Desenvolvimento de sensor biomimético para detecção de resíduos de pesticidas organofosforados e carbamatos em alimentos
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Data
2012-06-29
Autores
Santos, Glauco Pilon dos [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Enzimas têm sido empregadas na construção de dispositivos para quantificação de substratos por meio de reação catalítica. Tratando-se de moléculas de alto peso molecular e estrutura complexa, tais compostos biológicos podem apresentar instabilidade, além do alto custo de produção. Neste contexto, a utilização de um peptídeo artificial, proposto por estudos de modelagem molecular para mimetizar o sítio ativo da acetilcolinesterase (AChE), foi uma alternativa investigada para o desenvolvimento de metodologia analítica para detectar pesticidas organofosforados e carbamatos, que em sua maioria apresentam mutagenicidade, elevada toxicidade e são inibidores da AChE, essencial na transmissão de impulsos nervosos. O trabalho envolveu a síntese, purificação e caracterização de duas sequências peptídicas, SEQPEP1: NH3+-EHGGPS-COO- e SEQPEP2: NH3+-CEHGGPS-COO-, obtidas por meio de metodologia em fase sólida, que apresentaram índice de pureza acima de 95 %, possibilitando aplicação analítica. À sequência peptídica SEQPEP2, foi inserido o aminoácido cisteína com a finalidade de imobilizá-lo de forma ordenada na superfície de ouro. Os estudos espectrofotométricos demonstraram uma forte interação (K = 4,10 x 105 M-1) do peptídeo com o pesticida diclorvós e a formação de um complexo com absorção máxima em λ = 250 nm. Como suporte para a imobilização do peptídeo, foram utilizados eletrodos de ouro à base de discos compactos graváveis (CD-R) (Au-CDtrodos). As condições experimentais de imobilização do peptídeo foram otimizadas sendo os melhores resultados obtidos com uma concentração de peptídeo de 1 x 10-3 mol L-1 e um tempo de incubação de 1 h à 25 ºC. A estratégia de inserir cisteína para imobilizar o peptídeo indicou recobrimento de 68 % da superfície. Demonstrou-se pelos experimentos a viabilidade...
Enzymes have been used in the construction of devices to quantify substrate by catalytic reaction. The disadvantages of biological materials as instability and high production cost are well known. Therefore, the design and development of artificial oligopeptides as a mimic of the acethylcholinesterase (AChE) binding site, preserving the highly selective biological properties, was the approach used in this study on the development of an analytical methodology for the detection of organophosphate and carbamate pesticides, because of their mutagenicity, high toxicity and the inhibition of acetylcholinesterase enzyme, which is essential for the transmission of nerve impulses. This work have involved the synthesis, purification and characterization of two peptide sequences, SEQPEP1: NH3+-EHGGPS-COO- and SEQPEP2: NH3+-CEHGGPS-COO-, obtained by solid phase methodology, which have presented a purity degree above 95 %, enabling the analytical application. A cysteine amino acid was added to the peptide sequence SEQPEP2 in order to self assembly it on the gold electrode surface. The spectrophotometric studies have demonstrated a strong interaction (K = 4.10 x 105 M-1) between the peptide and pesticide dichlorvos, and the formation of a complex with absorption maximum at λ = 250 nm. As a support for the immobilization of peptide, were used gold electrodes based on recordable compact discs (CD-R) (Au-CDtrodes). The experimental conditions for the immobilization of the peptide sequence were optimized, being the best results achieved by using a peptide concentration of 1 x 10-3 mol L-1 and an incubation time of 1 h at 25 ºC. The strategy to add a cysteine amino acid for the immobilization of the peptide has indicated a surface recovery of 68 %. The experiments have shown the viability of monitoring the interaction... (Complete abstract click electronic access below)
Enzymes have been used in the construction of devices to quantify substrate by catalytic reaction. The disadvantages of biological materials as instability and high production cost are well known. Therefore, the design and development of artificial oligopeptides as a mimic of the acethylcholinesterase (AChE) binding site, preserving the highly selective biological properties, was the approach used in this study on the development of an analytical methodology for the detection of organophosphate and carbamate pesticides, because of their mutagenicity, high toxicity and the inhibition of acetylcholinesterase enzyme, which is essential for the transmission of nerve impulses. This work have involved the synthesis, purification and characterization of two peptide sequences, SEQPEP1: NH3+-EHGGPS-COO- and SEQPEP2: NH3+-CEHGGPS-COO-, obtained by solid phase methodology, which have presented a purity degree above 95 %, enabling the analytical application. A cysteine amino acid was added to the peptide sequence SEQPEP2 in order to self assembly it on the gold electrode surface. The spectrophotometric studies have demonstrated a strong interaction (K = 4.10 x 105 M-1) between the peptide and pesticide dichlorvos, and the formation of a complex with absorption maximum at λ = 250 nm. As a support for the immobilization of peptide, were used gold electrodes based on recordable compact discs (CD-R) (Au-CDtrodes). The experimental conditions for the immobilization of the peptide sequence were optimized, being the best results achieved by using a peptide concentration of 1 x 10-3 mol L-1 and an incubation time of 1 h at 25 ºC. The strategy to add a cysteine amino acid for the immobilization of the peptide has indicated a surface recovery of 68 %. The experiments have shown the viability of monitoring the interaction... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Quimica analitica, Pesticidas, Alimentos, Pesticides
Como citar
SANTOS, Glauco Pilon dos. Desenvolvimento de sensor biomimético para detecção de resíduos de pesticidas organofosforados e carbamatos em alimentos. 2012. 103 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Química, 2012.