A Biosensor Based on Immobilization of Horseradish Peroxidase in Chitosan Matrix Cross-linked with Glyoxal for Amperometric Determination of Hydrogen Peroxide

Abstract

An amperometric biosensor for hydrogen peroxide (H 2 O 2 ) was developed via an easy and effective enzyme immobilization method with the "sandwich" configuration: ferrocene-chitosan: HRP: chitosan-glyoxal using a glassy carbon electrode as the basic electrode. In order to prevent the loss of immobilized HRP under optimized conditions, the biosensor surface was cross-linked with glyoxal. Ferrocene was selected and immobilized on the glassy carbon electrode surface as a mediator. The fabrication procedure was systematically optimized to improve the biosensor performance. The biosensor had a fast response of less than 10 s to H 2 O 2 , with a linear range of 3.5×10 -5 to 1.1×10 -3 M, and a detection limit of 8.0×10 -6 M based on S/N = 3.

Imobilização da lacase em micropartículas de quitosana obtidas por spray drying e usadas na construção de biossensores

“…[10][11][12][13][14][42][43][44] A Figura 3 mostra uma proposta da imobilização da lacase em quitosana (a) reticulada com TPP (b) baseada na literatura. [10][11][12][13][14][42][43][44] O TPP é um íon pentavalente e reage com a quitosana formando ligações iônicas entre as cargas negativas dos oxigênios do TPP (P 3 O 10 5-) com os grupos amino protonados da quitosana (NH 3 + ) em valores de pH menores que 6,5, obtendo-se a estrutura representada em (c). Sugere-se que neste processo a lacase (d) esteja entrapeada entre as ligações iônicas da quitosana e do TPP, resultando na estrutura apresentada em (e).…”

“…A maior sensibilidade observada para este biossensor pode ser em decorrência da eficiência na formação da rede polimérica altamente organizada entre a quitosana e o glioxal, onde a enzima pode estar confinada e/ou imobilizada por ligações covalentes. 7,8,42 Além disso, as micropartículas obtidas no processo de reticulação usando o glioxal apresentaram uma morfologia esférica e, possivelmente, uma maior área superficial quando comparada com as partículas obtidas pelo processo de reticulação da quitosana com TPP. Porém, como a região linear das duas curvas analíticas foi praticamente a mesma, possivelmente foi imobilizada a mesma concentração da enzima lacase nos dois suportes de quitosana.…”

Effects of Temperature and pH on Immobilized Laccase Activity in Conjugated Methacrylate-Acrylate Microspheres

“…On the other hand, the optimum pH for immobilized laccase was shifted to pH 5.0. The microenvironment of the immobilized enzyme and bulk solution usually has unequal partitioning of H + and OH − concentrations due to electrostatic interactions with the matrix, which often leads to the displacements in the pH activity profile [25,27]. Furthermore, pH profiles of the immobilized laccase are broader than free enzyme, which means that the immobilization method preserved enzyme activity in a wider pH range [31,32].…”

“…When glutaric dialdehyde concentration was increased, the amount of free aldehyde groups on the microspheres' surface will be increased thus enhancing the laccase loading capacity. However as the concentration of glutaric dialdehyde was International Journal of Polymer Science more than 10%, the excessive cross-linking within the immobilization matrix may be blocked and change the enzyme conformation, hence resulting in the declination in the activity of the immobilized enzyme [27]. in Figure 7.…”

“…Long chain alkyl sulphate surfactant, which is amphiphilic, was used to stabilize the emulsion system and to prevent monomers from forming larger droplets by allowing small droplets to remain stable during emulsion phase [27,28]. Photopolymerization caused droplets that contain GMA and nBA monomers in the presence of photoinitiator to be converted into poly(GMAco-nBA) microspheres at room temperature.…”