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.

By using the technology of molecularly imprinted polymer (MIP), we propose to synthesize a protein conformational imprint that also acts as a plastic enzyme, inducing protein structural transitions. The imprint aims at MIP-induced stabilization and / or formation of bound protein secondary structure and the applications associated with analysis and correction of misfolded proteins. The screening of polymeric functional monomers being able to induce the conformational transitions in proteins is investigated in this report. The fluoro-silanes (3-heptafluoroisopropoxy)propalethoxysilane (7F) and 3,3,3-trifluoropropylmethoxysilane (3F) were employed as functional monomers for synthesis of this catalytic protein conformational imprint via sol-gel reactions. 3F was demonstrated superior to 7F for fluoro-modification of tetraethylorthosilicate (TEOS) gel in terms of retaining gel transparency and increasing hydrophobicity while maintaining a uniform distribution of encapsulated protein. Both hydrolyzed 3F and polymerized 3F exhibited strong influences on structure transitions of three template proteins: bovine serum albumin (BSA), beta-lactoglobulin (BLG), and bovine carbonic anhydrase (BCA). The formation of molten globule intermediates that stabilized by increased alpha-helices was induced by the trifluoro-silane in BLG and BCA. Additionally, 3F was effective at a lower concentration than the benchmark fluoro-alcohol 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), validating the application of 3F as a functional monomer for protein conformational imprinting.

L’interface entre deux solutions électrolytiques immiscibles (ITIES) peut agir comme un support pour l’assemblage de nanoobjets. Cela présente de nombreux avantages : les particules ne requièrent pas d’ingénierie particulière pour leur obtention, peuvent s’assembler dans des conditions qui leur sont propres, sont pratiquement non dégradables et facilement renouvelables. Les recherches actuelles portent tant sur leur utilisation potentielle en tant que plateformes pour des appareils optiques ajustables, pour des capteurs ou encore pour de la catalyse. L’adsorption de nanoparticules de silice, dense ou mésoporeuse, à l’interface liquide-liquide a été étudiée par voltammétrie en courant alternatif. L’interaction des nanoparticules de silice avec le bleu de méthylène et l’éosine B a été étudiée par voltammétrie cyclique et spectrophotométrie. Les constantes thermodynamiques d’adsorption du bleu de méthylène ont été déterminées à 1.66 105 et 3.68 103 sur les particules de silice dense et mésoporeuse respectivement. La variation de constante entre les deux types de silice repose essentiellement sur leur état d’ionisation respectif. L’énergie de Gibbs de transfert entre phase liquide est modifiée de 8.9 kJ mol-1 en présence de nanoparticules denses ce qui donne des indications sur le mécanisme de transfert du bleu de méthylène en présence de nanoparticules. Mettant à profit l’aptitude de la silice à accumuler le bleu de méthylène et à s’adsorber sur l’interface liquide il nous a été possible d’améliorer la sensibilité de la détection électrochimique. L’éosine B n’a aucune interaction avec les particules de silice. Nos efforts ont ensuite porté sur l’amélioration de la sélectivité du transfert électrochimie par l’utilisation de nanoparticules de silice à empreinte moléculaire. Des nanoparticules de silice dense à empreinte moléculaire de Diclofénac (DIN) ont été synthétisées. Cette molécule est un anti-inflammatoire non stéroïdien très largement utilisé et figurant sur la liste européenne des polluants émergents. Les constantes d’affinité du Diclofénac pour les DIN et les particules équivalentes sans empreinte sont de 7.47 108 et 2.96 107 respectivement ce qui démontre clairement la présence d’empreintes ayant une forte affinité pour le diclofénac au sein des particules. Des molécules analogues (Diclofénac acide, Aceclofenac, acide 4 phenyl-azo benzoique) ont été testées et ont une affinité faible pour les DIN. En électrochimie, l’ajout de DIN bloque le transfert de Diclofénac à l’interface liquide-liquide / The interface between two immiscible electrolyte solutions (ITIES) can act as a scaffold for the assembly of nanometer-sized objects. The assembly of nanoparticles at liquid-liquid interfaces has numerous advantages – the nanoparticles do not require engineering, can assemble given proper conditions, are practically non-degrading and easily renewable. Research is ongoing into their use as a platform for tunable optical devices, sensors and catalysis. The adsorption of both dense and mesoporous silica nanoparticles at the ITIES was studied by AC voltammetry. Their interactions with methylene blue (MB+) and Eosin B (EB-), selected as a model ions, were studied by cyclic voltammetry and UV/Vis absorption spectroscopy. The thermodynamic constants of adsorption of MB+ were found to be 1.66 105 and 3.68 103 onto dense and mesoporous silica nanoparticles respectively. The difference of adsorption constants for the two types of silica was explained by their differing ionisation states. The Gibbs energy of transfer of MB+ is shifted by -8.9 kJ mol-1 in the presence of dense silica nanoparticles, giving some insights to the transfer mechanism of MB+ in presence of nanoparticles. Combining the ability of silica to adsorb onto the ITIES and their affinity for MB+, MB+ was accumulated at the ITIES and so an increase in sensitivity of electrochemical detection was achieved. Eosin B demonstrated no affinity for the silica nanoparticles and its transfer at the ITIES was not influenced by their presence. Next the focus was placed on improving the selectivity of the interaction by synthesising imprinted silica nanoparticles, more specifically, Diclofenac-imprinted dense silica nanoparticles. This drug was chosen as it is a commonly used nonsteroidal anti-inflammatory drug which has been placed on the European watch list of emerging pollutants. The thermodynamic constants were calculated as 7.47 108 for Diclofenac-imprinted silica and only 2.96 107 for non-imprinted silica. Thus the presence of imprint cavities greatly influences the affinity of diclofenac for the silica nanoparticles. The analogues of Diclofenac (Aceclofenac, Acid diclofenac, 4-phenyl azo benzoic acid) were shown to have a very limited affinity for the imprinted particles. Electrochemical experiments at the liquid-liquid interface revealed that the diclofenac transfer is blocked by the presence of imprinted particles

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.

<p>An intrinsic mathematical model for simulation of fixed bed chromatography was demonstrated and compared to more simplified models. The former model was shown to describe variations in the physical, kinetic, and operating parameters better than the latter ones. This resulted in a more reliable prediction of the chromatography process as well as a better understanding of the underlying mechanisms responsible for the separation. A procedure based on frontal liquid chromatography and a detailed mathematical model was developed to determine effective diffusion coefficients of proteins in chromatographic gels. The procedure was applied to lysozyme, bovine serum albumin, and immunoglobulin γ in Sepharose™ CL-4B. The effective diffusion coefficients were comparable to those determined by other methods.</p><p>Molecularly imprinted polymers (MIPs) are traditionally prepared as irregular particles by grinding monoliths. In this thesis, a suspension polymerization providing spherical MIP beads is presented. Droplets of pre-polymerization solution were formed in mineral oil with no need of stabilizers by vigorous stirring. The droplets were transformed into solid spherical beads by free-radical polymerization. The method is fast and the performance of the beads comparable to that of irregular particles. Optimizing a MIP formulation requires a large number of experiments since the possible combinations of the components are huge. To facilitate the optimization, chemometrics was applied. The amounts of monomer, cross-linker, and porogen were chosen as the factors in the model. Multivariate data analysis indicated the influence of the factors on the binding and an optimized MIP composition was identified. The combined use of the suspension polymerization method to produce spherical beads with the application of chemometrics was shown in this thesis to drastically reduce the number of experiments and the time needed to design and optimize a new MIP.</p>

An intrinsic mathematical model for simulation of fixed bed chromatography was demonstrated and compared to more simplified models. The former model was shown to describe variations in the physical, kinetic, and operating parameters better than the latter ones. This resulted in a more reliable prediction of the chromatography process as well as a better understanding of the underlying mechanisms responsible for the separation. A procedure based on frontal liquid chromatography and a detailed mathematical model was developed to determine effective diffusion coefficients of proteins in chromatographic gels. The procedure was applied to lysozyme, bovine serum albumin, and immunoglobulin γ in Sepharose™ CL-4B. The effective diffusion coefficients were comparable to those determined by other methods. Molecularly imprinted polymers (MIPs) are traditionally prepared as irregular particles by grinding monoliths. In this thesis, a suspension polymerization providing spherical MIP beads is presented. Droplets of pre-polymerization solution were formed in mineral oil with no need of stabilizers by vigorous stirring. The droplets were transformed into solid spherical beads by free-radical polymerization. The method is fast and the performance of the beads comparable to that of irregular particles. Optimizing a MIP formulation requires a large number of experiments since the possible combinations of the components are huge. To facilitate the optimization, chemometrics was applied. The amounts of monomer, cross-linker, and porogen were chosen as the factors in the model. Multivariate data analysis indicated the influence of the factors on the binding and an optimized MIP composition was identified. The combined use of the suspension polymerization method to produce spherical beads with the application of chemometrics was shown in this thesis to drastically reduce the number of experiments and the time needed to design and optimize a new MIP.

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%.

Sample preparation has a critical role as a first step in analytical processes, especially in bioanalysis and environmental analysis. A good sample preparation technique should be robust and stable, regardless of the sample matrix. The aim of this thesis is to design and synthesize molecularly imprinted polymers that can be used in various sample preparation techniques, such as on-line MEPS, on-line SPE and on-line monolithic pre-columns used for the extraction of drugs, hormones, and cancer biomarkers from human plasma and urine samples. Additional aim was to provide full automation, on-line coupling, short sample preparation time and high-throughput. In this thesis MIP in MEPS was used on-line with liquid chromatography-tandem mass spectrometry (LC/MS/MS) for the determination of sarcosine in human urine and plasma samples. The method was fully automated and the packed sorbent could be used for about hundred extractions. In additional work a coated needle with MIP-Sol-Gel as thin layer was prepared and used for the microextraction of bilirubin from human plasma and urine. Small sample volumes could be handled and the validation of the method showed that the method was robust and selective. In a further work MIP-SPE on-line with HPLC was used for the extraction and determination of dextromethorphan in human plasma samples. MIP-SPE showed a good selectivity and high recovery (87% - 92%). On-line MIP monolithic pre-column was prepared and used in a coupled system for the extraction of tramadol in human plasma and urine samples. The MIP monolithic pre-column showed good selectivity and high extraction recovery was obtained (91-96%). The extraction and analysis of human insulin in plasma and pharmaceutical formulation solutions were carried out using MIP-SPE on-line with HPLC. The validation of the method showed that the method was accurate and robust. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Submitted.</p>

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.