Desenvolvimento de sensor bioinspirado em hexapeptídeo de enzima acetilcolinesterase para detecção de pesticidas.
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Data
2018-03-09
Autores
Rodrigues, Núbia Fernanda Marinho
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Universidade Estadual Paulista (Unesp)
Resumo
Os pesticidas estão entre os poluentes mais preocupantes, devido à toxicidade e presença significativa no ambiente. A sua toxicidade é baseada na capacidade de inibir irreversivelmente a enzima acetilcolinesterase (AChE) que é chave na transmissão de impulsos nervosos. Este trabalho descreve o desenvolvimento de sensor contendo hexapeptídeo, bioinspirado em enzima acetilcolinesterase, para detecção de pesticidas organofosforados e carbamatos. A sequência peptídica (NH3+ - His – Glu – Trp – Arg – Pro – Ser – COO-) foi imobilizada sobre nanopartículas magnéticas (Fe3O4) previamente sintetizadas, modificadas com quitosana e posteriormente funcionalizadas com 1,12-diaminododecano. As condições experimentais de imobilização do peptídeo foram otimizadas, sendo estas: concentração 5,0 x 10-5 mol L-1 e tempo de incubação de 30 minutos a 25 ºC. O grupo carboxílico presente na sequência peptídica foi ativado com o uso de agentes de acoplamento 1-etil-3-(3-dimetilaminopropil) carbodiimida (EDC) e N-hidróxisuccinimida (NHS). A razão de concentração otimizada de EDC/NHS foi de 18,6/12,5 mmol L-1, respectivamente, e tempo de ativação de 60 minutos. O sinal eletroquímico do peptídeo foi monitorado pelo pico de oxidação da histidina, cujo valor é diminuído ao interagir com o pesticida. O perclorato de sódio (NaClO4) 0,1 mol L-1 pH 7,5 foi selecionado como eletrólito suporte. Os parâmetros da voltametria de onda quadrada foram otimizados (frequência de 100 Hz, amplitude de 90 mV e incremento de varredura de 6 mV) utilizando uma matriz de planejamento fatorial. O tempo de pré-concentração do peptídeo com o pesticida foi fixado em 5 minutos. O sensor apresentou resposta linear nas faixas de concentração estudadas, com limites de detecção de 6,0 x 10-11 mol L-1 e 4,0 x 10-10 mol L-1 para carbofurano e clorpirifós, respectivamente. O armazenamento na geladeira a ±4 °C possibilitou 85% de estabilidade do peptídeo imobilizado após um período de 60 dias. O método desenvolvido foi aplicado em amostras reais de batata e tomate, sendo obtido um percentual de recuperação no intervalo de 95 a 100%. O método cromatográfico de análise (HPLC/UV) apresentou intervalo linear de 1,0 x 10-6 a 5,0 x 10-5 mol L-1 para carbofurano e de 5,0 x 10-6 a 5,0 x 10-5 mol L-1 para clorpirifós, com recuperação de 95 a 105%. O sensor bioinspirado, permitiu o desenvolvimento de metodologia analítica alternativa para detecção de pesticida, mostrando-se promissor e com viabilidade de substituição de métodos cromatográficos utilizados para determinação de pesticidas em amostras de alimentos.
Pesticides are among the most worrying pollutants due to toxicity and significant presence in the environment. Its toxicity is based on the ability to irreversibly inhibit the enzyme acetylcholinesterase (AChE) which is key in the transmission of nerve impulses. This work describes the development of a sensor containing hexapeptide, bioinspiring enzyme acetylcholinesterase, for the detection of organophosphorus pesticides and carbamates. The peptide sequence (NH3+ - His - Glu - Trp - Arg - Pro - Ser - COO-) was immobilized on previously synthesized magnetic nanoparticles (Fe3O4), modified with chitosan and subsequently functionalized with 1,12 - diaminododecane. The experimental conditions of immobilization of the peptide were optimized, being: 5,0 x 10-5 mol L-1 concentration and incubation time of 30 minutes at 25 ºC. The carboxyl group present in the peptide sequence was activated with the use of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) coupling agents. The optimum concentration ratio of EDC / NHS was 18.6 / 12.5 mmol L-1, respectively, and activation time of 60 minutes. The electrochemical signal of the peptide was monitored by the histidine oxidation peak, whose value is decreased when interacting with the pesticide. Sodium perchlorate (NaClO4) 0.1 mol L-1 pH 7.5 was selected as supporting electrolyte. The parameters of the square wave voltammetry were optimized (frequency of 100 Hz, amplitude of 90 mV and increment of sweep of 6 mV) using a matrix of factorial planning. The preconcentration time of the peptide with the pesticide was fixed in 5 minutes. The sensor presented linear response in the studied concentration ranges, with detection limits of 6.0 x 10-11 mol L-1 and 4.0 x 10- 10 mol L- 1 for carbofuran and chlorpyrifos, respectively. The storage in the refrigerator at ± 4 °C allowed 85% stability of the immobilized peptide after a period of 60 days. The developed method was applied in real samples of potato and tomato, obtaining a percentage of recovery in the range of 95 to 100%. The chromatographic (HPLC/UV) analysis showed a linear range of 1.0 x 10-6 to 5.0 x 10-5 mol L-1 for carbofuran and 5.0 x 10-6 to 5.0 x 10-5 mol L-1 for chlorpyrifos, with recovery of 95 to 105%. The bio-inspired sensor allowed the development of an alternative analytical methodology for pesticide detection, showing promise and feasibility of replacement of chromatographic methods used to determine pesticides in food samples.
Pesticides are among the most worrying pollutants due to toxicity and significant presence in the environment. Its toxicity is based on the ability to irreversibly inhibit the enzyme acetylcholinesterase (AChE) which is key in the transmission of nerve impulses. This work describes the development of a sensor containing hexapeptide, bioinspiring enzyme acetylcholinesterase, for the detection of organophosphorus pesticides and carbamates. The peptide sequence (NH3+ - His - Glu - Trp - Arg - Pro - Ser - COO-) was immobilized on previously synthesized magnetic nanoparticles (Fe3O4), modified with chitosan and subsequently functionalized with 1,12 - diaminododecane. The experimental conditions of immobilization of the peptide were optimized, being: 5,0 x 10-5 mol L-1 concentration and incubation time of 30 minutes at 25 ºC. The carboxyl group present in the peptide sequence was activated with the use of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) coupling agents. The optimum concentration ratio of EDC / NHS was 18.6 / 12.5 mmol L-1, respectively, and activation time of 60 minutes. The electrochemical signal of the peptide was monitored by the histidine oxidation peak, whose value is decreased when interacting with the pesticide. Sodium perchlorate (NaClO4) 0.1 mol L-1 pH 7.5 was selected as supporting electrolyte. The parameters of the square wave voltammetry were optimized (frequency of 100 Hz, amplitude of 90 mV and increment of sweep of 6 mV) using a matrix of factorial planning. The preconcentration time of the peptide with the pesticide was fixed in 5 minutes. The sensor presented linear response in the studied concentration ranges, with detection limits of 6.0 x 10-11 mol L-1 and 4.0 x 10- 10 mol L- 1 for carbofuran and chlorpyrifos, respectively. The storage in the refrigerator at ± 4 °C allowed 85% stability of the immobilized peptide after a period of 60 days. The developed method was applied in real samples of potato and tomato, obtaining a percentage of recovery in the range of 95 to 100%. The chromatographic (HPLC/UV) analysis showed a linear range of 1.0 x 10-6 to 5.0 x 10-5 mol L-1 for carbofuran and 5.0 x 10-6 to 5.0 x 10-5 mol L-1 for chlorpyrifos, with recovery of 95 to 105%. The bio-inspired sensor allowed the development of an alternative analytical methodology for pesticide detection, showing promise and feasibility of replacement of chromatographic methods used to determine pesticides in food samples.
Descrição
Palavras-chave
Pesticidas, Eletroquímica, Nanopartículas, Materiais bioinpirados, Inibidores de colinesterase, Pesticides, Electrochemistry, Nanoparticles, Bioinspired materials, Cholinesterase Inhibitors