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.

This work is devoted to the study of boron doped diamond as electrode material, its properties and use in electroanalytical methods - in voltammetric and subsequently amperometric methods in combination with high performance liquid chromatography. The series of boron doped diamond films was tested with respect to the effect of boron concentration on their morphology, quality, electrochemical and spectral properties using scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and cyclic voltammetry and differential pulse voltammetry. Further, the effect of boron concentration on the determination of selected substances was investigated, both for their oxidation (2-aminobiphenyl, benzophenone-3) and for their reduction (5-nitroquinoline). Furthermore, a voltammetric and amperometric method was developed for the determination of a mixture of aminobiphenyls and aminonaphthalenes using a boron doped diamond electrode. The effects of activation cleaning programs on the signal of benzophenone-3 were investigated using a boron doped diamond electrode, and the determination of benzophenone-3 on boron doped diamond electrode in the presence of the selected surfactant was studied. Boron doped diamond as carbon-based material was compared with other selected carbon materials such as glassy...

This diploma thesis deals with the determination of creatinine using a combination of flow injection analysis (FIA) or high-performance liquid chromatography (HPLC) with pulse amperometry, an electrochemical technique based on the application of potential pulses on a gold working electrode. The determination was performed in a basic environment of borate buffer with creatinine concentration of 1∙10-4 mol∙l-1 . The lenght of the cleaning and activation pulse was optimized as well as the pH of the running buffer. A cleaning pulse of +1.8 V was first applied to the electrode for 100 ms, then an activation potential of -0.5 V was applied for 150 ms and then a measuring potential of +0.2 V for 300 ms. The optimal pH was selected as pH=9,4. Methanol and acetonitrile were added to the borate buffer to test whether creatinine could be determined in presence of these organic solvents and whether flow injection analysis could be transformed into HPLC. Methanol in the system caused peak deformation, acetonitrile did not cause the peak deformation in the system, at higher contents the baseline was destabilized. Furthermore, the calibration dependence in the range of concentrations from 2.5∙10-4 mol∙l-1 to 5∙10-6 mol ∙ l-1 was measured using PAD in combination with FIA. At higher concentrations, peaks splitted....

Research on the cure and early detection of diseases such as diabetes, Alzheimer's, and Parkinson's is becoming of great interest due to the increasing number of people affected by them every year. An accurate and quick detection of various damaging species is highly critical in treatments of such diseases not only for exploring possible cures but also for early detection. If these diseases are detected during the initial stages than the possibility of curing them is much higher. Motivated by this, many researchers today have developed numerous types of sensing devices that can detect various physiological and biological compounds. However, most of these sensors are enzyme based. They have several setbacks such as the lack of sensitivity, restricted selectivity, short shelf life, and biological fouling. To overcome these obstacles, we examine the use of nanoceria modified Pt and Au electrodes for the detection of glucose and reactive oxygen species such as hydrogen peroxide. Amperometric detection of glucose and hydrogen peroxide is critical for biological applications for diabetes and possible Alzheimer's and Parkinson's patients. This dissertation focuses on the exploration of non-enzymatic detection of glucose and reactive oxygen species which has the prospective to be used for biological applications, in addition to an investigation of an odor control technology that uses these reactive oxygen species for the treatment of wastewater plants. The combination of bi-metallic composites with nanoceria showed increased oxidation ability towards glucose and hydrogen peroxide. The following dissertation expands on the relationship between bi-metallic nanoceria composite materials and its electro-oxidation of glucose and hydrogen peroxide towards biological sensing along with an investigation of an odor control technology that utilizes generates hydroxyl radical fine particle mist for the degradation of hydrogen sulfide odor in wastewater treatment plants.

>Magister Scientiae - MSc / Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin(hCG) is presented. This involves the modification of a gold surface with a thiolated β-cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.

Le projet de ma thèse a consisté en la mise au point de biomatériaux capables d'agir en tant que capteurs moléculaires pour la construction de biocapteurs d'affinité tels que des immunocapteurs, aptacapteurs et capteurs d'ADN, basés sur la lecture électrochimique. Les biocapteurs électrochimiques deviennent une technique intéressante pour l'identification des biomolécules en raison de possibilités de miniaturisation, de faible coût et de la lecture directe des signaux électriques. Toutefois, le choix d'un transducteur, qui permet d'obtenir un signal électrochimique, est crucial dans la construction du biocapteur. Au cours de ma thèse, j'ai eu l'occasion de comparer l'efficacité de différents matériaux conductrices tels que les conducteurs polymères (polypyrrole), les nanotubes de carbone et des nanoparticules d'or. Pour obtenir une réponse électrochimique intense, j'ai associé ces plateformes avec un marqueur redox-ferrocène. Les biocapteurs ont été basés sur la détection directe, généralement avec un «signal off» (diminution de la réponse électrochimique lors de la détection). J'ai travaillé sur différents types de reconnaissance biologique comme anticorps/antigène, aptamer/protéine, sonde ADN/ADN cible. Ces biocapteurs sont particulièrement intéressants dans le domaine de la biologie et de la santé publique. Au début je me suis intéressée à la nouvelle protéine impliquée dans le virus de la grippe et démontrée son évolution dans le cycle viral avec l'objectif de développer de nouveaux médicaments pour cette maladie ainsi que de nouveaux outils de détection. J'ai construit ces biocapteurs basés sur polymère conductrice-polypyrrole associé avec le marqueur redox, ferrocène pour l'immobilisation des anticorps spécifique pour les protéines impliquées dans le virus de la grippe. De nouveaux biorécepteurs - aptamères et des techniques électrochimiques ont été ensuite développés pour concevoir un système sensible capable de détecter la protéine prion cellulaire au niveau pM dans les échantillons de plasma humaine. Les aptamères sont associés sur la plateforme composée de nanotubes de carbone, conjuguées avec des dendrimères poly(amidoamine) PAMAM. Les composites combinent les performances électriques de nanotubes mais permet simultanément l'attachement de nombreux biomolécules, en raison des nombreux groupes amines portant par des dendrimères. Puis j'étais aussi intéressé par la détection de l'ADN par le développement de biocapteurs à base de nanotubes de carbone pour deux maladies infectieuses telles que l'hépatite C avec des cibles d'ADN synthétiques et l'ADN de M. tuberculosis provenant d'échantillons PCR. Ces exemples ont été utilisés pour démontrer que le capteur d'ADN pourrait être généralisé à toutes les maladies infectieuses et utilisé dans le système «point of care». Des études précédentes ont consisté dans le dépôt de nanotubes de carbone sur la surface par adsorption et j'ai trouvé que c'était problématique en termes de reproductibilité. Alors, j'ai utilisé polypyrrole comme une matrice pour l'association des nanotubes de carbone. Cette méthode semble être la plus efficace et a permis de combiner les propriétés des nanotubes avec celles de polypyrrole conducteurs. Au cours de ma thèse, j'ai démontré que les capteurs électrochimiques d'affinité à base de polymères conducteurs et les nanomatériaux peuvent être appliqués dans différents domaines concernant les problèmes de santé. Ces biocapteurs sont prêts pour être intégrés dans les microsystèmes ainsi que dans les systèmes «point of care». / The project of my thesis consisted on the development of biomaterials that are able to act as molecular transducers for the construction of affinity biosensors such as immunosensors, aptasensors and DNA sensors, based on electrochemical reading. Electrochemical biosensors become an attractive technique for the identification of biomolecules due to miniaturization possibilities, low cost and direct lecture of electric signals. However the choice of a transducer, which allows obtaining electrochemical signal, is crucial in biosensor construction. During my thesis I had the opportunity to compare the efficacy of different conducting materials such as conducting polymers (polypyrrole), carbon nanotubes. To obtain an intense electrochemical response, I associated these platforms with a redox marker – ferrocene. The biosensors which I constructed were based on direct detection, usually with “signal off” (decrease in electrochemical response during detection). I worked on different types of biological recognition such as antibody/antigen, aptamer/protein, DNA probe/DNA target. These biosensors are especially attractive in the biological field and public health. First, I was interested in the new protein involved in Influenza virus and demonstrated its evolution in viral cycle with the objective to develop new drugs for this disease as well as new tools for detection. I constructed biosensors based on conducting polypyrrole which was studied extensively in our group. I used this polypyrrole matrix associated with redox marker, ferrocene for immobilization of antibody specific for protein involved in Influenza virus. By this approach I demonstrate that electrochemical biosensors can become effective tools in the daily laboratory work, especially useful for biologists who are often limited by commercially available methods. Then new bioreceptors - aptamers and electrochemical techniques have been developed to design a sensitive system able to detect cellular prion protein at pM level in plasma samples. Aptamers were associated on the platform composed of polypyrrole or carbon nanotubes conjugated with dendrimers poly(amidoamine) PAMAM. Composite combines the high electrical performance of transducers but simultaneously allows attachment of high number of biomolecules, due to numerous amine groups bearing by dendrimers. I was also interested in DNA detection and in the development of biosensors based on carbon nanotubes for two infectious diseases like hepatitis C with synthetic DNA targets and M. tuberculosis DNA from PCR samples. Such examples were used to demonstrate that DNA sensor could be generalised to all infectious diseases and used in point-of-care system. My previous studies consisted on the deposition of carbon nanotubes on the surface by adsorption and I found that it was problematic in terms of reproducibility, so important in biosensor construction. I used polypyrrole as a matrix for carbon nanotubes association. This method seems to be effective and allowed combination of nanotubes properties with those of conducting polypyrrole. During my thesis I demonstrated that electrochemical affinity sensors based on conducting polymers and nanomaterials can be applied in different fields concerning health problems. These biosensors are ready for integration in microsystems for application as analytical tools as well as in point-of-care systems.

Hämoglobin-A1c (HbA1c) ist ein Hämoglobin (Hb)-Subtypus, der durch nicht-enzymatische Glykierung des N-terminalen Valinrestes der Hämoglobin-beta-Kette entsteht. Das gemessene Verhältnis von HbA1c zum Gesamt-Hämoglobin (5-20 % bei Diabetikern) repräsentiert den Mittelwert der Blutglucosekonzentration über einen zweimonatigen Zeitraum und stellt zur Beurteilung der diabetischen Stoffwechsellage eine Ergänzung zur Akutkontrolle der Glukosekonzentration dar.<br /> Ziel der vorliegenden Arbeit war es, einen amperometrischen Biosensor für die Bestimmung des medizinisch relevanten Parameters HbA1c zu entwickeln. Durch Selektion geeigneter Bioerkennungselemente und deren Immobilisierung unter Erhalt der Bindungsfunktion für die Zielmoleküle Hämoglobin bzw. HbA1c wurden spezifische, hochaffine und regenerationsstabile Sensoroberflächen geschaffen. Für die Entwicklung des HbA1c-Biosensors wurden zwei Konzepte - Enzymsensor und Immunosensor - miteinander verglichen. <br /> Die enzymatische Umsetzung von HbA1c erfolgte mit der Fructosylamin Oxidase (FAO) aus Pichia pastoris N 1-1 unter Freisetzung von H2O2, welches sowohl optisch über eine Indikatorreaktion als auch elektrochemisch nach Einschluss der FAO in PVA-SbQ und Fixierung des Immobilisats vor einer H2O2-Elektrode nachgewiesen wurde. Die Kalibration des Enzymsensors mit der HbA1c-Modellsubstanz Fructosyl-Valin ergab Nachweisgrenzen, die ausserhalb des physiologisch relevanten HbA1c-Konzentrationsbereich lagen. Aus der Umsetzung von glykierten Peptiden mit einer nicht HbA1c analogen Aminosäurensequenz, z.B. Fructosyl-Valin-Glycin wurde zudem eine geringe HbA1c-Spezifität abgeleitet.<br /> Für den Immunosensor wurden zwei heterogene Immunoassay-Formate unter Verwendung von hochaffinen und spezifischen Antikörpern in Kombination mit Glucose Oxidase (GOD) als Markerenzym zum Nachweis von HbA1c untersucht. Beim indirekt-kompetitiven Immunoassay wurde anstelle des kompletten HbA1c-Moleküls das glykierte Pentapeptid Fructosyl-Valin-Histidin-Leucin-Threonin-Prolin (glkPP) als Kompetitor und Affinitätsligand immobilisiert und so eine regenerierfähige Oberfläche geschaffen. Beim Sandwich-Immunoassay wurde im ersten Schritt Gesamt-Hämoglobin an die mit Haptoglobin (Hp) modifizierte Festphase angereichert und im zweiten Schritt der gebundene HbA1c-Anteil nachgewiesen. <br /> Für die Konstruktion des HbA1c-Immunosensors wurden Affinitätsmatrizen durch Modifizierung von Cellulose-Dialysemembranen mit glkPP bzw. Hp hergestellt. Grundlegend studiert wurde die Aktivierung der Cellulose-Membranen mit 1,1'-Carbonyldiimidazol (CDI) und 1-Cyano-4-dimethylaminopyridintetrafluoroborat (CDAP) als Aktivierungsagenzien. Eine gerichtete Immobilisierung der Liganden wurde realisiert, indem glkPP über dessen C-Terminus (einzige Carboxylatgruppe) und Hp über dessen periodat-oxidiertem Kohlenhydratrest an die amino- oder hydrazidfunktionalisierte Membranen kovalent gekoppelt wurden. <br /> Mit dem Einsatz der glkPP- und Hp-modifizierten Membranen in der elektrochemischen Messzelle war erstmalig der biosensorische Nachweis von HbA1c möglich. Als Transduktor diente eine Pt-Elektrode, an der das von der GOD generierte H2O2 umgesetzt und ein mit der HbA1c-Konzentration korrelierendes Stromsignal erzeugt wurde. Die Immunosensoren zeigten Ansprechzeiten von 3 s. Mit dem Immunosensor auf Basis des indirekt-kompetitiven Testprinzips wurde eine Kalibrationskurve für HbA1c im Bereich von 0,25-30 &#181;g/ml (3,9-465 nM, CV 3-9 %) mit Assayzeiten von 60 min und mit dem Immunosensor im Sandwich-Format eine Kalibrationskurve im Bereich von 0,5-5 &#181;g/ml (7,8-78 nM; 5-50 % HbA1c vom Gesamt-Hb, CV 6-10 %, 3 h) aufgenommen. / Hemoglobin-A1c (HbA1c) is a hemoglobin subtype formed by non-enzymatic reaction of glucose with the N-terminus of the beta-polypeptide chains. As it reflects the glycemic status of diabetics over the preceding 8-12 weeks, the determination of HbA1c has become an established procedure in the management of diabetes mellitus. It is measured as the percentage of total hemoglobin. Up to 5 % HbA1c are considered as normal whereas in diabetic subjects it could be elevated from 5-20 %. In addition to amperometric biosensors for glucose self monitoring which have been successfully applied in diabetes management, biosensors for HbA1c would be an useful supplement for a comprehensive diabetes control. <br /> <br /> Objective of this work was to develop and compare amperometric biosensors for determination of HbA1c based on enzymatic and immunochemical methods. <br /> <br /> For the enzyme based HbA1c assay a novel fructosamine oxidase (FAO) derived from marine yeast Pichia pastoris, strain N1-1 was utilized. It recognizes and oxidatively degrades fructosyl-valine (FV) which corresponds to the glycated N-terminus of the beta-chain of HbA1c and therefore is regarded as a model compound for HbA1c. Hydrogen peroxide which is liberated by the FAO during FV conversion was indicated optically in a horseradish peroxidase (POD) coupled reaction and electrochemically. For the biosensor the FAO was embedded in polyvinyl alcohol-stylbazole (PVA-SbQ) and fixed it in front of a Pt-electrode. So far, the measuring range of FV did not cover the clinically relevant range of HbA1c. Low specificity was assumed since enzyme activity also was obtained with glycated peptides, e.g. fructosyl-valine-glycine, not corresponding to the glycated N-terminus of the hemoglobin-beta-chain.<br /> <br /> For the immunosensor two immunoassays formats - heterogeneous sandwich and heterogeneous competitive - were tested. The assays were designed as follows: The competitive immunoassay was based on the immobilized synthetic glycated pentapeptide fructosyl-valine-histidine-leucine-threonine-proline (glkPP) utilized as HbA1c analogue. The peptide has an amino acid sequence corresponding to the N-terminus of the hemoglobin beta-chains and is capable for competition together with the HbA1c of the sample for the amount of a glucose oxidase (GOD)-labelled anti-HbA1c antibody. In the sandwich-type assay haptoglobin (Hp), a natural hemoglobin binding molecule with antibody characteristic properties, was used as bioreceptor for enrichment of total hemoglobin onto the surface. In a subsequent step the HbA1c fraction was quantified by a GOD-labelled HbA1c specific antibody. <br /> <br /> Cellulose dialysis membrane was used as the solid support for immobilization of Hp and glkPP near the sensor surface. For activation of the membrane two reagents, 1,1&prime;-carbonyldiimidazole (CDI) and 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP), were compared with respect to the degree of activation and coupling efficiency. Site-directed immobilization of Hp and glkPP was achieved by coupling Hp via its carbohydrate residue and glkPP via its C-terminus to the activated membrane using a bis-amine or bis-hydrazide spacer. <br /> <br /> The affinity membranes were placed in front of a modified Clark-type hydrogen peroxide electrode in an electrochemical measuring cell and HbA1c analysis was carried out within the stirred cell. Detection of the bound GOD-label was achieved by measurement of the electrocatalytic oxidation of hydrogen peroxide at +600 mV vs. Ag/AgCl. The indication was done in only 3 s. For the competitive principle a typical inhibition curve with a linear range between 0,25-30 &#181;g/ml (3,9-465 nM, CV 3-9 %, 60 min per sample) HbA1c was obtained. Due to the high functional stability of the peptide multiple regeneration of the affinity surface was possible without loss of binding capacity. With the sandwich assay configuration the clinically relevant range could easily be covered (calibration curve: 5-50 % HbA1c corresponding to 7,8-78 nM, CV 6-10 %, 3 h per sample).

>Magister Scientiae - MSc / Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin(hCG) is presented. This involves the modification of a gold surface with a thiolated β-cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.

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

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this work was realized characterization studies of a new material, poly (3- hidroxyphenylacetic acid) and its application for the construction of an amperometric immunosensor for detection of Acute Myocardial Infarction (AMI). Initially, it was carried out the electropolymerization of 3-hidroxyphenylacetic acid at three different pH's (0.0, 6.5 and 12.0) which could be assessed, electrochemically, the relationship between the behavior of the polymer and the reaction s pH. It was found that in the acid solutions (pH 0.0) the formation of electrochemically active material is more evident. Moreover, using electrochemical techniques, were carried out investigations on the structure of the polymeric material by using cationic and anionic probes. Monomer and polymer were characterized by infrared spectroscopy (FTIR), Ultra-Violet (UV/Vis.) and fluorescence; thermal analysis (DTA and TGA) and structural analysis (XDR). These studies were extremely important to highlight the main differences between the starting material (monomer) and electropolymerized material (polymer), and in particular assess the main characteristics of the polymer in order to enable its use as a platform to the proposed immunosensor. Studies of the immunosensor were conducted using two substances that acted as indicators of the reaction between the specific antibody for the AMI (anti-troponin T) and specific antigen for AMI (troponin T), hexaaminruthenium chloride and ferro/ferricyanide potassium. The results showed that the hexaaminruthenium chloride showed the best performance to indicate the formation of antibody-antigen complex wich occurs in the AMI. / Neste trabalho foram realizados estudos de caracterização de um novo material, o poli(ácido 3-hidroxifenilacético) e sua aplicação para construção de um imunossensor amperométrico para detecção do Infarto Agudo do Miocárdio (IAM). Inicialmente, foi realizada a eletropolimerização do ácido 3-hidroxifenilacético em três pH s diferentes (0,0; 6,5 e 12,0) onde foi possível avaliar, eletroquimicamente, a relação entre o comportamento do polímero formado e o pH do meio reacional. Foi possível constatar que em meio ácido (pH 0,0) a formação do material eletroquimicamente ativo é mais evidenciada. Além disso, com o auxílio de técnicas eletroquímicas, foram realizadas investigações sobre a estrutura do material polimérico formado, utilizando sondas catiônicas e aniônicas. Monômero e polímero foram caracterizados por técnicas espectroscópicas de Infravermelho (FTIR), Ultra-Violeta (UV/Vis.) e Fluorescência; análises térmicas (TGA e DTA) e análises estruturais (DRX). Estes estudos foram de extrema importância para destacar as principais diferenças entre o monômero e o material eletropolimerizado e, principalmente, avaliar as principais características do polímero, no sentido de viabilizar a sua utilização como plataforma do imunossensor proposto. Os estudos do imunossensor foram conduzidos utilizando-se duas substâncias que atuaram como indicadores da reação entre anticorpo específico para o IAM (antitroponina T) e antígeno específico para o IAM (troponina T), o cloreto de hexaaminrutênio II e o ferro/ferricianeto de potássio. Os resultados obtidos mostraram que o cloreto de hexaaminrutênio II teve melhor desempenho para indicar a formação do complexo anticorpo-antígeno, característico no evento do IAM. / Mestre em Química