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Cholesterol and ethanol biosensors based on conducting polypyrrole (PPy), poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(3,4-ethylenedioxypyrrole) (PEDOP) were constructed. Cholesterol oxidase (ChOx, from Pseudomonas fluorescens) and alcohol oxidase (AlcOx, from Pichia pastoris) were physically entrapped during electropolymerization of the monomers (Py, EDOT, EDOP) in phosphate buffer containing sodium dodecylsulfate (SDS) as the supporting electrolyte. The amperometric responses of the enzyme electrodes were measured monitoring oxidation current of H2O2 at +0.7 V in the absence of a mediator. Kinetic parameters, such as Km and Imax, operational and storage stabilities, effects of pH and temperature were determined for all entrapment supports. Based on Michaelis-Menten (Km) constants, it can be interpreted that both enzymes immobilized in PEDOT showed the highest affinities towards their substrates. Before testing the alcohol biosensors on alcoholic beverages, effects of interferents (glucose, acetic acid, citric acid, L-ascorbic acid) which might be present in beverages were determined. The alcohol content of the distilled beverages (vodka, dry cin, whisky, raki) was measured with these biosensors. A good match with the chromatography results (done by the companies) was observed.

In this thesis, six different biosensors based on conducting polymers of poly 4-(2,5-di(thiophen-2-yl)-1H-pyrrole-1-l) benzenamine [poly(SNSNH2)] and poly(1- (4-nitrophenyl)-2,5-di(2-thienyl)-1H-pyrrole [poly(SNSNO2)] were prepared. Electrochemical technique was used for polymerization of conducting polymers and two different immobilization techniques / crosslinking and adsorption were used for immobilizing enzyme or microbial in the conducting polymer matrices. The proposed biosensors were characterized and optimized. Optimum pH, thickness of conducting polymer and biological material amount were determined. Linearity, repeatability and operational stability experiments were performed. Carbon nanotubes and gold nanoparticles were also added to the biosensing system to see the effects of nanoparticles. The biosensors also used for ethanol and/or glucose biosensing in commercial samples. In the first part of thesis, a biosensor was designed by immobilizing Gluconobacter oxydans in poly(SNSNH2) matrix on graphite electrode. The biosensor preparation method was a two-step procedure where the cells were immobilized by adsorption on the surface after the electropolymerization step.Use of dialysis membrane to cover the surface after immobilization conserves the bioactive surface during the operation. The preparation is simple and not time consuming. Systems proposed showed good linearity and repeatability as well as high operational stability. Glucose amount in fruit juice, ethanol amount in vodka and whisky were determined. In the second part of thesis, a second biosensor was designed with electrochemical polymerization of 1-(4-nitrophenyl)-2,5-di(2-thienyl)- 1H-pyrrole via cyclic voltammetry on graphite electrode. Afterwards, Pseudomonas fluorescens and Gluconobacter oxydans were immobilized successfully on the conducting polymer matrix separately. The proposed biosensors showed good linear range, and repeatability as well as high operational stability. In the third and fourth parts, gold nanoparticle and carbon nanotube effects were studied on poly(SNSNH2)/glucose oxidase biosensor, respectively. Covalent binding of glucose oxidase was achieved to poly(SNSNH2) by the help of glutaraldehyde on the top of graphite and carbon paste electrodes. Nanoparticle amount and optimum pH were determined for both biosensors. After analytical characterization, glucose amount in two fruit juices were determined with poly(SNSNH2)/GOx/AuNP and poly(SNSNH2)/ GOx/CNT biosensors. In the last part, biosensor was designed with immobilizing alcohol oxidase in poly(SNSNH2) matrix via crosslinking with glutaraldehyde on platinum electrode. The proposed biosensor was characterized and optimized in terms of thickness, enzyme loading, pH, AuNPs, CNTs, linear range, repeatability and operational stability.

Made available in DSpace on 2016-08-19T12:56:35Z (GMT). No. of bitstreams: 1 DARLAN FERREIRA DA SILVA.pdf: 808482 bytes, checksum: cec93c6177a364a96efd7df7673d26a0 (MD5) Previous issue date: 2010-10-11 / Organophosphate insecticides, especially those based on the active ingredient methyl parathion, have been extensively used in crop protection of rice in the State of Maranhão. In this work, an optimized chromatographic method for determination of the methyl parathion insecticide by using solid phase microextraction in a confined environment (headspace) followed by analysis employing gas chromatography with mass spectrometric detection (HS-SPME-GC/MS). Electroanalytical methods using amperometric biosensors were also developed and used to analyze the composite samples of fresh rice. The optimized method using SPME resulted in satisfactory results for sensitivity (limit of detection: 0.026 mg/L-1), precision (coefficients of variation between 6.1 and 22.4%, 8.8% and 32.5 and 19.7 and 24.6%, for milled rice samples, rice with shells and full rice, respectively) and accuracy (recoveries ranging from 73.2 to 90% for milled rice samples, from 88.7 to 89.5%, for paddy rice samples and from 59.5 to 60.6% for full rice samples). In the case of biosensors, these have been built based on acetylcholinesterase enzyme (AChE), commercially obtained or genetically modified, having the sensors prepared with carbon paste modified with TCNQ (tetracyanoquinodimethane) mediator. Better sensitivities were observed with sensors based on AChE enzymes extracted from Drosophila melanogaster. Detection limits of 10.0, 0.05 and 0.001 μg.L-1 were found for the sensors based on AChE (ee), AChE (eb) and mutant enzymes (AChE (Dros) B08 e B12), respectively. The efficiency of the detection method of the methyl parathion insecticide in rice samples without any previous treatment was evidenced by an average recovery of 103.9%, 101%, 102.3% and 105.6%, for the sensors prepared with AChE (ee), AChE (eb), AChE (Dros) B08 and B12 enzymes, respectively. In general, the chromatographic technique used was practical and efficient for direct detection of insecticide residues in rice samples in a wider range of concentration (0.1 a 1 mg.Kg-1). Since the biosensor was more sensitive and effective in smaller amounts (0.0005 a 0.01 mg.Kg-1) of such insecticide in rice, considering the additional advantage of speed and decreasing cost of the analytical technology. / Os inseticidas organofosforados, em especial aqueles à base do princípio ativo paration metílico, têm sido extensivamente usados na proteção de cultivos de arroz no Estado do Maranhão. Neste trabalho, foi otimizado um método cromatográfico para determinação do inseticida paration metílico usando microextração em fase sólida em ambiente confinado (headspace), seguido de análise empregando cromatografia a gás com detecção por espectrometira de massas (HS-SPME-GC/MS). Métodos eletroanalíticos empregando biossensores amperométricos também foram desenvolvidos, tendo sido empregados para análise do composto em amostras de arroz in natura. O método otimizado empregando SPME resultou em resultados satisfatórios quanto à sensibilidade (limite de detecção: 0,026 mg/L-1), precisão (coeficientes de variação entre 6,1 e 22,4%; 8,8 e 32,5% e 19,7 a 24,6%, para as amostras de arroz polido, arroz com casca e arroz integral respectivamente) e exatidão (recuperações na faixa de 73,2 a 90%, para as amostras de arroz polido; de 88,7 a 89,5 %, para as amostras de arroz com casca e de 59,5 a 60,6%, para as amostras de arroz integral). No caso dos biossensores, estes foram construídos à base de enzimas acetilcolinesterase (AChE), comerciais e geneticamente modificadas, tendo sido os sensores preparados com pasta de carbono modificada com o mediador TCNQ (tetracianoquinodimetano). Melhores sensibilidades foram verificadas com sensores preparados à base de enzimas AChE extraídas da Drosophila Melanogaster. Limites de detecção de 10; 0,05 e 0,001μg.L-1 foram encontrados para os sensores preparados com as enzimas AChE (ee), AChE (eb) e mutantes (AChE (Dros) B08 e B12), respectivamente. A eficiência do método de detecção do inseticida paration metílico em amostras de arroz sem nenhum prévio tratamento foi comprovada pelas recuperações médias de 103,9%; 101 %; 102,3% e 105,6 % para os sensores preparados com as enzimas AChE (ee), AChE (eb), AChE (Dros) B08 e B12 respectivamente. De modo geral, a técnica cromatográfica empregada foi prática e eficiente para detecção direta de resíduos do inseticida em amostras de arroz, em uma faixa maior de concentração (0,1 a 1 mg.Kg-1). Já os biossensores foram mais sensíveis e eficientes em teores menores (0,0005 a 0,01 mg.Kg-1) do inseticida no arroz, considerando ainda a vantagem adicional da rapidez e menor custo da técnica analítica.

Dans cette thèse, des travaux de recherche ont été menés pour immobiliser différentes enzymes (oxydases et déshydrogénases) au sein d'une matrice de silice dans le but de construire un biocapteur ampérométrique. Des matériaux nanostructurés ont ensuite été introduit dans ce système afin d'améliorer les caractéristiques analytiques de ces biocapteurs. La méthode de dépôt sol-gel par assistance électrochimique a été choisie pour l'immobilisation des enzymes à la surface des électrodes et des nanomatériaux car elle donne la possibilité de contrôler finement l'épaisseur du film déposé afin de couvrir individuellement ces objets. La faisabilité de cette approche a été montrée par la modification de nanofibres de platine présentant une grande surface active pour l'oxydation de H2O2 produit en présence de biocomposite silice-glucose oxidase. Le dépôt sol-gel électrochimiquement assisté permet également la modification d'électrodes imprimées en or par un biocomposite silice-choline oxidase, ce qui donne la possibilité de construire rapidement un biocapteur à choline présentant de très bonnes caractéristiques analytiques. Les nanotubes de carbone ont également été choisis comme matrice pour l'immobilisation de déshydrogénases car ils permettent d'obtenir une grande aire spécifique et des propriétés catalytiques intéressantes pour l'oxydation du co-facteur enzymatique NADH. La méthode de dépôt électrophorétique a été utilisée pour créer des couches de nanotubes poreuses ayant une épaisseur contrôlée à la surface d'un support de carbone vitreux. Les électrodes ainsi préparées présentent de bonne performances électrochimiques, permettant notamment de déplacer le potentiel d'oxydation de NADH et d'augmenter la sensibilité de détection. Un biocomposite silice-sorbitol déshydrogénase a ensuite été déposé à la surface de la couche de nanotubes de carbone en utilisant la méthode de dépôt sol-gel assisté par électrochimie pour construire un biocapteur à sorbitol. La méthode de dépôt électrophorétique a enfin été appliquée pour la première fois à l'élaboration d'assemblages de nanotubes de carbone macroporeux. De tells assemblages ont été utilisés comme support pour co-immobiliser la sorbitol déshydrogénase et le co-facteur enzymatique au sein des macropores, ce qui a permis d'augmenter la sensibilité de la détection du sorbitol par comparaison avec un assemblage de nanotubes de carbone non macroporeux / In this thesis, the research work was focused on the immobilization of different enzymes (oxidases and dehydrogenases) into biocomposite silica matrix with the aim of amperometric biosensors construction. Then, the structured nanomaterials were introduced in the system in order to improve the characteristics of biosensors. The method of electrochemically-assisted deposition was chosen for the immobilization of enzymes on the surface of nanomaterials as it provides possibility of fine tuning of film thickness allowing covering each individual nanoobject. The feasibility of this was shown while modifying the platinum nanofibers, which demonstrate high electroactive surface and H2O2 oxidation rate, with silica-glucose oxidase biocomposite. The electrochemically-assisted deposition also allows the express modification of gold screen-printed electrodes with silica-choline oxidase biocomposite making possible the quick fabrication of cheap choline biosensors with high analytical characteristics. The carbon nanotubes was chosen as matrix for the immobilization of dehydrogenases as they have high surface area and electrocatalytic properties towards the oxidation of enzymatic co-factor NADH. The method of electrophoretic deposition was used for the creation of porous CNT-layer with controllable thickness on the surface of glassy carbon electrode. Created thereby matrix demonstrated good electrochemical performance significantly shifting the potential and increasing sensitivity of NADH oxidation. Then the biocomposite silica-sorbitol dehydrogenase film was deposited on the CNT-layer by means of electrochemically-assisted deposition for the construction of high-performance sorbitol biosensor. The method of electrophoretic deposition was also applied for the first time for the construction of thick macroporous CNT-assemblies with uniform pore size using template approach. Such macroporous CNT-electrode was used for the co-immobilization of sorbitol dehydrogenase and enzymatic co-factor inside the pores demonstrating higher sensitivity of sorbitol detection in comparison with nonporous CNT-electrode