Avaliação da imobilização de anticorpos anti-enterotoxina estafilocócica A em óxido de grafeno reduzido
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Data
2023-03-29
Autores
Rocha, Giovanna da Silva
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Universidade Estadual Paulista (Unesp)
Resumo
O óxido de grafeno reduzido (rGO) é um material alótropo de carbono que apresenta características estruturais que o torna uma alternativa relevante como suporte de imobilização de anticorpos para a construção de biossensores. A estratégia de construção de imunossensores deve levar em consideração as características da superfície do transdutor (rGO), assim como a do componente biológico (antígeno/anticorpo) imobilizado no material de suporte, principalmente para a manutenção da atividade imunológica dos mesmos juntamente a sua especificidade. Neste trabalho, foram avaliados diferentes métodos de imobilização do anticorpo anti-Enterotoxina Estafilocócica A (anti-SEA), com o intuito de construir um imunossensor para detecção da SEA em amostras de leite. A SEA é uma toxina secretada por Staphylococcus aureus que está associada a intoxicações alimentares decorrentes da ingestão de alimentos contaminados por essa bactéria. Desta forma, utilizando eletrodos de carbono vítreo modificados com rGO e com os agentes de bioconjugação pireno-NHS e EDC-NHS avaliou-se a capacidade de imobilização do anticorpo anti-SEA na superfície do transdutor. A caracterização eletroquímica dos biossensores foi realizada utilizando as técnicas de Voltametria Cíclica (CV), no intervalo de potencial de - 0,3 V a + 0,9 V vs. Ag/AgCl com velocidade de varredura de 0,1 V s-1 e por Espectroscopia de Impedância Eletroquímica (EIS), com frequência variante do espectro de 105 a 101 Hz e 5 mV de amplitude. Ambas as técnicas foram realizadas em uma solução 0,1 PBS (pH 7,0) contendo 0,1 mol L-1 KCl e 5,0 mmol L-1 da sonda redox [Fe(CN)6] 3-/4-. Assim, comparou-se três métodos de imobilização: 1) Adsorção física; 2) Bioconjugação com pireno-NHS e 3) Bioconjugação com EDC-NHS. Os resultados evidenciaram que, apesar dos agentes de bioconjugação serem vantajosos para auxiliar na imobilização orientada, para o anticorpo estudado o método adsortivo teve um melhor desempenho. Dessa forma, a utilização do rGO se apresenta como uma alternativa vantajosa ao processo de construção de biossensores eletroquímicos, evidenciando o potencial de aplicação em escala industrial em imunossensores, visto que a manutenção da estabilidade e atividade dos anticorpos imobilizados na superfície do rGO sem a utilização de agentes de imobilização implicam na redução de etapas e de custo na fabricação do imunossensor.
Reduced graphene oxide (rGO) is an allotrope carbon derived material that presents structural characteristics that make it a relevant alternative as an antibody immobilization support for the construction of biosensors. The construction strategy of immunosensors must consider the characteristics of the transducer surface (rGO), as well as the biological component (antigen/antibody) immobilized on the support material, mainly for the maintenance of their immunological activity together with their specificity. In this work, different methods of immobilization of the anti- Staphylococcal Enterotoxin A (anti-SEA) antibody were evaluated, with the aim of building an immunosensor to detect SEA in milk samples. SEA is a toxin secreted by Staphylococcus aureus that is associated with food intoxication. Thus, using glassy vitreous carbon (GC) electrodes modified with rGO and with the bioconjugation agents pyrene- NHS and EDC-NHS, the immobilization capacity of the anti-SEA antibody on the surface of the transducer was evaluated. The electrochemical characterization of the biosensors was performed using Cyclic Voltammetry (CV) techniques, in the potential range from -0.3 V to +0.9 V vs. Ag/AgCl with a sweep speed of 0.1 V s-1 and by Electrochemical Impedance Spectroscopy (EIS), with a frequency varying from 105 to 101 Hz and 5 mV of amplitude. Both techniques were performed in a 0.1 PBS solution (pH 7.0) containing 0.1 mol L-1 KCl and 5.0 mmol L-1 of the redox probe [Fe(CN)6] 3-/4-. Thus, three methods of immobilization were compared: 1) Physical adsorption; 2) Bioconjugation with Pyrene-NHS and 3) Bioconjugation with EDC-NHS. The results showed that the adsorptive method was more advantageous for the immobilization of anti-SEA on rGO. Thus, the use of rGO presents itself as an advantageous alternative to the construction process of electrochemical biosensors, showing the potential for application on an industrial scale in immunosensors, since the maintenance of the stability and activity of antibodies immobilized on the surface of rGO without the use of immobilization agents imply in the reduction of steps and cost in the manufacture of the immunosensor.
Reduced graphene oxide (rGO) is an allotrope carbon derived material that presents structural characteristics that make it a relevant alternative as an antibody immobilization support for the construction of biosensors. The construction strategy of immunosensors must consider the characteristics of the transducer surface (rGO), as well as the biological component (antigen/antibody) immobilized on the support material, mainly for the maintenance of their immunological activity together with their specificity. In this work, different methods of immobilization of the anti- Staphylococcal Enterotoxin A (anti-SEA) antibody were evaluated, with the aim of building an immunosensor to detect SEA in milk samples. SEA is a toxin secreted by Staphylococcus aureus that is associated with food intoxication. Thus, using glassy vitreous carbon (GC) electrodes modified with rGO and with the bioconjugation agents pyrene- NHS and EDC-NHS, the immobilization capacity of the anti-SEA antibody on the surface of the transducer was evaluated. The electrochemical characterization of the biosensors was performed using Cyclic Voltammetry (CV) techniques, in the potential range from -0.3 V to +0.9 V vs. Ag/AgCl with a sweep speed of 0.1 V s-1 and by Electrochemical Impedance Spectroscopy (EIS), with a frequency varying from 105 to 101 Hz and 5 mV of amplitude. Both techniques were performed in a 0.1 PBS solution (pH 7.0) containing 0.1 mol L-1 KCl and 5.0 mmol L-1 of the redox probe [Fe(CN)6] 3-/4-. Thus, three methods of immobilization were compared: 1) Physical adsorption; 2) Bioconjugation with Pyrene-NHS and 3) Bioconjugation with EDC-NHS. The results showed that the adsorptive method was more advantageous for the immobilization of anti-SEA on rGO. Thus, the use of rGO presents itself as an advantageous alternative to the construction process of electrochemical biosensors, showing the potential for application on an industrial scale in immunosensors, since the maintenance of the stability and activity of antibodies immobilized on the surface of rGO without the use of immobilization agents imply in the reduction of steps and cost in the manufacture of the immunosensor.
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Palavras-chave
Óxido de grafeno, Enterotoxinas, Imunossensor, Nanotecnologia, Staphylococcus aureus, Anticorpos