiu a obtenção de limite de detecção de 4,58 x 10-8 mol L-1 usando espectrofotometria de absorção no UV-vis com recuperações superiores a 90 porcento e separação efetiva de outros flavonóides como a flavona e naringenina. O MIP preparado com piraclostrobina foi usado na análise de amostra de água de rio e urina e permitiu o alcance de limite de detecção de 4,6 x 10-9 mol L-1, usando detecção por espectrofluorimetria, e recuperações maiores que 90 porcento na presença de outros pesticidas da classe das estrobilurinas. Para o MIP preparado com kanamicina, o limite de detecção alcançado usando detecção com sonda de nanopartículas fluorescentes foi 9 ng mL-1 (1,5 x 10-8 mol L-1) com aplicação na análise de urina e vacina contra febre amarela com recuperações maiores a 90 porcento. / [en] In this work, molecularly imprinted polymers (MIPs) have been developed for application procedures for solid phase extraction (SPE) in order to separate and concentrate quercetin (flavonoid), pyraclostrobin (pesticide) and kanamycin (antibiotic) in samples containing interfering substances in the determination of target analytes. The selectivity in respect of analytes has been achieved by specific binding of these chemical species with the recognition sites of the polymers. The production of MIP polymerization was based on "bulk" and in order to prove the effectiveness thereof, the performance of these polymers were compared with non-printed (NIP) thereof, that were produced without the use of template molecule. The procedures for synthesis are simple and relatively low cost and is chemically resistant material produced under the conditions of use. The characterization of the material produced was done by scanning electron microscopy (MEV), absorption spectroscopy in the infrared region and CHN elemental analysis. The MIP prepared with quercetin was used for the selective extraction of flavonoids in urine samples and dietary supplement and allowed to obtain a detection limit of 4,58 x 10-8 mol L-1 using absorption spectrophotometry in the UV-vis with recoveries exceeding 90 percent and effective separation of other flavonoids such as naringenin and flavone. The MIP prepared with pyraclostrobin was used to analyze samples of urine and river water and allowed to reach the detection limit of 4,6 x 10-9 mol L-1, detection using spectrofluorimetry and recoveries greater than 90 percent in presence of other pesticides from the class of strobilurins. For the MIP prepared with kanamycin, the detection limit using detection with fluorescent nanoparticles probe was 9 ng mL-1 (1,5 x 10-8 mol L-1) application to the analysis of urine and yellow fever vacine with recoveries greater than 90 percent.

L’impression moléculaire de polymères est une technique utile pour créer des récepteurs artificiels capables de lier sélectivement des substrats. Cette propriété de reconnaissance rend les polymères à empreintes moléculaires particulièrement intéressants pour développer des catalyseurs macromoléculaires. Notre objectif a ainsi été d’accéder à un catalyseur supramoléculaire recyclable et capable d’oxyder des dérivés du soufre dans des conditions hétérogènes en présence d’un oxydant doux et éco-compatible tel que le peroxyde d’hydrogène. Compte-tenu de l’engagement du laboratoire dans la lutte contre les armes chimiques, nous avons décidé d’orienter nos travaux vers la mise au point d’un système catalytique qui serait utile pour la décontamination de surfaces ou de matériels, suite à l’exposition à certains composés dont la détoxification passe par l’oxydation sélective d’un sulfure. Nous avons ainsi préparé un MIP à base de porphyrine dont nous avons testé l'efficacité dans l'oxydation de divers sulfures d’intérêt. Les conditions douces utilisées pour la catalyse ont ainsi permis de contrôler le degré d’oxydation d’un simulant de l’ypérite, en obtenant avec une très grande sélectivité le dérivé sulfoxyde, sans que le dérivé sulfone qui serait tout aussi toxique que l’arme chimique elle-même ne se forme. Aves des structures plus proches de celle du VX, nous avons montré qu’il était possible d’oxyder régiosélectivement le soufre, et mis en évidence l’intérêt de ce procédé pour faciliter le clivage d’une liaison C-S. Cette approche représente donc une stratégie de décontamination intéressante qu’il conviendra d’évaluer dans le cas de véritables agents de guerre chimique. / Molecular imprinting polymer is a useful technique for creating artificial receptors able of selectively binding substrates. These recognition properties are particularly interesting for developing macromolecular catalysts. Our aim was to access to a reusable supramolecular catalyst able of oxidizing sulfur derivatives under heterogeneous conditions in the presence of a mild and eco-sustainable oxidant such as hydrogen peroxide. Given the expertise of the team, we decided to focus our work on the development of a catalytic system for the decontamination of surfaces or equipment, after an exposure to compounds that can be detoxified by a selective oxidation of sulfides. Thus, we prepared a porphyrin-based MIP that we tested in the oxidation of various sulfides. The mild conditions used for the catalysis allowed a control of the oxidation degree of a simulant of yperite, leading to a great selectivity the sulfoxide derivative, and avoiding the toxic sulfone formation. With other structures similar to VX, we showed that it is possible to regioselectively oxidize the sulfur atom, and highlighted the interest of this process to facilitate the cleavage of a C-S bond. This approach is then an interesting decontamination strategy that will be evaluated in the future to the case of true chemical warfare agents.

Lysophosphatidic acid (LPA) influences many physiological processes, such as brain and vascular development. It is associated with several diseases including ovarian cancer, breast cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, multiple myeloma atherosclerotic diseases, cardiovascular diseases, pulmonary inflammatory diseases and renal diseases. LPA plasma and serum levels have been reported to be important values in diagnosing ovarian cancer and other diseases. However, the extraction and quantification of LPA in plasma are very challenging because of the low physiological concentration and similar structures of LPA to other phospholipids. Many previous studies have not described the separation of LPA from other phospholipids, which may make analyses more challenging than necessary. We developed an SPE extraction method for plasma LPA that can extract LPA at high purity. We also developed an HPLC post-column fluorescence detection method that allows the efficient quantification of LPA. These methods were used in a clinical study for ovarian cancer diagnosis to help validate LPA as a biomarker of ovarian cancer. Moreover, molecular imprinted polymers (MIPs) were designed and synthesized as material for the improved extraction of LPA. Compared to the commercially available materials, the MIP developed shows enhanced selectivity for LPA. The extraction was overall relatively more efficient and less labor-intensive.

[pt] Nanopartículas de ouro dispersas em água (AuNPs-H) e nanopartículas de ouro na presença dos surfactantes brometo de cetiltrimetilamônio (AuNPs-CTAB) e brometo de didecildimetilamônio (AuNPs-C10DAB) foram sintetizadas e caracterizadas por diversas técnicas. As variações nos perfis espectrais no UV-Vis dessas nanopartículas foram estudadas levando-se em consideração a intensidade e o comprimento de onda máximo da banda de ressonância plasmônica de superfície localizada (LSPR); foram acompanhadas em função do tempo para nanopartículas estocadas sob refrigeração (4 graus celsius) com as mantidas na temperatura ambiente (27 graus celsius). Um comportamento anômalo (mais instável) foi observado para AuNPs-C10DAB na concentração 1,0 ×10-4 mol L-1. As suas atividades catalíticas também foram avaliadas na presença de 4-nitrofenol e verificou-se que a cinética mais efetiva foi do sistema sem surfactante. Finalmente, um estudo de interação com aminoglicosídeos (AMG) foi feito visando o uso das nanopartículas como sonda analítica. Todavia, somente AuNPs-H apresentaram resultados satisfatórios, o que indica que o surfactante impede que o AMG se aproxime da superfície das nanopartículas, visto que, em concentrações mais elevadas de CTAB ou C10DAB, não há variação significativa no sinal original da sonda quando o AMG está presente. Um método analítico foi desenvolvido baseado na interação AuNPs-H-gentamicina e AuNPs-H-neomicina a partir da otimização dos parâmetros: concentração de AuNPs-H, tampão, tempo de medição e faixa de concentração de AMG. Na condição ajustada para sondagem quantitativa, cada dispersão de trabalho foi constituída por 40 porcento v/v (1,2 × 10-9 mol L-1) de AuNPs-H 1,0 mL de tampão citrato pH 4,0 (1,0 × 10-2 mol L-1) e volume final de 5,0 mL ajustado pela adição água ultrapura, após micro-volumes apropriados de soluçãoestoque de AMG (exceto na dispersão branco) ou de amostras adicionadas. O monitoramento da variação dos perfis espectrais foi feito em 511 e em 681 nm, habilitando a construção de curvas analíticas em 681, e na razão (681/511). Para gentamicina, a faixa linear variou de 0,6 a 600 microgramas L-1 com o limite de detecção (LD) de 0,06 microgramas L-1. Para neomicina, faixa linear foi de 7,3 a 550 microgramas L-1, tendo LD de 6,2 microgramas L-1. A aplicação do método foi feita determinando-se gentamicina (fortificação) em leite integral, e neomicina em solução controle (amostra aquosa simulada), medicamento e em saliva. Para habilitar a seletividade do método foi necessária a utilização de extração em fase sólida (SPE) em cartuchos comercias empacotados com polímero de impressão molecular de AMG. Em amostras de leite (40 microlitros), após limpeza e SPE, foi possível quantificar 1,72 mais ou menos 0,03 microgramas de gentamicina, o equivale a 39,1 mais ou menos 0,2 porcento. As recuperações para neomicina em amostras de medicamento foram de aproximadamente 45 mais ou menos 3,0 porcento (após SPE), indicando interferências de outros componentes. Neomicina também foi recuperada em amostras de saliva após uso do medicamento obtendo valores próximos a 0,36 mais ou menos 0,02 microgramas. Em contraste, as recuperações em amostras simuladas chegaram até 102,6 mais-menos 1,3 porcento (sem necessidade de SPE). Por fim foi feita uma avaliação comparativa de interação de AuNPs-H e nanopartículas de ouro sintetizadas com redução com citrato (AuNPs-citrato) que indicou diferença de comportamento na interação com AMG. / [en] Water-dispersed gold nanoparticles (AuNPs-H) and gold nanoparticles in the presence of surfactants cetyltrimethylammonium bromide (AuNPs-CTAB) and didecyldimethylammonium bromide (AuNPs-C10DAB) were synthesized and characterized using several techniques. The differences in the UV-Vis spectral profiles of these nanoparticles were studied monitoring the intensity and the localized surface plasmon resonance band (LSPR) maximum wavelength; these were monitored as a function of time taking into account storage conditions: under refrigeration (4 Celsius degrees) and at room-temperature (27 Celsius degrees). Anomalous (more unstable) profile was observed for AuNPs-C10DAB at 1.0 × 10-4 mol L-1. Their catalytic activities were also evaluated in the presence of 4-nitrophenol and it was shown that the most effective kinetics was observed for the system without surfactant. Finally, an interaction study with aminoglycosides (AMG) was conducted aiming the application of nanoparticles as analytical probe. Only AuNPs-H produced adequate results, indicating that the surfactant prevents interaction between AMG and the surface of the nanoparticles since at higher concentrations of CTAB or C10DAB there is no significant variation in the original probe signal in presence of AMG. An analytical method was developed based on the AuNPs-H-gentamicin and AuNPs-H-neomycin interaction taking into consideration the optimization of the parameters: AuNPs-H concentration, buffer, signal stabilization time and the AMG concentration range. In the adjusted condition for quantitative probing, each dispersion consisted of 40 percent v/v (1.2 × 10-9 mol L-1) of AuNPs-H, 1.0 mL of citrate buffer pH 4.0 (1.0 × 10-2 mol L-1) and final volume of 5.0 mL adjusted with ultrapure water addition, after appropriate micro-volumes of a stock solution of AMG (except in blank dispersion) or sample added. The spectral profiles were monitored at 511 and 681 nm, allowing theconstruction of analytical curves at 681, and at the ratio (681/511). For gentamicin, the linear range reached from 0.6 to 600 microgram L-1 with the limit of detection (LD) of 0.06 microgram L-1 . For neomycin, the linear range was 7.3 to 550 microgram L-1, with LD of 6.2 microgram L-1. The application of the method was made by determining gentamicin(fortification) in whole milk, and neomycin in control solution (simulated aqueous sample), pharmaceutical samples and saliva. In order to enable selectivity of the method it was necessary to use solid phase extraction (SPE) using a commercial SPE cartridge packed with an AMG molecular imprinted polymer. In milk samples (40 microlitre), after clean up and SPE, it was possible to quantify 1,72 plus-minus 0,03 microgram gentamicin, which is equivalent to 39,1 plus-minus 0,2 percent). Recoveries for neomycin in drug samples were approximately 45 plus-minus 3.0 percent (after SPE), indicating interferences of other components. Neomycin was also recovered in saliva samples after drug use obtaining values close to 0.36 plus-minus 0.02 microgram. In contrast, the recoveries in simulated samples reached up to 102.6 plus-minus 1.3 percent (no need for SPE). Finally, a comparative evaluation of the interaction of AuNPs-H and gold nanoparticles synthesized with reduction with citrate (AuNPs-citrato) was performed, indicating differences in terms of the interaction with AMG.

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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.