Utilização da montmorilonita na construção de sensores eletroquímicos baseados em acetilcolinesterase para detecção de clorpirifós
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Data
2019-01-29
Autores
Lupi, Caroline Polini [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A produção agrícola tem recebido inquestionáveis benefícios devido ao uso de pesticidas, entretanto, essas substâncias podem causar sérios danos aos organismos vivos, devido sua toxicidade, baixa biodegrabilidade e alto potencial carcinogênico. Dessa forma, a determinação de pesticidas, em nível de traços, se faz de extrema importância, sendo necessário encontrar abordagens analíticas simples e efetivas para seu monitoramento. Nesse sentido, diversos tipos de biossensores enzimáticos, têm sido investigados e fundamentados na inibição de acetilcolinesterase, enzima envolvida na transmissão dos impulsos nervosos. Os pesticidas das classes dos organofosforados interagem com essa classe de enzimas, inativando-as e provocando intoxicações agudas, podendo levar, como consequência, à morte do indivíduo. A montmorilonita é um argilomineral do grupo das esmectitas, que possui excelentes propriedades de intercalação e inchamento, alto potencial de adsorção e elevada afinidade com diversas substâncias orgânicas e inorgânicas. Essas características as constituem como um adequado meio de imobilização de moléculas orgânicas/inorgânicas e biológicas, tais como enzimas e nanopartículas metálicas. Recentemente, eletrodos modificados com montmorilonita têm atraído considerável atenção na área de eletroanalítica/eletroquímica, revelando como propriedades: alta sensibilidade, boa seletividade, custo relativamente baixo, resposta rápida e possibilidade de miniaturização, dentre outras vantagens. Nesse sentido, este trabalho buscou evidenciar a possibilidade de modificação da montmorilonita com três sequências peptídicas sintéticas, propostas por modelagem molecular, que mimetizam o sítio ativo da acetilcolinesterase, e a aplicação dessa argila modificada na construção de sensores, utilizados na detecção eletroquímica, por espectroscopia de impedância eletroquímica, de clorpirifós, pesticida da classe dos organofosforados. As sequências peptídicas utilizadas foram (NH3+ – Glu – His – Gly – Gly – Pro – Ser – COO-); (NH3+ – His – Glu – Trp – Arg – Pro – Ser – COO-); (NH3+ – His – Glu – Tyr – Lys – Pro – Ser – COO-). Essas sequências biomiméticas, que já haviam sido propostas e estudadas anteriormente, apresentaram uma alta constante de afinidade pelos pesticidas da classe dos organofosforados. A modificação da montmorilonita foi demonstrada pelas técnicas de caracterização análise térmica, difração de raios X, infravermelho e microscopia eletrônica de varredura. A interação das argilas modificadas com o clorpirifós foi demonstrada por DSC, e as três sequências peptídicas apresentaram comportamentos diferentes frente ao pesticida, podendo, duas delas, serem utilizadas satisfatoriamente em substituição à enzima original na construção de sensores impidimétricos. Os sensores construídos apresentaram boa linearidade na faixa de concentração de 1 x 10-8 a 1 x 10-7 mol L-1 (sensor 1: R = 0,9997; sensor 3: R = 0,9999) e também de 1 x 10-7 a 1 x 10-6 mol L-1 (sensor 3: R = 0,9950), limites de detecção entre 1,41 x 10-10 e 2,40 x 10-10 mol L-1, e foram satisfatoriamente aplicados na detecção da clorpirifós em amostra de tomate.
Agricultural production has received unquestionable benefits due to the use of pesticides; however, these substances can cause serious damage to living organisms due to their toxicity, low biodegradability and high carcinogenic potential. Thus, the determination of pesticides at trace level is extremely important, and it is necessary to find simple and effective analytical approaches for its monitoring. In this sense, several types of enzymatic biosensors have been investigated and based on the inhibition of acetylcholinesterase, an enzyme involved in the transmission of nerve impulses. The pesticides of the classes of organophosphates interact with this class of enzymes, inactivating them and provoking acute intoxications, which can lead, as a consequence, to the death of the individual. The montmorillonite is a clay of the smectite group, which has excellent intercalation and swelling properties, high adsorption potential and high affinity with various organic and inorganic substances. These characteristics constitute them as an adequate means of immobilizing organic/inorganic and biological molecules, such as enzymes and metal nanoparticles. Recently, montmorillonite modified electrodes have attracted considerable attention in the field of electroanalytical/electrochemistry, revealing as properties: high sensitivity, good selectivity, low cost, rapid response and possibility of miniaturization, among other advantages. In this sense, this work aimed to show the possibility of modifying the montmorillonite with three synthetic peptide sequences, proposed by molecular modeling, that mimic the active site of acetylcholinesterase, and the application of this modified clay in the construction of sensors, used in electrochemical detection, by spectroscopy electrochemical impedance, chlorpyrifos, pesticide of the organophosphorus class. The peptide sequences used were (NH3+ – Glu – His – Gly – Gly – Pro – Ser – COO-); (NH3+ – His – Glu – Trp – Arg – Pro – Ser – COO-); (NH3+ – His – Glu – Tyr – Lys – Pro – Ser – COO-). These biomimetic sequences, which had already been proposed and studied previously, had a high affinity constant for organophosphorus class pesticides. The modification of montmorillonite was demonstrated by the techniques of thermal analysis, X-ray diffraction, infrared and scanning electron microscopy. The interaction of the modified clays with the chlorpyrifos was demonstrated by DSC, and the three peptide sequences presented different behaviors against the pesticide, and two of them could be used satisfactorily in substitution of the original enzyme in the construction of imidimetric sensors. The constructed sensors presented good linearity in the concentration range of 1 x 10-8 to 1 x 10-7 mol L-1 (sensor 1: R = 0,9997; sensor 3: R = 0,9999) and also from 1 x 10-7 to 1 x 10-6 mol L-1 (sensor 3: R = 0,9950), detection limits between 1,41 x 10-10 and 2,40 x 10-10 mol L-1, and were satisfactorily applied in the detection of chlorpyrifos in a tomato sample.
Agricultural production has received unquestionable benefits due to the use of pesticides; however, these substances can cause serious damage to living organisms due to their toxicity, low biodegradability and high carcinogenic potential. Thus, the determination of pesticides at trace level is extremely important, and it is necessary to find simple and effective analytical approaches for its monitoring. In this sense, several types of enzymatic biosensors have been investigated and based on the inhibition of acetylcholinesterase, an enzyme involved in the transmission of nerve impulses. The pesticides of the classes of organophosphates interact with this class of enzymes, inactivating them and provoking acute intoxications, which can lead, as a consequence, to the death of the individual. The montmorillonite is a clay of the smectite group, which has excellent intercalation and swelling properties, high adsorption potential and high affinity with various organic and inorganic substances. These characteristics constitute them as an adequate means of immobilizing organic/inorganic and biological molecules, such as enzymes and metal nanoparticles. Recently, montmorillonite modified electrodes have attracted considerable attention in the field of electroanalytical/electrochemistry, revealing as properties: high sensitivity, good selectivity, low cost, rapid response and possibility of miniaturization, among other advantages. In this sense, this work aimed to show the possibility of modifying the montmorillonite with three synthetic peptide sequences, proposed by molecular modeling, that mimic the active site of acetylcholinesterase, and the application of this modified clay in the construction of sensors, used in electrochemical detection, by spectroscopy electrochemical impedance, chlorpyrifos, pesticide of the organophosphorus class. The peptide sequences used were (NH3+ – Glu – His – Gly – Gly – Pro – Ser – COO-); (NH3+ – His – Glu – Trp – Arg – Pro – Ser – COO-); (NH3+ – His – Glu – Tyr – Lys – Pro – Ser – COO-). These biomimetic sequences, which had already been proposed and studied previously, had a high affinity constant for organophosphorus class pesticides. The modification of montmorillonite was demonstrated by the techniques of thermal analysis, X-ray diffraction, infrared and scanning electron microscopy. The interaction of the modified clays with the chlorpyrifos was demonstrated by DSC, and the three peptide sequences presented different behaviors against the pesticide, and two of them could be used satisfactorily in substitution of the original enzyme in the construction of imidimetric sensors. The constructed sensors presented good linearity in the concentration range of 1 x 10-8 to 1 x 10-7 mol L-1 (sensor 1: R = 0,9997; sensor 3: R = 0,9999) and also from 1 x 10-7 to 1 x 10-6 mol L-1 (sensor 3: R = 0,9950), detection limits between 1,41 x 10-10 and 2,40 x 10-10 mol L-1, and were satisfactorily applied in the detection of chlorpyrifos in a tomato sample.
Descrição
Palavras-chave
Acetilcolinesterase, Clorpirifós, Montmorilonita, Espectroscopia de impedância eletroquímica, Análise térmica