Este trabalho descreve o desenvolvimento de novos métodos cromatográficos em conjunto com técnicas de microextração para determinação de parabenos em produtos cosméticos e leite humano e rifampicina em amostras de plasma. Destaca-se também o desenvolvimento da fase extratora molecularmente impressa para extração em fase sólida (SPE) de parabenos em amostras de leite humano. O capítulo I descreve a avaliação da extração sortiva em barra de agitação (SBSE) em conjunto com a cromatografia líquida (LC) com detecção espectrofotométrica (LC-UV) para análises de parabenos em produtos cosméticos para fins de controle de qualidade. As variáveis do processo de extração SBSE, tais como tempo e temperatura de extração, pH e força iônica da amostra e condições de dessorção foram otimizadas para aumentar a sensibilidade analítica do método e diminuir o tempo de análise. O método SBSE/LC-UV desenvolvido apresentou faixa linear correspondente ao limite de quantificação (LQ) a 2,50 µg. mg-1 e coeficientes de determinação maiores que 0,993. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 5,0% para todos os parabenos analisados e a exatidão variou de 90 a 100%. O método desenvolvido foi aplicado para análises de amostras de produtos cosméticos comerciais (cremes hidratantes, antitranspirantes em creme e filtros solares). O método SBSE/LC-UV padronizado e validado resultou nas seguintes vantagens em relação aos métodos convencionais: simplicidade no preparo de amostra, redução do volume de solvente orgânico e da quantidade de amostra utilizada. O capítulo II descreve o desenvolvimento da fase extratora molecularmente impressa para SPE de parabenos em amostras de leite humano e análises por cromatografia em fase líquida com detecção espectrofotométrica (SPE/LC-UV). Os polímeros de impressão molecular, materiais sintéticos baseados em sistemas biomiméticos, foram sintetizados através do processo sol-gel tendo como molde, o benzilparabeno. Após a remoção do molde, cavidades seletivas (sítios de reconhecimento molecular) ao benzilparabeno e análogos estruturais foram reveladas. O método SPE/LC desenvolvido apresentou faixa linear correspondente ao limite de quantificação a 150 ng. mL-1 e coeficientes de determinação 0,992. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 13% e a exatidão variou de 86 a 117%. A aplicabilidade do método SPE/LC foi demonstrada através das análises de amostras de leite humano de lactantes. Segundo os parâmetros de validação analítica avaliados, o método padronizado SPE/LC-UV é adequado para análises de parabenos em amostras de leite humano. O capítulo III descreve a avaliação da microextraçao em fase sólida no capilar (in-tube SPME) acoplada à cromatografia líquida (LC) com detecção espectrofotométrica (LC-UV) para análises de rifampicina em amostras de plasma para fins de monitorização terapêutica. As variáveis in-tube SPME, tais como fase estacionária da coluna capilar, volume de amostra, ciclos aspirar/dispensar e vazão foram otimizadas visando obter maior eficiência do processo. O método in-tube SPME/LC desenvolvido apresentou faixa linear correspondente ao limite de quantificação (0,1 µg. mL-1) a 100 µg. mL-1 e coeficientes de determinação igual a 0,998. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 2% e a exatidão variou de 83 a 92%. A aplicabilidade do método in-tube SPME/LC, padronizado e validado foi comprovada através das análises de amostras de plasma de pacientes em tratamento com rifampicina. O método in-tube SPME/LC resultou na automação do processo de análise, maior precisão analítica, menor tempo de análise, menor volume de solvente orgânico e de fluido biológico. / This work describes the development of chromatographic methods in combination with microextraction techniques for the determination of parabens in cosmetic and human milk samples and of rifampicin in plasma samples. The design of a molecularly imprinted extraction phase for the solid-phase extraction (SPE) analysis of parabens in human milk samples is also reported. Chapter I describes the evaluation of the stir bar sorptive extraction (SBSE) in combination with liquid chromatography (LC) with UV detection (LC-UV) for the analysis of parabens in cosmetics aiming at quality control. The SBSE variables such as extraction time, temperature, pH of the matrix, ionic strength, and desorption conditions have been optimized, in order to establish the parabens partition equilibrium in a short analysis time. The linear range of the SBSE/LC method lay from LOQ to 2.5 µg. mg-1, with a coefficient of determination higher than 0.993. The interday precision of the SBSE/LC method presented a coefficient of variation lower than 5%, and the accuracy ranged from 90 to 99%. The effectiveness of the proposed method for the analysis of commercial cosmetic products such as body creams, antiperspirant creams, and sunscreens has been proven. Therefore, the combination of SBSE with liquid desorption, followed by LC analysis, provides a simple, and selective tool for the analysis of parabens in cosmetic products using minimized volume of organic solvent as well as very small amount of the target sample. Chapter II presents the development of a molecularly imprinted extraction phase for the SPE analysis of parabens in human milk samples. The molecularly imprinted polymers, which are synthetic materials based on biomimetic systems, were synthesized by the sol-gel technology in the presence of the template (benzilparaben). After template removal, selective cavities (benzilparaben) were revealed for parabens selective extraction. The linear range of the SPE/LC method lay from LOQ to 150 ng. mL-1, with a coefficient of determination higher than 0.992. The interday precision of the SPE/LC method presented a coefficient of variation lower than 13%, and the accuracy ranged from 86 to 117%. The effectiveness of the proposed method for the analysis of human milk samples has been demonstrated. According to the evaluated analytical validation parameters, the SPE/LC-UV method is suitable for the analysis of parabens in human milk samples. Chapter III depicts the evaluation of solid-phase microextraction in the capillary (in-tube SPME) in combination with liquid chromatography (LC) with UV detection (LC-UV) for the analysis of rifampicin in plasma samples for therapeutic drug monitoring. The in-tube SPME/LC method was linear over the LOQ (0.1) to 100 g. mL-1 range, with a linear coefficient value (r2) of 0.998. The inter-assay precision presented coefficient of variation 2%, and the accuracy ranged from 83 to 92%. The effectiveness and practicability of the proposed method have been by analysis of plasma samples from ageing patients undergoing therapy with rifampicin. The in-tube SPME/LC method allowed for automated continuous sample preparation (extraction, concentration, desorption, and injection of analytes) and minimized the analysis time as well as the volumes of organic solvent and biological fluid.

As sulfonamidas são antibióticos amplamente utilizados na medicina humana e animal. Por apresentarem um custo relativamente baixo, além de seu uso medicinal são comumente utilizadas como aditivos nos alimentos para promover o crescimento de animais. O alto consumo das sulfonamidas é preocupante, pois grandes quantidades desses compostos são descartadas no ambiente por meio de excreções humanas, excreções animais e por águas residuárias industriais e hospitalares. A presença de sulfonamidas no ambiente, mesmo em níveis de traços, pode gerar uma toxicidade direta nas espécies expostas bem como a proliferação seletiva de bactérias resistentes, que podem transferir os genes de resistência para outras espécies bacterianas. O impacto ambiental negativo ocasionado por esse tipo de composto exige o monitoramento adequado e o consequentemente desenvolvimento de técnicas e métodos de análise e de preparo de amostra que consigam atingir suficientes intervalos e limites de detecção. Na atualidade a SPE é uma técnica bastante utilizada para extração de analitos em amostras ambientais, pois permite trabalhar com um consumo de solvente reduzido, sua reprodutibilidade é alta, o tempo de preparo de amostra é curto e permite a automatização. No formato automatizado on-line a fase extratora é empacotada em uma pré-coluna e ligada ao sistema cromatográfico geralmente por meio de uma válvula de seis pórticos. Dentre as possíveis fases para a SPE os polímeros molecularmente impressos (MIP) têm tornado-se atrativos por causa de sua alta seletividade em comparação com as fases extratoras tradicionais; além disso, permitem, mediante procedimentos simples, associar a tecnologia MIP com a tecnologia de materiais de acesso restrito - RAM (do inglês: restricted access media) obtendo assim os materiais RAM-MIP. Nesse trabalho foi sintetizado e avaliado o desempenho de um polímero de impressão molecular (MIP), e três polímeros de impressão molecular restritos à ligação de macromoléculas (RAM-MIP, BSA-MIP e RAM-MIP-BSA) para a pré-concentração seletiva de sete sulfonamidas (SNs) em amostras complexas para análise em sistema HPLC-bidimensional. Os materiais obtidos foram avaliados em termos de fator de retenção, seletividade, seletividade competitiva e capacidade de eliminação de macromoléculas. O polímero BSA-MIP apresentou os melhores resultados, demonstrando-se muito mais seletivo para sulfonamidas do que para alguns interferentes como fluoroquinolonas, cafeína, ácido acetilsalicílico entre outros, também demonstrou uma capacidade de exclusão de macromoléculas de 97,63% ao ser testado com uma solução de 44 mg mL-1 de BSA. / Sulfonamides are antibiotics widely used in human and veterinary medicine. Due to their relatively low cost and their therapeutic effects, they are frequently employed as growth promoting additive in animal food. This high consumption causes that high amounts of them are being ejected to the environment through the animal and human excrements or industrial and hospital wastewater. Even in trace amount, these compounds can produce direct toxicity in several species and the selective spread of the antibiotic resistant bacteria that can transfer the resistance genes to others bacteria species. This negative impact has to be adequately monitored and, thus, there is a claim to the development of analytical and sample preparation techniques and methods that permit reaching to very low detection limits and intervals. Currently solid phase extraction (SPE) is the technique commonly used for isolation of analytes from environmental samples, since it allows working with smaller volumes of solvent, shorter sample preparation times, achieving high reproducibility and allowing automation. In automated on-line SPE the extracted phase is packed into a precolumn and linked to the chromatographic system, generally by a 6-port valve. Among the possible phases for SPE the molecular imprinted polymer (MIP) has become interesting because it is more selective than traditional extraction; moreover, through simple procedure it can be associated with the restricted acess media (RAM) producing the RAM-MIP and the MIP-BSA materials. Here we synthesized and evaluated the performance of one MIP and three molecular imprinted polymers linked to macromolecules (RAM-MIP, BSA-MIP e RAM-MIP-BSA) for a selective preconcentration of seven sulfonamides in complex samples for two-dimensional HPLC analysis. The synthesized materials was evaluated regarding the retention factor, competitive selectivity and elimination capacity of macromolecules. The BSA-MIP option showed the best results, achieving high selectivity for sulfonamides against others interferences such as fluoroquinolone, caffeine and acetylsalicylic acid; additionally presenting an exclusion capacity of 97,63% of macromolecules when was tested with a BSA solution at 44 mg ml-1.

Food quality control is a mandatory task in the food industry, and for coffee manufacturers, one of the key target compounds is caffeine, because of its well-known biological effects and currently expensive analytical methods are employed for caffeine quantification. This project was thought with the aim of developing a cost-effective caffeine sensor for application in industrial environments. Two main approaches were investigated: i) the use of imprinted polymers; ii) electrochemical using bare carbon electrodes. Chapter 1 is the main introduction, it focuses on the importance of food quality control, the limitation of the methodologies currently employed and it presents the advantages of using imprinted polymers recognition and electrochemistry signal transduction. Chapter 2 presents data related to studies of self-association of caffeine in water using a novel computational approach. The wider applicability of the method was assessed by studying self-association of paraxanthine and the results were validated via a collaboration with Miss R. Anastasiadi who employed isothermal titration calorimetry. Chapter 3 describes the synthesis of MIPs in water, starting with the selection of the functional monomers: HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt). The polymerisation conditions were optimised in terms of solvent to be used, initiator content, temperature and total monomer concentration. Afterwards, the polymers were synthesised, characterised, and preliminary data on the rebinding ability towards caffeine were presented. Chapter 4 focuses on electrochemical methods for the detection of caffeine using bare carbon electrodes. A voltammetric method for accurate determination of caffeine in beverages was developed. Based on the encouraging results obtained, further studies on the applicability of electrochemical methods for coffee quality analysis were done. A second method was developed for simultaneous detection of caffeine and polyphenols and an amperometric chemosensor for sucrose determination was acquired and its applicability for the analysis of green coffee beans was evaluated.

As sulfonamidas são antibióticos amplamente utilizados na medicina humana e animal. Por apresentarem um custo relativamente baixo, além de seu uso medicinal são comumente utilizadas como aditivos nos alimentos para promover o crescimento de animais. O alto consumo das sulfonamidas é preocupante, pois grandes quantidades desses compostos são descartadas no ambiente por meio de excreções humanas, excreções animais e por águas residuárias industriais e hospitalares. A presença de sulfonamidas no ambiente, mesmo em níveis de traços, pode gerar uma toxicidade direta nas espécies expostas bem como a proliferação seletiva de bactérias resistentes, que podem transferir os genes de resistência para outras espécies bacterianas. O impacto ambiental negativo ocasionado por esse tipo de composto exige o monitoramento adequado e o consequentemente desenvolvimento de técnicas e métodos de análise e de preparo de amostra que consigam atingir suficientes intervalos e limites de detecção. Na atualidade a SPE é uma técnica bastante utilizada para extração de analitos em amostras ambientais, pois permite trabalhar com um consumo de solvente reduzido, sua reprodutibilidade é alta, o tempo de preparo de amostra é curto e permite a automatização. No formato automatizado on-line a fase extratora é empacotada em uma pré-coluna e ligada ao sistema cromatográfico geralmente por meio de uma válvula de seis pórticos. Dentre as possíveis fases para a SPE os polímeros molecularmente impressos (MIP) têm tornado-se atrativos por causa de sua alta seletividade em comparação com as fases extratoras tradicionais; além disso, permitem, mediante procedimentos simples, associar a tecnologia MIP com a tecnologia de materiais de acesso restrito - RAM (do inglês: restricted access media) obtendo assim os materiais RAM-MIP. Nesse trabalho foi sintetizado e avaliado o desempenho de um polímero de impressão molecular (MIP), e três polímeros de impressão molecular restritos à ligação de macromoléculas (RAM-MIP, BSA-MIP e RAM-MIP-BSA) para a pré-concentração seletiva de sete sulfonamidas (SNs) em amostras complexas para análise em sistema HPLC-bidimensional. Os materiais obtidos foram avaliados em termos de fator de retenção, seletividade, seletividade competitiva e capacidade de eliminação de macromoléculas. O polímero BSA-MIP apresentou os melhores resultados, demonstrando-se muito mais seletivo para sulfonamidas do que para alguns interferentes como fluoroquinolonas, cafeína, ácido acetilsalicílico entre outros, também demonstrou uma capacidade de exclusão de macromoléculas de 97,63% ao ser testado com uma solução de 44 mg mL-1 de BSA. / Sulfonamides are antibiotics widely used in human and veterinary medicine. Due to their relatively low cost and their therapeutic effects, they are frequently employed as growth promoting additive in animal food. This high consumption causes that high amounts of them are being ejected to the environment through the animal and human excrements or industrial and hospital wastewater. Even in trace amount, these compounds can produce direct toxicity in several species and the selective spread of the antibiotic resistant bacteria that can transfer the resistance genes to others bacteria species. This negative impact has to be adequately monitored and, thus, there is a claim to the development of analytical and sample preparation techniques and methods that permit reaching to very low detection limits and intervals. Currently solid phase extraction (SPE) is the technique commonly used for isolation of analytes from environmental samples, since it allows working with smaller volumes of solvent, shorter sample preparation times, achieving high reproducibility and allowing automation. In automated on-line SPE the extracted phase is packed into a precolumn and linked to the chromatographic system, generally by a 6-port valve. Among the possible phases for SPE the molecular imprinted polymer (MIP) has become interesting because it is more selective than traditional extraction; moreover, through simple procedure it can be associated with the restricted acess media (RAM) producing the RAM-MIP and the MIP-BSA materials. Here we synthesized and evaluated the performance of one MIP and three molecular imprinted polymers linked to macromolecules (RAM-MIP, BSA-MIP e RAM-MIP-BSA) for a selective preconcentration of seven sulfonamides in complex samples for two-dimensional HPLC analysis. The synthesized materials was evaluated regarding the retention factor, competitive selectivity and elimination capacity of macromolecules. The BSA-MIP option showed the best results, achieving high selectivity for sulfonamides against others interferences such as fluoroquinolone, caffeine and acetylsalicylic acid; additionally presenting an exclusion capacity of 97,63% of macromolecules when was tested with a BSA solution at 44 mg ml-1.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-09-19T17:06:54Z No. of bitstreams: 1 JosimarAraujo.pdf: 950712 bytes, checksum: 6ec752a1c9c3751ffeba0d040eb09762 (MD5) / Made available in DSpace on 2017-09-19T17:06:54Z (GMT). No. of bitstreams: 1 JosimarAraujo.pdf: 950712 bytes, checksum: 6ec752a1c9c3751ffeba0d040eb09762 (MD5) Previous issue date: 2017-09-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The present work describes a study of the construction of an electrochemical sensor involving sol-gel process and molecular imprinting for detection of triclosan. The sol-gel process is a process in which a network is formed from a solution, by progressively changing a liquid precursor, from a sol to a gel and, in most cases, finally to a dry network. In turn, molecular imprinting is a method of induced formation of some predetermined selectivity recognition element for some template molecule. Thus, for the determination of the template molecule triclosan, 5-chloro-2-(2,4-dichlorophenoxy)-phenol, the following steps were performed: 1) verification of a glassy carbon electrode modified with chitosan and multi-walled carbon nanotubes, and molecularly imprinted siloxanes, would be suitable for the voltammetric detection of triclosan; 2) to observe if a network would be formed with the sol-gel process and to evaluate the mechanical stability of the electrochemical sensor constructed; 3) to evaluate the modified electrode as an electrochemical sensor with selectivity, specificity and detection limit for the determination of triclosan in aqueous solutions; 4) to verify the applicability of the proposed sensor by detecting of triclosan oxidation in real samples. The results showed that glassy carbon electrode modified with chitosan, multi-walled carbon nanotubes and molecularly imprinted siloxanes, constructed by sol-gel and molecular imprinting processes, presented mechanical stability, sensitivity, selectivity, specificity and applicability as an electrochemical sensor for the detection of triclosan in commercial samples of physical moisturizer and of toothpaste. / O presente trabalho descreve um estudo da construção de um sensor eletroquímico envolvendo processo sol-gel e impressão molecular para detecção de triclosan. O processo sol-gel é um procedimento no qual uma rede é formada a partir de uma solução. Esta formação é por meio de uma mudança progressiva deste líquido inicial, de um sol para um gel e, na maioria dos casos, finalmente para uma rede seca. Por sua vez, a impressão molecular é um método de formação induzida de elementos de reconhecimento com seletividade predeterminada para alguma molécula molde. Assim, para a determinação da molécula molde triclosan, 5-cloro-2- (2,4-diclorofenoxi)-fenol, as seguintes etapas foram executadas: 1) verificar se um eletrodo de carbono vítreo modificado com quitosana, nanotubos de carbono de paredes múltiplas e siloxanos molecularmente impressos seria adequado para a detecção voltamétrica do triclosan; 2) averiguar a formação de uma rede com o processo sol-gel e avaliar a estabilidade mecânica do sensor eletroquímico construído; 3) avaliar a sensibilidade do eletrodo modificado como um sensor eletroquímico com seletividade, especificidade e limite de detecção para a determinação de triclosan em soluções aquosas; e 4) comprovar a aplicabilidade do sensor proposto mediante a detecção de sinal de oxidação de triclosan em amostras reais. Os resultados mostraram que eletrodos de carbono vítreo modificados com quitosana, nanotubos de carbono de paredes múltiplas e siloxanos molecularmente impressos, construídos por processo sol-gel e impressão molecular, apresentaram estabilidade mecânica, sensibilidade, seletividade, especificidade e aplicabilidade como um sensor eletroquímico para a detecção de triclosan em amostras comerciais de hidratante corporal e de creme dental.

As Sulfonamidas (SAs) são antibióticos de uso muito comum na medicina veterinária, sendo também aplicadas na medicina humana. Os resíduos dessas substâncias, ou dos seus metabolitos na carne e outros alimentos, podem causar efeitos adversos para a saúde dos consumidores como, por exemplo, resistências a antibióticos e alergias. Este trabalho apresenta o desenvolvimento e aplicação de métodos modernos de preparo de amostra para determinação de multiresíduo de sulfonamidas em alimentos por cromatografia líquida acoplada a espectrometria de massas e ultravioleta visível. Dentre os métodos de preparo de amostra, aplicou-se o método de extração QuEChERS (Quicky, Easy, Cheap. Effective, Rugged, Safe) modificado, em combinação com a cromatografia liquida acoplada a espectrometria de massas, para a análise de dez sulfonamidas em amostras de músculo de frango e bovino. No segundo estudo, seguindo a tendência de miniaturização, sintetizou-se um polímero molecularmente impresso (Sulfadimetoxina-MIP) para uso como sorbente em dispositivos de Microextração com Sorbentes Empacotado (MEPS). O método, denominado SDM-MIP-MEPS, foi otimizado e aplicado em amostras de músculo de frango usando a cromatografia liquida com detecção por ultravioleta-visivel. Embora o uso do polímero molecularmente impresso (MIP) como sorbente seletivo para o MEPS já tenha sido reportada em dois trabalhos, pela primeira vez é descrito a aplicação de um MIP impresso com sulfdimetoxina (SDM), usando MEPS para extração de sulfonamidas em músculo de frango. Portanto, a novidade neste caso, é o uso da nova técnica extração miniaturizada (MEPS) com o polímero sintetizado sulfadimetoxina-MIP como sorbente de empacotamento. As metodologias foram validadas com sucesso de acordo com as diretrizes 657/2002/EU. / Sulfonamides are widely used in veterinary and human medicine. Residues of these compounds, or their metabolites in animal meats and other foods, are toxic and can cause side effects in human\'s health, such as resistance to antibiotics and allergic reactions. This study describes the development and application of a modern sample preparation approach for sulfonamides multiresidue determination in food by chromatographic methods coupled to mass spectrometry and ultraviolet-visible spectroscopy. Among those sample preparation methods, the modified QuEChERS extraction in combination with liquid chromatography in tandem with mass spectrometric detection was applied to the analysis of residues of 10 sulfonamides in chiken and cattle muscle. In the second study, following the miniaturization trends, it was synthesized a molecularly imprinted polymer (Sulfadimethoxine-MIP), for application as sorbent in Microextraction by Packed Sorbents (MEPS). The extraction method was optimized and successfully applied to chicken muscle samples in combination with highperformance liquid chromatography by ultraviolet-visible detection. Although the use of MIPs as selective packing materials for MEPS has already been reported in two papers, is first time that application of a MIP imprinted with Sulfadimethoxine is evaluated for the extraction of sulfonamides in chicken muscle. Therefore the novelty of the present work is the use of this new miniaturization extraction technique with synthesized sulfadimethoxine-MIP polymer as packing sorbent. The methods were successfully validated according to the 2002/657/EC guidelines.

Molecularly imprinted polymers (MIPs) have been compared to natural antibodies in that they can specifically bind target compounds in a similar way that antibodies specifically bind to an antigen. The attraction of the MIPs technology is the ease of creating binding elements which are relatively cheap compared with the process of isolating natural antibodies. In this research monoterpenes, such as α-terpineol, were chosen to be the model compounds for investigating the molecular imprinting of small, poorly functionalised organic compounds. The conventional non-covalent approach was mainly used to synthesise these MIPs, but the sacrificial-spacer semi-covalent approach was also investigated. A less widely used method, porogen-imprinting - a variant of non-covalent imprinting - was adapted for α-terpineol. The latter novel terpene MIP appeared to specifically bind α-terpineol, by hydrogen bonding, so the polymer was characterised in detail. The main parameters which were altered for preparing non-covalent MIPs included the template (α-terpineol, (-)-menthol or trans-terpin); the functional monomer (methacrylic acid, 2-hydroxyethyl methacrylate, bilirubin and phenol [for the semi-covalent MIP]); the cross-linking monomer (ethylene glycol dimethacrylate, divinylbenzene and trimethylolpropane trimethacrylate); and also the polymerisation method (block or precipitation polymerisation). The binding specificity and cross-reactivity for all the polymers were tested using a liquid batch-binding setup. The batch-binding setup required the detection of analyte that was not bound in order to calculate by difference the fraction of analyte bound to the polymer. Initially the terpenes were to be detected by a colorimetric method; however attempts to make the method sensitive and reliable were not successful. In comparison, gas chromatography was more reliable for the detection of terpenes and was used for the experiments presented in this thesis. 1H-NMR studies of the interaction between α-terpineol and acetic acid (as a non-polymerisable analogue of methacrylic acid) were investigated as a basis for understanding the binding to the carboxyl functional group moiety employed in many of the non-covalent MIPs that were made. The interaction between (-)-menthol and phenol was also investigated because the phenol moiety was employed in the semi-covalent MIP. Only selected MIPs, which appeared to specifically bind the template, were physically characterised. This included optimising the batch-binding parameters, scanning electron microscopy imaging, surface area and pore radius analysis and in some cases Fourier transform-infrared spectroscopy of the polymers.

The aim of this study was to develop methods for sample preparation for 8-OHdG in blood plasma samples with different solid phase extraction techniques using HPLC with an elec- trochemical detector. The solid phase extraction cartridges used were Chromabond® C18, Oasis® MAX, and three types of SupelMIPTM cartridges for chloramphenicol, riboflavin, and nitroimidazoles. The SupelMIPTM cartridges are based on molecularly imprinted polymers- technique. The separation of 8-OHdG in samples extracted from blood plasma was carried out with a Thermo Quest Hypersil Division ODS column (250 mm × 4 mm, 3μm I.D.) and methanol:buffer (10:90, v/v) as mobile phase. Recovery and selectivity was studied for the different solid phase extraction methods. The highest recovery was obtained using the Chromabond C18 cartridge with a recovery of 92%, and CV coefficient 9.5% (n = 4). 8-OHdG could not be extracted on MIP-cartridges for chloramphenicol or riboflavin, but was retained on MIP columns for nitroimidazoles, and the highest recovery was 49%.

Bulk imprinted polymers were synthesized using traditional small molecular weight imprinting techniques for the recognition of bovine serum albumin (BSA). Reproducibility and capacity concerns prompted the use of circular dichroism to investigate the potential effects that conditions commonly employed have on the structure of the protein prior to polymerization. These studies clearly showed a substantial change in the secondary structure of three common model protein templates when in the presence of various monomers and crosslinkers. Molecular docking was used to further examine the interactions taking place at the molecular level. Docking simulations revealed that significant amounts of non-covalent interactions are occurring between the amino acid side chains and ligands; although, the interactions taking place amongst the analyte and polypeptide backbone are responsible for the experimentally observed conformational change. The computational studies also showed that several of the ligands preferentially ‘docked’ to the same amino acids in the protein, indicating that if multiple monomers are employed, this competition for similar binding sites will potentially result in non-specific recognition. These findings are important as they offer insight into the fundamental reasons why recognition of macromolecular templates has proven difficult as well as provide guidance for future success in the field. Using this information, novel surface imprinted polymers were synthesized via a facile technique for the specific recognition of BSA. Thin films based on 2-(dimethylamino)ethyl methacrylate (DMAEMA) as the functional monomer and varying amounts of either N,N’ methylenebisacrylamide (MBA) or poly(ethylene glycol) (400) dimethacrylate (PEG400DMA) as crosslinker were synthesized via UV free-radical polymerization. A clear and reproducible increase in recognition of the template was demonstrated for these systems as 1.6-2.5 times more BSA was recognized by the MIP sample relative to the control polymers. Additionally, these polymers exhibited specific recognition of the template relative to similar competitor proteins with up to 2.9 times more BSA adsorbed than either glucose oxidase or bovine hemoglobin. These synthetic antibody mimics hold significant promise as the next generation of robust recognition elements in a wide range of bioassay and biosensor applications. / text

Molecularly imprinted polymers (MIPs) are biomimetic sensing elements that combine the accuracy and highly specific binding affinity of a biosensor, with the robustness and reusability associated with artificial electrochemical sensors. This thesis investigates the application of a MIP sensor to address the challenge of phosphate detection in precision agriculture. Traditional chemical sensing approaches using portable electrochemical sensors display a significant cross-interference between inorganic phosphate and other nutrient ions. This is due to the low position of phosphate in the Hofmeister Selectivity Series for anions, its high electronegativity and its pH dependent structure, resulting in a molecule that is very difficult to detect. To address this challenge, a sensor was created by spin coating a phosphate selective MIP onto a substrate containing a series of electrodes. These electrode devices allowed for electrical measurements to be taken using an inductance, capacitance and resistance (LCR) testing station, and to observe the change in the materials dielectric constant as the binding sites become occupied by the target analyte. The devices underwent several design reiterations to produce an optimised setup consisting of 100 interdigitated chrome electrodes with a width of 1 μm and a separation distance of 1 μm on a quartz substrate. The final electrode design was used to carry out a nutrient cross-interference study across several polymer permutations. The purpose of this was to develop an optimised MIP formulation for binding specifically to inorganic phosphate ions. From this study, an optimal phosphate selective MIP was identified, based upon a binding site constructed from methacrylic acid around a diphenyl phosphate template molecule. During capacitance measurements, this MIP formulation demonstrated a clear preferential response to phosphate (1610 pF) over the average capacitance results observed following exposure to the competing nitrate (1286 pF) and sulphate (1212 pF) nutrients tested in the cross-interference study.