La technique de l’impression moléculaire permettant d'obtenir des polymères hautement sélectifs est utiliséedans de nombreuses applications. Dans le cadre de cette thèse, cette technique est employée pour préparer despolymères spécifiques du benzo(a)pyrène (BaP) en vue d’une application capteur. L’introduction d’une sonderédox au sein même du réseau de polymère offre la possibilité d’une détection directe de la cible, par unetechnique électrochimique. Dans le domaine des MIPs, ce travail est le premier à décrire l’utilisation d’unmonomère participant à la formation du réseau tridimensionnel tout en apportant une propriété rédox. Lespolymères synthétisés par polymérisation par précipitation ont été caractérisés en termes de composition, destabilité thermique et de propriétés texturales. Les propriétés de rétention ont été évaluées en mode batch parHPLC-UV en présence de BaP et d'interférents de type hydrocarbures aromatiques polycycliques. Ces MIPsprésentent de bonnes propriétés d’adsorption vis-à-vis du BaP avec des facteurs d’empreinte supérieurs à 1montrant l’efficacité de l’impression. Ces propriétés sont conservées en présence d’une matière organique (seld’acide humique), mais également après plusieurs cycles d’utilisation. Les analyses électrochimiques ontdémontré que les propriétés électrochimiques de ces MIPs diffèrent selon la présence ou pas de la cible avecune limite de détection de 0,09 μM en BaP, ouvrant la porte à la réalisation de capteurs. / Molecularly Imprinted Polymers show highly selective affinity towards the target molecule and are used inmany applications. In this context, the technology is used for preparing selective polymers for benzo(a)pyrene(BaP) with the aim of sensor fabrication. The addition of a redox tracer inside the polymer allows thepossibility of direct target detection by an electrochemical technique. In the MIP field, this work is the firstreporting the use of a functional monomer that not only participates in the creation of the polymer network butalso provides a redox property. The polymers synthesized using precipitation polymerization technique werecharacterized in terms of composition, thermal stability and textural properties. The adsorption properties wereevaluated in batch mode by HPLC-UV in the presence of BaP and interfering polycyclic aromatichydrocarbons. These MIPs show good adsorption behavior towards BaP with imprinting factors superior to 1showing the efficiency of the molecular imprinting. These properties were also preserved even in presence ofan organic matter (humic acid salt), and also after several uses. Finally, electrochemical analysis showed thatthese MIPs had a different electrochemical behavior depending on the presence or the absence of the targetwith a detection limit of 0.09 μM for BaP. The use of such electrochemical MIPs can thus be interestinglyconsidered in sensor devices.

Propõe-se método novo e completo para análise de acetona em ar exalado envolvendo coleta com pré-concentração em água, derivatização química e determinação eletroquímica assistida por novo algoritmo de processamento de sinais. Na literatura recente a acetona expirada vem sendo avaliada como biomarcador para monitoramento não invasivo de quadros clínicos como diabetes e insuficiência cardíaca, daí a importância da proposta. Entre as aminas que reagem com acetona para formar iminas eletroativas, estudadas por polarografia em meados do século passado, a glicina apresentou melhor conjunto de características para a definição do método de determinação por voltametria de onda quadrada sem a necessidade de remoção de oxigênio (25 Hz, amplitude de 20 mV, incremento de 5 mV, eletrodo de gota de mercúrio). O meio reacional, composto de glicina (2 mol·L-1) em meio NaOH (1 mol·L-1), serviu também de eletrólito e o pico de redução da imina em -1,57 V vs. Ag|AgCl constituiu o sinal analítico. Para tratamento dos sinais, foi desenvolvido e avaliado um algoritmo inovador baseado em interpolação de linha base por ajuste de curvas de Bézier e ajuste de gaussiana ao pico. Essa combinação permitiu reconhecimento e quantificação de picos relativamente baixos e largos sobre linha com curvatura acentuada e ruído, situação em que métodos convencionais falham e curvas do tipo spline se mostraram menos apropriadas. A implementação do algoritmo (disponível em http://github.com/batistagl/chemapps) foi realizada utilizando programa open source de álgebra matricial integrado diretamente com software de controle do potenciostato. Para demonstrar a generalidade da extensão dos recursos nativos do equipamento mediante integração com programação externa em linguagem Octave (open source), implementou-se a técnica da cronocoulometria tridimensional, com visualização de resultados já tratados em projeções de malha de perspectiva 3D sob qualquer ângulo. A determinação eletroquímica de acetona em fase aquosa, assistida pelo algoritmo baseado em curvas de Bézier, é rápida e automática, tem limite de detecção de 3,5·10-6 mol·L-1 (0,2 mg·L-1) e faixa linear que atende aos requisitos da análise em ar exalado. O acetaldeído, comumente presente em ar exalado, em especial, após consumo de bebidas alcoólicas, dá origem a pico voltamétrico em -1,40 V, contornando interferência que prejudica vários outros métodos publicados na literatura e abrindo possibilidade de determinação simultânea. Resultados obtidos com amostras reais são concordantes com os obtidos por método espectrofotométrico, em uso rotineiro desde o seu aperfeiçoamento na dissertação de mestrado do autor desta tese. Em relação à dissertação, também se otimizou a geometria do dispositivo de coleta, de modo a concentrar a acetona num volume menor de água gelada e prover maior conforto ao paciente. O método completo apresentado, englobando o dispositivo de amostragem aperfeiçoado e o novo e efetivo algoritmo para tratamento automático de sinais voltamétricos, está pronto para ser aplicado. Evolução para um analisador portátil depende de melhorias no limite de detecção e facilidade de obtenção eletrodos sólidos (impressos) com filme de mercúrio, vez que eletrodos de bismuto ou diamante dopado com boro, entre outros, não apresentaram resposta. / A new and complete method for analyzing acetone in exhaled breath is proposed, involving sample collection by preconcentration in water, chemical derivatization and electrochemical determination assisted by a new signal processing algorithm. Breath acetone is under investigation in the medical literature as a biomarker for noninvasive monitoring of clinical conditions such as diabetes and heart failure and better analyzers are demanded. Some amines that react with acetone to form electroactive imines, already studied by polarography in the middle of the last century, were evaluated here as derivatizing agents and glycine presented the best compromise for the definition of a determination method by square wave voltammetry without purging of dissolved oxygen (20 mV pulses at 25 Hz with 5 mV scanning steps, mercury drop electrode). The derivatization medium of glycine (2 mol·L-1) and NaOH (1 mol·L-1) also serves as electrolyte and the imine reduction peak at -1.57 V vs. Ag|AgCl constitutes the analytical signal. For signal processing a novel algorithm based on baseline automatic prediction using Bézier curves was developed in combination with Gaussian peak fitting. This innovative combination enables the recognition and quantification of relatively low and broad peaks on a curved and noisy baseline while standard procedures fail and fitting of splines is less appropriate. The algorithm (available at http://github.com/batistagl/chemapps) was implemented using open source matrix algebra software directly integrated with the potentiostat control software. The generality of this approach to extend the native resources of commercial equipment was evidenced by proceeding the implementation of 3D chronocoulometry with help of the open source Octave language. The processed charge vs. potential vs. time function 3-D surfaces for the forward and backward steps can be observed from any angle on the screen. The fast automatic electrochemical determination of acetone assisted by the Bézier based curve fitting algorithm, with an acetone detection limit of 3.5·10-6 mol·L-1 (0.2 mg·L-1) and a wide linear range is entirely satisfactory for breath analysis. Acetaldehyde, an expected component of breath, especially after ingestion of alcohol, and a common interferent for some other analytical methods, generates a peak at -1.40 V that does not interfere with the acetone determination and that is possibly suitable for simultaneous determination of this analyte. Results obtained with real samples are in agreement with spectrophotometric determinations routinely applied to breath analysis, formerly developed in the master dissertation of the author of this thesis. The geometry of the impinger sampling device was also improved (in comparison with the dissertation) in order to collect the acetone in a smaller volume of cooled water and to allow the patient to breath more freely. The complete method at its current state of development, encompassing the improved sampler and the new effective signal processing algorithm for voltammetric signals is ready for use. Evolution to a portable analyzer can be envisioned if the shortcomings of detection limits of solid (screen-printed) electrode preparation with a mercury film are overcome, once the imine signal was not observed on solid electrodes like bismuth and boron doped diamond.

nÃo hà / Este trabalho descreve o desenvolvimento de uma proposta de procedimento eletroanalÃtico para a determinaÃÃo do antidepressivo tricÃclico imipramina (IMP) em formulaÃÃes farmacÃuticas comerciais, utilizando eletrodo de diamante dopado com boro (EDDB) e voltametria de onda quadrada (VOQ). Os estudos da oxidaÃÃo eletroquÃmica da IMP foram realizados em soluÃÃo de tampÃo Britton-Robbinson (BR) 0,04 mol L-1. Os resultados utilizando VOQ mostraram dois picos de oxidaÃÃo bem definidos, com pico 1 em 0,04 V e pico 2 em 0,82 V versus Ag/AgCl/Cl- 3 mol L-1. Para o desenvolvimento dos estudos foi utilizado o pico 1 por ter se apresentado mais sensÃvel e seletivo. O efeito dos parÃmetros experimentais e voltamÃtricos foram avaliados e as melhores condiÃÃes foram obtidas em pH 7,4, frequÃncia de aplicaÃÃo de pulsos de potencial de 100 s-1, incremento de potencial de 2 mV e amplitude de 50 mV. Sob estas condiÃÃes, foram construÃdas curvas analÃticas com resposta linear na faixa de concentraÃÃo de 1,73 x 10-7 mol L-1 a 2,53 x 10-6 mol L-1 (r = 0,9984), com um limite de detecÃÃo e de quantificaÃÃo de 4,35 x 10-8 mol L-1 e 1,45 x 10-7 mol L-1, respectivamente. O mÃtodo proposto foi aplicado com sucesso na determinaÃÃo de IMP em formulaÃÃes farmacÃuticas comerciais e validado por comparaÃÃo com mÃtodo padrÃo de determinaÃÃo de imipramina. Os resultados obtidos estiveram de acordo, em um nÃvel de confianÃa de 95%, com aqueles obtidos usando o mÃtodo oficial da FarmacopÃia BritÃnica. / This research describes the development of an electroanalytical procedure proposal for the determination of tricyclic antidepressant imipramine (IMP) in commercial pharmaceutical formulations, using Boron-Doped Diamond Electrode (BDDE) and Square-Wave Voltammetry (SWV). The electrochemical oxidation of imipramine was studied in 0.04 mol L-1 Britton-Robbinson buffer solution (BR). The results using VOQ showed two well-defined oxidation peaks with potentials of 0.04 V and 0.82 V versus Ag/AgCl/Cl- 3 mol L-1 for peaks 1 and 2, respectively. For the studiesâ development was used the peak 1 because it demonstrated to be more sensitive and selective. The effect of the experimental and voltammetric parameters were evaluated and the best performance was obtained in pH 7.4, pulses application frequency of potential of 100 s-1, potential increment of 2 mV and amplitude of 50 mV. Under these conditions, the analytical curves were obtained in the linear range of concentration from 1.73 x 10-7 mol L-1 to 2.53 x 10-6 mol L-1 (r = 0,9984), with detection and quantitation limits 4.35 x 10-8 mol L-1 e 1.45 x 10-7 mol L-1, respectively. The proposed method was applied with success in the determination of IMP in commercial pharmaceutical formulations and validated by comparison with standard method for determination of imipramine. The obtained results were in close agreement, at a 95% confidence level, with those obtained using an official method of the British Pharmacopoeia.

[pt] O cloridrato de sibutramina está entre os sacietógenos mais prescritos para o controle da obesidade e tem sido adicionado, de forma não declarada, a alimentos protéicos para esportistas. Nenhuma metodologia analítica é descrita em compêndios oficiais para a determinação de sibutramina. O presente trabalho objetiva o desenvolvimento e a validação de métodos analíticos baseados na voltametria de pulso diferencial (DPV) e na voltametria de onda quadrada (SWV) para quantificação do cloridrato de sibutramina em fármacos e em alimentos protéicos. A influência dos parâmetros eletroquímicos foi estudada para selecionar as melhores condições de trabalho como: eletrólito suporte (tampão Mcllvaine) pH (4,0), velocidade de varredura (40 mV s(-1) para DPV e 240 mV s(-1) para SWV) e amplitude de pulso (50 mV). Não foi possível pré-concentrar a sibutramina no eletrodo de trabalho (gota de mercúrio). O comportamento eletroquímico desta substância no eletrodo de mercúrio também foi investigado a partir de estudos com voltametria cíclica. A redução da sibutramina é aparentemente reversível envolvendo a participação de 1 próton e 1 elétron. O potencial de pico da sibutramina é cerca de - 100 mV (Ag/AgCl 3 mol L-1). No processo de validação foram avaliadas as faixas de resposta linear (do limite de quantificação até 33,3 mg L(-1) em ambos os métodos), o limite de detecção (0,4 para DPV e 0,3 mg L(-1) para SWV), e o limite de quantificação (1,4 para DPV e 1,1 mg L(-1) para SWV). A precisão do método foi satisfatória. Testes de recuperação indicaram 90,4 % em ambos os métodos. O desempenho das técnicas voltamétricas foi comparado estatisticamente com a técnica CLAE e não houve diferença significativa entre os resultados obtidos. / [en] Sibutramine hydrochloride is among the most prescribed satiety inducer for obesity control and it has also been added, in a fraudulent way, in proteinrich food complement for athletes. There is no analytical method for sibutramine described in the official literature. The goal of this work is to develop and validate electroanalytical methods for the determination of sibutramine in pharmaceuticals and in protein-rich food complements. Differential pulse voltammetry (DPV) and square wave voltammetry (SWV) were the techniques employed. The influence of electrochemical parameters was studied in order to select the best working conditions such as supporting electrolyte (Mcllvaine buffer), pH (4.0), scan rate (40 mV s(-1) for DPV and 240 mV s(-1) for SWV) and pulse amplitude (50 mV). The pre-concentration of sibutramine in the working electrode (mercury drop) was not possible. The electrochemical behavior in the mercury electrode was also investigated using cyclic voltammetry. The reduction of sibutramine is probably reversible and involves of 1 proton and 1 electron. The peak potential of sibutramine is about - 100 mV (Ag/AgCl3 mol L(-1)). In the validation process, the linear range of the analytical response (from the limit of quantification to 33.3 mg L-1, in both methods), the limit of detection (0.6 for DPV and 0.4 mg L(-1) for SWV) and the limit of quantification (1.8 for DPV and 1.1 mg L(-1) for SWV) were evaluated. Satisfactory precision was achieved. Recovery tests produced results of 90.4% for both methods. These results were compared with the ones achieved by HPLC and no significant statistical difference was found between them.

Este trabalho descreve o comportamento eletroquÃmico dos antibiÃticos cefuroxima (CFX) e cefalexina (CFA) por voltametria cÃclica (VC) e o desenvolvimento de metodologia eletroanalÃtica por voltametria de onda quadrada (VOQ) para a determinaÃÃo de CFX e de CFA sobre eletrodo de gota pendente de mercÃrio (HMDE, do inglÃs Hanging Mercury Drop Electrode). CFX apresentou dois processos catÃdicos irreversÃveis em -0,35 V (pico 1) e em -1,03 V (pico 2), com a participaÃÃo de dois elÃtrons e dois prÃtons tanto para o pico 1 como para o pico 2. Sugere-se que a reaÃÃo de reduÃÃo da molÃcula de CFX ocorre no grupo â C = N â para o pico 1 e no grupo â CH2 â R para o pico 2. Os parÃmetros experimentais e voltamÃtricos foram otimizados, com as respostas sendo obtidas em tampÃo BR pH 2,0,f = 90 s-1, a = 30 mV e &#916;Es = 2 mV. Os limites de detecÃÃo (LD) e de quantificaÃÃo (LQ) foram calculados utilizando-se o pico 1, com LD = 4,92x10-9 mol L-1 e LQ = 1,64x10-8 mol L-1 e recuperaÃÃo 109,15% com desvio padrÃo relativo (RSD, do inglÃs Relative Standart Deviation) de 2,39% (n = 3) em eletrÃlito de suporte. A metodologia desenvolvida foi aplicada em amostras de medicamentos, leite bovino e Ãguas naturais, com recuperaÃÃes variando de 96,40 a 129,4%. Para CFA constatou-se a presenÃa de processos catÃdicos em -0,97 V (pico 1) e em -1,2 V (pico 2). Nesse caso, tem-se que a reduÃÃo ocorre na dupla ligaÃÃo do grupo etilÃnico envolvendo a transferÃncia de dois elÃtrons e dois prÃtons. Os parÃmetros experimentais e voltamÃtricos foram avaliados com as respostas otimizadas sendo obtidas em tampÃo BR pH 2,0, f = 100 s-1, a = 30 mV e &#916;Es = 4 mV. Considerando-se o pico em -1,2 V, curvas analÃticas foram construÃdas, a partir delas os limites de detecÃÃo e de quantificaÃÃo foram calculados, LD = 1,27x10-8 mol L-1 e LQ = 4,23x10-8 mol L-1 com recuperaÃÃo de 89,19% com RSD = 1,51% (n = 3) em eletrÃlito de suporte. A metodologia foi aplicada na quantificaÃÃo de amostras de medicamentos, leite bovino e Ãguas naturais, com percentuais de recuperaÃÃo variando de 78,45 a 92,13%. Nas metodologias desenvolvidas, foram obtidos baixos valores de LD e de LQ, recuperaÃÃo entre 78,00-130,0% e precisÃo intermediÃria com RSD < 5,00%, mostrando que estas metodologias apresentam Ãtima sensibilidade, eficiÃncia e precisÃo. / The present work describes the electrochemical behavior of the antibiotic cefuroxime (CFX) and cephalexin (CFA) by cyclic voltammetry (CV) and the development of an electroanalytical methodology for CFX and CFA determination on mercury electrodes allied to the square wave voltammetry (SWV). In this work, CFX showed two irreversible reduction peak at -0.35 V (peak 1) and -1.03 V (peak 2), with the participation of two electrons and two protons for both peaks. It is suggested that the reduction reaction of the CFX occurs on the group â C = N â for the peak one and on the group â CH2 â R for the peak two. Under optimized conditions, the results were obtained in BR buffer pH 2.0, f = 90 s-1, a = 30 mV and &#916;Es = 2 mV. Considering the peak 1, analytical curves were constructed and the limits of detection (LOD) and quantitation (LOQ) were calculated with LOD = 4.92Ã10-9mol L-1and LOQ = 1.64Ã10-8mol L-1. The recovery in electrolyte was 109.15% with RSD = 2.39% (n = 3). The methodology was applied in raw natural waters, commercial formulations and bovine milk obtaining recovery values from 96.40 to 129.4%. CFA reductions waves are observed at -0.97 V (peak 1) and -1.2 V (peak 2). These reduction waves are attributed to the reduction of ethylenic bond of CFA involving the transfer of two electrons and two protons. The optimized conditions were obtained in BR buffer pH 2.0, f = 100 s-1, a = 30 mV and &#916;Es = 4 mV. Considering the peak at -1.2 V, analytical curves were constructed and were calculated with LOD = 1.27Ã10-8mol L-1and LOQ = 4.23Ã10-8 mol L-1. The recovery in electrolyte was 89.19% with RSD = 1.51% (n = 3). The methodology was applied to quantify in raw natural waters, commercial formulations and bovine milk obtaining recovery values from 78.45 to 92.13%. In developed methodologies, we obtained low values of LOD and LOQ, recovery between 78.00 to 130.00% and reproducibility below 5.00%, showing that those methods had excellent sensitivity, precision and efficiency.

This research had as main objective to develop an electroanalytical procedure for the determination of amodiaquine in human milk, using a carbon paste electrode modified with hemin, hemoglobin functional group responsible for transporting oxygen. The amodiaquine (a derivative 4-aminoquinoquinolínico) is a drug used to treat malaria with anti-inflammatory and antipyretic properties. The characterization and preparation of carbon paste electrode modified with hemin was investigated. The electrochemical behavior of modified electrode was explored using differential pulse voltammetry. The best voltammetric response was obtained for the composition of carbon paste 10% hemin solution in Britton-Robson buffer pH 7.0. To study the electrochemical behavior of amodiaquine was used the technique of cyclic voltammetry, which presented in a neutral medium oxidation process at 0.054 V vs. Ag / AgCl at a rate of 20 mV s-1. The voltammetric techniques used to develop the methodology where the differential pulse voltammetry and square wave voltammetry. In this study the optimized conditions were: pH 7, scan rate = 40 mV s-1, amplitude = 200 mV and frequency = 150 Hz, where the parameters indicated the square wave voltammetry as the best technique for determining the voltammetric amodiaquine has a minor detection limit. The limits of detection and quantification were obtained in the determination of 8.7 x 10-7 mol L-1 and 2.9 x 10-6 mol L-1, respectively. After optimization of all relevant parameters, the analytical method developed was successfully applied in samples of breast milk. The concentration of amodiaquine obtained was 1.7 x 10-5 mol L-1. The method was efficient and reproducible in the determination of amodiaquine present in breast milk, there is no need any pretreatment of the sample. / Este trabalho teve como objetivo desenvolver uma metodologia eletroanalítica para a determinação de amodiaquina em leite materno, utilizando um biossensor a base de hemina, uma porfirina presente na hemoglobina e responável pelo transporte de oxigênio. A amodiaquina, por sua vez, é um derivado 4-aminoquinoquinolínico amplamente utilizada no tratamento da malária, com propriedades antiinflamatória e antipirética. Sua transferência para o leite materno é moderada, porém mesmo em baixas dosagens pode ser considerada prejudicial ao lactante. A caracterização e a preparo do biossensor foi a primeira etapa deste trabalho. O comportamento eletroquímico do biossensor foi explorado pelo emprego da técnica voltametria de pulso diferencial. As melhores respostas voltamétricas foram obtidas com a composição da pasta de carbono contendo 10% de hemina em solução-tampão Britton-Robson de pH 7,0. Para o estudo do comportamento eletródico da amodiaquina foi utilizada a técnica de voltametria cíclica, que apresentou em meio neutro um processo de oxidação em 0,054 V vs. Ag/AgCl na velocidade de 20 mV s-1 em pH 7,0. As técnicas voltamétricas utilizadas para o desenvolvimento da metodologia foram a voltametria de pulso diferencial e a voltametria de onda quadrada. Nesse estudo as condições otimizadas encontradas foram: pH = 7; velocidade de varredura = 40 mV s-1; amplitude = 200 mV e freqüência = 150 Hz, onde os parâmetros indicaram a voltametria de onda quadrada como a melhor técnica para a determinação voltamétrica da amodiaquina por apresentar melhor desempenho. Os limites de detecção e quantificação obtidos na determinação da amodiaquina por voltametria de onda quadrada foram de 8,7 x 10-7 mol L-1 e 2,9 x 10-6 mol L-1, respectivamente. Após a otimização de todos os parâmetros relevantes, o método analítico desenvolvido foi aplicado com sucesso em amostras de leite materno. A concentração de amodiaquina analisada foi de 1,7 x 10 -5 mol L-1. O método desenvolvido mostrou-se eficiente e reprodutível na determinação de amodiaquina presente no leite materno, não sendo necessário qualquer tratamento prévio da amostra.

Propõe-se método novo e completo para análise de acetona em ar exalado envolvendo coleta com pré-concentração em água, derivatização química e determinação eletroquímica assistida por novo algoritmo de processamento de sinais. Na literatura recente a acetona expirada vem sendo avaliada como biomarcador para monitoramento não invasivo de quadros clínicos como diabetes e insuficiência cardíaca, daí a importância da proposta. Entre as aminas que reagem com acetona para formar iminas eletroativas, estudadas por polarografia em meados do século passado, a glicina apresentou melhor conjunto de características para a definição do método de determinação por voltametria de onda quadrada sem a necessidade de remoção de oxigênio (25 Hz, amplitude de 20 mV, incremento de 5 mV, eletrodo de gota de mercúrio). O meio reacional, composto de glicina (2 mol·L-1) em meio NaOH (1 mol·L-1), serviu também de eletrólito e o pico de redução da imina em -1,57 V vs. Ag|AgCl constituiu o sinal analítico. Para tratamento dos sinais, foi desenvolvido e avaliado um algoritmo inovador baseado em interpolação de linha base por ajuste de curvas de Bézier e ajuste de gaussiana ao pico. Essa combinação permitiu reconhecimento e quantificação de picos relativamente baixos e largos sobre linha com curvatura acentuada e ruído, situação em que métodos convencionais falham e curvas do tipo spline se mostraram menos apropriadas. A implementação do algoritmo (disponível em http://github.com/batistagl/chemapps) foi realizada utilizando programa open source de álgebra matricial integrado diretamente com software de controle do potenciostato. Para demonstrar a generalidade da extensão dos recursos nativos do equipamento mediante integração com programação externa em linguagem Octave (open source), implementou-se a técnica da cronocoulometria tridimensional, com visualização de resultados já tratados em projeções de malha de perspectiva 3D sob qualquer ângulo. A determinação eletroquímica de acetona em fase aquosa, assistida pelo algoritmo baseado em curvas de Bézier, é rápida e automática, tem limite de detecção de 3,5·10-6 mol·L-1 (0,2 mg·L-1) e faixa linear que atende aos requisitos da análise em ar exalado. O acetaldeído, comumente presente em ar exalado, em especial, após consumo de bebidas alcoólicas, dá origem a pico voltamétrico em -1,40 V, contornando interferência que prejudica vários outros métodos publicados na literatura e abrindo possibilidade de determinação simultânea. Resultados obtidos com amostras reais são concordantes com os obtidos por método espectrofotométrico, em uso rotineiro desde o seu aperfeiçoamento na dissertação de mestrado do autor desta tese. Em relação à dissertação, também se otimizou a geometria do dispositivo de coleta, de modo a concentrar a acetona num volume menor de água gelada e prover maior conforto ao paciente. O método completo apresentado, englobando o dispositivo de amostragem aperfeiçoado e o novo e efetivo algoritmo para tratamento automático de sinais voltamétricos, está pronto para ser aplicado. Evolução para um analisador portátil depende de melhorias no limite de detecção e facilidade de obtenção eletrodos sólidos (impressos) com filme de mercúrio, vez que eletrodos de bismuto ou diamante dopado com boro, entre outros, não apresentaram resposta. / A new and complete method for analyzing acetone in exhaled breath is proposed, involving sample collection by preconcentration in water, chemical derivatization and electrochemical determination assisted by a new signal processing algorithm. Breath acetone is under investigation in the medical literature as a biomarker for noninvasive monitoring of clinical conditions such as diabetes and heart failure and better analyzers are demanded. Some amines that react with acetone to form electroactive imines, already studied by polarography in the middle of the last century, were evaluated here as derivatizing agents and glycine presented the best compromise for the definition of a determination method by square wave voltammetry without purging of dissolved oxygen (20 mV pulses at 25 Hz with 5 mV scanning steps, mercury drop electrode). The derivatization medium of glycine (2 mol·L-1) and NaOH (1 mol·L-1) also serves as electrolyte and the imine reduction peak at -1.57 V vs. Ag|AgCl constitutes the analytical signal. For signal processing a novel algorithm based on baseline automatic prediction using Bézier curves was developed in combination with Gaussian peak fitting. This innovative combination enables the recognition and quantification of relatively low and broad peaks on a curved and noisy baseline while standard procedures fail and fitting of splines is less appropriate. The algorithm (available at http://github.com/batistagl/chemapps) was implemented using open source matrix algebra software directly integrated with the potentiostat control software. The generality of this approach to extend the native resources of commercial equipment was evidenced by proceeding the implementation of 3D chronocoulometry with help of the open source Octave language. The processed charge vs. potential vs. time function 3-D surfaces for the forward and backward steps can be observed from any angle on the screen. The fast automatic electrochemical determination of acetone assisted by the Bézier based curve fitting algorithm, with an acetone detection limit of 3.5·10-6 mol·L-1 (0.2 mg·L-1) and a wide linear range is entirely satisfactory for breath analysis. Acetaldehyde, an expected component of breath, especially after ingestion of alcohol, and a common interferent for some other analytical methods, generates a peak at -1.40 V that does not interfere with the acetone determination and that is possibly suitable for simultaneous determination of this analyte. Results obtained with real samples are in agreement with spectrophotometric determinations routinely applied to breath analysis, formerly developed in the master dissertation of the author of this thesis. The geometry of the impinger sampling device was also improved (in comparison with the dissertation) in order to collect the acetone in a smaller volume of cooled water and to allow the patient to breath more freely. The complete method at its current state of development, encompassing the improved sampler and the new effective signal processing algorithm for voltammetric signals is ready for use. Evolution to a portable analyzer can be envisioned if the shortcomings of detection limits of solid (screen-printed) electrode preparation with a mercury film are overcome, once the imine signal was not observed on solid electrodes like bismuth and boron doped diamond.

Philosophiae Doctor - PhD / South African Carpobrotus species have been found to contain hydrolysable tannins,various flavonoids e.g. rutin and hyperoside, phytosterols and aromatic acids which have a diverse range of pharmacological properties including antimicrobial and, antioxidant activities. The main aim of the thesis was to determine the natural products in C. mellei and C. quadrifidus using chromatographic techniques and electrochemical analysis. The antioxidant activity of both Carpobrotus species was determined by using a superoxide dismutase (SOD) biosensor. ESI-LC-MS was used to separate and determine flavonoids in C. mellei and C. quadrifidus. 8 flavonoid compounds: catechin, epicatechin, epicatechin-epicatechin, coumarylquinic acid, isorhamnetin, quercetin-hexose (hyperoside), rutin and myricetin-deoxyhexose were identified. Cyclic and square wave voltammetry were used to detect flavonoids from C. mellei and C. quadrifidus. Catechin was detected in the ethyl acetate extract of C. mellei and C. quadrifidus. The oxidation potential of the plant extracts were observed at +150.6 mV to +1072.6 mV. The oxidation mechanism proceeds in sequential steps, related to the catechol moiety, -OH groups in C ring and the resorcinol group. The oxidation process of the catechol moiety involves a two electron - two proton reversible reaction and forms o-quinone. This occurs first at low potential and is a reversible reaction. The hydroxyl group in the C ring and resorcinol group oxidise there after and undergo an irreversible reaction. UV-vis and FTIR spectroscopy were used to confirm the presence of catechin in the ethyl acetate extract of both plants.UV-visible spectroelectrochemistry confirmed the oxidation process of catechin at constant potential. Since C. mellei and C. quadrifidus were confirmed to contain flavonoids by ESI-LC-MS and electrochemical analysis, the antioxidant activity was further investigated using a SOD biosensor. The superoxide dismutase (SOD) enzyme was immobilised with 1% Nafion on a platinum electrode. Detection limit and sensitivity of the SOD biosensor were found to be 0.03918 μmol L-1 and 1.44 μA(μmol L-1)-1, respectively. The results showed that C. mellei and C. quadrifidus have antioxidant activity, with relative antioxidant capacity (RAC) of 24% and 42%, respectively. May 2009

La détection de molécules odorantes représente un enjeu important dans divers domaines tels que l’industrie alimentaire, le diagnostic médical et la sécurité du territoire, par exemple. En effet, les odorants, présents par milliers dans notre environnement, véhiculent de nombreuses informations, via leur nature chimique ou leur concentration. Notre système olfactif est capable de discriminer des milliers de molécules différentes via des mécanismes biochimiques impliquant l’association de nombreux partenaires protéiques et un codage combinatoire de l’information. Ces biomolécules, qui englobent notamment les récepteurs olfactifs et les protéines de liaison aux odorants (OBP), constituent une source intéressante d’éléments de détection pour la conception de biocapteurs. Les OBP sont de petites protéines solubles présentent dans le mucus nasal à des concentrations de l’ordre du millimolaire. Leur poche de liaison hydrophobe leur confère la capacité de lier de façon réversible les molécules odorantes. Leur robustesse et leur manipulation aisée en font de bonnes candidates pour l’élaboration de biocapteurs. Au cours de ce travail, nous nous sommes intéressés à la détection de molécules odorantes en associant des OBP comme biorécepteur et l’électrochimie comme méthode de transduction. Par une méthode de mutagenèse dirigée, nous avons montré qu’en modifiant un seul des acides aminés dans la poche de liaison de deux OBP de rat (rOBP2 et rOBP3), il était possible de moduler leurs affinités envers les odorants. En parallèle, nous avons décrit la détection qualitative et quantitative de molécules volatiles à partir d’OBP. Nous avons montré que rOBP3 lie la 2-méthyl-1,4-naphtoquinone (MNQ), une sonde électrochimique. La quantité de MNQ déplacée de la poche de liaison de rOBP3 par la 3-isobutyl-2-méthoxypyrazine (IBMP), un odorant modèle, a été mesurée par voltammétrie cyclique à vagues carrées. Nous avons déterminé les constantes de dissociation des complexes rOBP3 / MNQ et rOBP3 / IBMP. Les valeurs mesurées par électrochimie ont été confirmées par compétition avec une sonde fluorescente et par titration calorimétrique isotherme. En combinant cette nouvelle méthode analytique à des variants de rOBP3 qui présentent des profils de liaison différents et complémentaires, nous avons détecté sélectivement chacun des constituants d’un mélange ternaire d’odorants. Ces travaux, qui allient ingénierie des OBP et électrochimie, offrent des perspectives intéressantes dans le domaine des nez électroniques. / The detection of odorant molecules has become an important challenge in different research area, such as the food industry, medical diagnostics and homeland security. Indeed, the thousands of odorants in our environment provide information on their chemical nature or their concentration. Human olfactory system is capable of discriminating thousands of different molecules thanks to biochemical mechanisms involving multiple protein receptor partners and a combinatorial coding. These biomolecules that include olfactory receptors and odorant-binding proteins (OBP) represent an interesting source of detectors for the design of biosensors. OBPs are small soluble proteins present in nasal mucus at millimolar concentrations. Their hydrophobic binding pocket gives them the ability to reversibly bind odorant molecules. OBPs are robust and easy to produce and are thus good candidates for the design of biosensors. In this work, we focused on the detection of odorant molecules associating OBPs as a bioreceptor and electrochemistry as a transduction method. Using site-directed mutagenesis, we have shown that by substituting a single amino acid in the binding pocket of two rat OBPs (rOBP2 and rOBP3), it is possible to modulate their binding affinities towards odorants. In parallel, we described a qualitative and quantitative method for the detection of volatile molecules using OBPs. We have shown that rOBP3 binds 2-methyl-1,4-naphtoquinone (MNQ), an electrochemical probe. The amount of MNQ displaced from the binding pocket of rOBP3 by the model odorant 3-isobutyl-2-methoxypyrazine (IBMP), was measured using square-wave voltammetry. We determined the dissociation constants of the rOBP3 / MNQ and rOBP3 / IBMP complexes. These values measured by electrochemistry were confirmed by a competitive fluorescent assay and isothermal titration calorimetry. By combining this new analytical method to rOBP3 variants with different and complementary binding profiles, we were able to selectively detect each of the components of a ternary mixture of odorants. This work, that combines the engineering of OBPs and electrochemistry, offers us interesting perspectives in the field of electronic noses.

Philosophiae Doctor - PhD / South African Carpobrotus species have been found to contain hydrolysable tannins, various tlavonoids e.g. rutin and hyperoside, phytosterols and aromatic acids which have a diverse range of pharmacological properties including antimicrobial and, antioxidant activities. The main aim of the thesis was to determine the natural products in C. mellei and C. quadrifidus using chromatographic techniques and electrochemical analysis. The antioxidant activity of both Carpobrotus species was determined by using a superoxide dismutase (SOD) biosensor. ESI-LC-MS was used to separate and determine tlavonoids in C. mellei and C. quadrifidus. 8 tlavonoid compounds: catechin, epicatechin, epicatechin-epicatechin, coumarylquinic acid, isorhamnetin, quercetin-hexose (hyperoside), rutin and myricetin-deoxyhexose were identified. Cyclic and square wave voltammetry were used to detect tlavonoids from C. mellei and C. quadrifidus. Catechin was detected in the ethyl acetate extract of C. mellei and C. quadrifidus. The oxidation potential of the plant extracts were observed at +150.6 mV to +1072.6 mV. The oxidation mechanism proceeds in sequential steps, related to the catechol moiety, -OH groups in C ring and the resorcinol group. The oxidation process of the catechol moiety involves a two electron - two proton reversible reaction and forms o-quinone. This occurs first at low potential and is a reversible reaction. The hydroxyl group in the C ring and resorcinol group oxidise there after and undergo an irreversible reaction. UV-vis and FTIR spectroscopy were used to confirm the presence of catechin in the ethyl acetate extract of both plants. UV -visible spectroelectrochemistry confirmed the oxidation process of catechin at constant potential. Since C. mellei and C. quadrifidus were confirmed to contain flavonoids by ESI-LC-MS and electrochemical analysis, the antioxidant activity was further investigated using a SOD biosensor. The superoxide dismutase (SOD) enzyme was immobilised with 1% Nafion on a platinum electrode. Detection limit and sensitivity of the SOD biosensor were found to be 0.03918 umol L-' and 1.44 !lA (umol i.'):', respectively. The results showed that C. mellei and C. quadrifidus have antioxidant activity, with relative antioxidant capacity (RAC) of 24% and 42%, respectively.