Avaliação do uso da hemoglobina extracelular de Amynthas gracilis (HbAg) na elaboração de detectores óticos de contaminação ambiental
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2023-07-07
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Universidade Estadual Paulista (Unesp)
Resumo
Diversos estudos com enzimas e até mesmo hemoproteínas tem mostrado um grande avanço na fabricação de biossensores para monitorar e quantificar moléculas em diferentes meios. Dentre as hemoproteínas, as hemoglobinas gigantes são uma classe que detém características muito interessantes para o uso neste tipo de ferramenta biotecnológica, como é o caso do biossensoriamento de metais pesados. No presente trabalho, foi verificada a interação da hemoglobina extracelular de Amynthas gracilis (HbAg) com os metais cobre, cádmio e zinco, visando um protótipo de biossensor de metais pesados. Os ensaios biofísicos em solução da HbAg frente aos metais cobre, cádmio e zinco foram monitorados através de diversas técnicas espectroscópicas. A imobilização da HbAg foi realizada através da técnica de Layerby- Layer (LBL) com o auxílio do polímero polietilenoimina (PEI) em quartzo para observar as mudanças espectrais no UV-Vis, e em eletrodo de carbono vítreo, para avaliação das alterações eletroquímicas da HbAg frente a adição dos metais. Além disso, a morfologia dos filmes também foi verificada com microscopia eletrônica de varredura e microbalança de cristal de quartzo. Verificou-se que a HbAg não se oxida na presença de cádmio e, frente ao zinco, a oxidação parcial é visualizada acima de 20 μmol L-1, além disso, nesta concentração, a hemoglobina sofre dissociação e agregação. A partir de 30 μmol L-1 de cobre em pH 7,0 a HbAg é oxidada, dissocia-se e perde parte de sua estrutura secundária. Em pH 5,0, sofre maior oxidação. Foi observado que o processo de imobilização não altera as propriedades óticas da hemoproteína e que, a partir da interação de 20 μmol L-1 do metal cobre sobre as bicamadas PEI/HbAg por 8 minutos, há um significativo deslocamento da banda de Soret, deslocando o máximo da banda de Soret para 409 nm na concentração de 70 μmol L-1. Ademais, a interação do eletrodo contendo PEI/HbAg com cobre, cádmio e zinco, monitorados por voltametria cíclica, mostraram curvas semelhantes entre si com saturação do eletrodo em torno de 10 μmol L-1 dos metais. Um estudo comparativo entre a HbAg e a hemoglobina humana (HHb) imobilizada no quartzo foi realizado com o intuito de verificar se a detecção com a HbAg seria melhor monitorada quando comparada com a HHb. Porém, não foram observadas alterações significativas entre os dois sistemas. Todavia, através dos estudos óticos foi possível propor que a HbAg é sensível ao cobre e zinco nas concentrações analisadas em solução e desta forma a potencialidade da HbAg como sensor ótico foi verificada. Não há seletividade da molécula quando utilizada em sensor eletroanalítico nas condições analisadas.
Several studies with enzymes and even hemoproteins have shown great advances in the manufacture of biosensors for monitoring and quantifying molecules in different media. Among the hemoproteins, giant hemoglobins are a class that possesses very interesting characteristics for use in this type of biotechnological tools, such as the biosensing of heavy metals. In this study, the interaction of HbAg with the metals copper, cadmium, and zinc was examined, with the aim of developing a prototype biosensor for heavy metals. The biophysical assays of HbAg in solution with copper, cadmium, and zinc metals were monitored using various spectroscopic techniques. The immobilization of HbAg was performed using the Layer-by-Layer (LBL) technique, with the assistance of the polyethyleneimine polymer (PEI), on quartz to observe UV- Vis spectral changes and on a glassy carbon electrode to evaluate the electrochemical changes of HbAg upon the addition of metals. Additionally, the morphology of the films was also examined using scanning electron microscopy and quartz crystal microbalance. It was found that HbAg does not oxidize in the presence of cadmium, while in the presence of zinc, partial oxidation is observed at concentrations above 20 μmol L-1; moreover, at this concentration, hemoglobin undergoes dissociation and aggregation. At 30 μmol L-1 of copper and pH 7.0, HbAg is oxidized, dissociates, and loses part of its secondary structure. At pH 5.0, it undergoes further oxidation. It was observed that the immobilization process does not alter the optical properties of the hemoprotein and that, starting from the interaction of 20 μmol L-1 of copper with the PEI/HbAg bilayers for 8 minutes, there is a significant shift of the Soret band towards 409 nm at a concentration of 70 μmol L-1. Furthermore, the interaction of the PEI/HbAg- containing electrode with copper, cadmium, and zinc, monitored by cyclic voltammetry, showed similar curves with electrode saturation occurring at around 10 μmol L-1 of the metals. A comparative study between HbAg and human hemoglobin (HHb) immobilized on quartz was performed to investigate if HbAg would provide better detection capabilities compared to HHb. However, no significant differences were observed between the two systems. Nevertheless, the optical studies suggest that HbAg is sensitive to copper and zinc at the analyzed concentrations in solution, thereby proving the potential of HbAg as an optical sensor. However, there is no selectivity of the molecule when used in an electroanalytical sensor under the analyzed conditions.
Several studies with enzymes and even hemoproteins have shown great advances in the manufacture of biosensors for monitoring and quantifying molecules in different media. Among the hemoproteins, giant hemoglobins are a class that possesses very interesting characteristics for use in this type of biotechnological tools, such as the biosensing of heavy metals. In this study, the interaction of HbAg with the metals copper, cadmium, and zinc was examined, with the aim of developing a prototype biosensor for heavy metals. The biophysical assays of HbAg in solution with copper, cadmium, and zinc metals were monitored using various spectroscopic techniques. The immobilization of HbAg was performed using the Layer-by-Layer (LBL) technique, with the assistance of the polyethyleneimine polymer (PEI), on quartz to observe UV- Vis spectral changes and on a glassy carbon electrode to evaluate the electrochemical changes of HbAg upon the addition of metals. Additionally, the morphology of the films was also examined using scanning electron microscopy and quartz crystal microbalance. It was found that HbAg does not oxidize in the presence of cadmium, while in the presence of zinc, partial oxidation is observed at concentrations above 20 μmol L-1; moreover, at this concentration, hemoglobin undergoes dissociation and aggregation. At 30 μmol L-1 of copper and pH 7.0, HbAg is oxidized, dissociates, and loses part of its secondary structure. At pH 5.0, it undergoes further oxidation. It was observed that the immobilization process does not alter the optical properties of the hemoprotein and that, starting from the interaction of 20 μmol L-1 of copper with the PEI/HbAg bilayers for 8 minutes, there is a significant shift of the Soret band towards 409 nm at a concentration of 70 μmol L-1. Furthermore, the interaction of the PEI/HbAg- containing electrode with copper, cadmium, and zinc, monitored by cyclic voltammetry, showed similar curves with electrode saturation occurring at around 10 μmol L-1 of the metals. A comparative study between HbAg and human hemoglobin (HHb) immobilized on quartz was performed to investigate if HbAg would provide better detection capabilities compared to HHb. However, no significant differences were observed between the two systems. Nevertheless, the optical studies suggest that HbAg is sensitive to copper and zinc at the analyzed concentrations in solution, thereby proving the potential of HbAg as an optical sensor. However, there is no selectivity of the molecule when used in an electroanalytical sensor under the analyzed conditions.
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Palavras-chave
Hemoglobina, Anelídeo, Biossensores, Metais pesados, Voltametria