Construção de genossensor amperométrico para diagnóstico da hepatite C baseado em monocamadas auto montadas e detecção não marcada
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Data
2009-04-27
Autores
Marques, Paulo Roberto Brasil de Oliveira [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O presente trabalho descreve estudos de construção de biossensores modificados com DNA para detecção de vírus da hepatite C. Utilizaram-se como superfície eletródica substratos de ouro oriundos de CDs graváveis, já descritos na literatura. O procedimento de construção deste tipo específico de eletrodo foi modificado e esta modificação foi avaliada por meio de técnicas eletroquímicas, de microscopias de varredura eletrônica de fluorescência. Duas propostas de imobilização foram avaliadas, sendo: a construção de monocamadas auto organizadas com moléculas de ácido mercaptopropiônico, com posterior ligação de material biológico via metodologia biotina estreptavidina e a construção de monocamadas a partir de moléculas de DNA modificadas com grupamentos tióis. As metodologias foram comparadas e a que fez uso do DNA tiolado foi escolhida para efetuar os procedimentos de imobilização, que foram então otimizados. Para monitorar o sinal de hibridização do genossensor, dois métodos foram estudados, sendo: detecção via oxidação da base nitrogenada guanina e detecção via interação do material genético com indicadores eletroanalíticos, sendo este último o que apresentou melhores resultados sendo selecionado. Como indicadores foram avaliados os corantes azul de metileno e azul de meldola e os compostos de cobre Cu(NO3)2(en)2 e Cu(NO3)2(NN)2, que foram caracterizados e avaliados por técnicas eletroanalíticas e espectrofotométricas. Estes foram estudados frente aos processos de interação com o material genético nativo e degradado, por meio do método da razão molar. O azul de metileno e o Cu(NO3)2(en)2 apresentaram forte interação com o DNA de Calf Thymus, com constantes de interação de 1,5 x 10 4 e 1,0 x 10 5 L mol -1. Estes foram utilizados como indicadores eletroanalíticos na etapa de hibridização, sendo eficientes para detecção de genótipos da hepatite C em amostras amplificadas de soro sanguineo.
This paper describes a studies of biosensors modified DNA construction for hepatitis C virus detection. It was used gold from recordable CDs as a electrodic substrates just described in the literature. The procedure of construction of this particular electrode was modified and this modification was evaluated using electrochemical, scanning electron microscopy and fluorescence techniques. Two detention proposals were evaluated: first, the construction of self assembly monolayers with mercaptopropionic acid and subsequent binding of biological material by streptavidin biotin method and second, the construction of monolayers from molecules of DNA modified with thiol groups. The methods were compared and the DNA thiol was chosen to perform the detention procedures, which were then optimized. To monitoring the hybridization signal genosensor, two methods were studied,: guanine oxidation detection and interaction of genetic material with electroanalytical indicators, the latter being the one with better results, and selected. As indicators were evaluated the methylene blue and meldola blue dye, and copper compounds Cu(NO3)2(en)2 and Cu(NO3)2(NN)2, which were characterized and evaluated by spectrophotometric and electroanalytical techniques. These coumpounds were studied before the interaction process with the native and degraded material by the molar ratio method. The methylene blue and Cu(NO3)2(en)2 showed strong interaction with the Calf Thymus DNA with constant of 1.5 x 10 4 and 1.0 x 10 5 L mol -1. These were used as indicators of hybridization in electroanalytical step and they were efficient for detection of hepatitis C genotypes in amplified blood serum samples.
This paper describes a studies of biosensors modified DNA construction for hepatitis C virus detection. It was used gold from recordable CDs as a electrodic substrates just described in the literature. The procedure of construction of this particular electrode was modified and this modification was evaluated using electrochemical, scanning electron microscopy and fluorescence techniques. Two detention proposals were evaluated: first, the construction of self assembly monolayers with mercaptopropionic acid and subsequent binding of biological material by streptavidin biotin method and second, the construction of monolayers from molecules of DNA modified with thiol groups. The methods were compared and the DNA thiol was chosen to perform the detention procedures, which were then optimized. To monitoring the hybridization signal genosensor, two methods were studied,: guanine oxidation detection and interaction of genetic material with electroanalytical indicators, the latter being the one with better results, and selected. As indicators were evaluated the methylene blue and meldola blue dye, and copper compounds Cu(NO3)2(en)2 and Cu(NO3)2(NN)2, which were characterized and evaluated by spectrophotometric and electroanalytical techniques. These coumpounds were studied before the interaction process with the native and degraded material by the molar ratio method. The methylene blue and Cu(NO3)2(en)2 showed strong interaction with the Calf Thymus DNA with constant of 1.5 x 10 4 and 1.0 x 10 5 L mol -1. These were used as indicators of hybridization in electroanalytical step and they were efficient for detection of hepatitis C genotypes in amplified blood serum samples.
Descrição
Palavras-chave
Biotecnologia, Hepatite C, Genossensores, Biotechnology
Como citar
MARQUES, Paulo Roberto Brasil de Oliveira. Construção de genossensor amperométrico para diagnóstico da hepatite C baseado em monocamadas auto montadas e detecção não marcada. 2009. 171 f. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Química, 2009.