Effects of glutaraldehyde cross-linking methods on enzyme immobilization in Ag nanoparticle/chitosan matrices

Abstract

Amperometric phenol biosensors were constructed by immobilization of Horseradish peroxidase (HRP, EC 1.11.1.7) in Ag-chitosan composite modified on glassy carbon electrodes. Five different methods of enzyme immobilization were investigated and compared: (A) HRP entrapment in Ag nanoparticle/chitosan composite; (B) HRP entrapment in glutaraldehyde solution/Ag nanoparticle/chitosan composite; (C) HRP entrapment in Ag nanoparticle/chitosan composite and then cross-linked with glutaraldehyde solution; (D) HRP entrapment in Ag nanoparticle/chitosan composite and then cross-linked with saturated glutaraldehyde vapor and (E) HRP adsorption in glutaraldehyde solution/Ag nanoparticle/chitosan composite. In this study, the experiments were divided into two parts. Firstly, the effects of Ag nanoparticle on electrochemical responses were studied. It was revealed that Ag nanoparticle enhanced the biosensor response and helped enzyme dispersion. However, too concentrated of Ag nanoparticles resulted in limited substrate or product mass transfer and led to lower responses. Secondly, effects of glutaraldehyde cross-linking methods on response currents and repeatabilities were determined and compared. It was found that the response currents of electrode A, B, C, D and E were 7.9x10-6, 0.66x10-6, 3.3x10-6, 1.2x10-6 and 4.3x10-6 A respectively. The response current was found to decrease when using glutaraldehyde sine glutaraldehyde could cause enzyme inactivation. But the repeatability was found to be improved by cross-linking with saturated glutaraldehyde vapor.