Low-Denaturazing Glucose Oxidase Immobilization onto Graphite Electrodes by Incubation in Chitosan Solutions

Abstract

In this work, glucose oxidase (GOx) has been immobilized onto graphite rod electrodes through an assisted-chitosan adsorption reaching an enzyme coverage of 4 nmol/cm2. The direct and irreversible single adsorption of the Flavine Adenine Dinucleotide (FAD) cofactor has been minimized by electrode incubation in a chitosan (CH) solution containing the enzyme GOx. Chitosan keeps the enzyme structure and conformation due to electrostatic interactions preventing FAD dissociation from the protein envelope. Using chitosan, both the redox cofactor FAD and the protein envelope remain in the active form as demonstrated by the electrochemistry studies and the enzymatic activity in the electrochemical oxidation of glucose up to a concentration of 20 mM. The application of the modified electrodes for energy harvesting delivered a power density of 119 µW/cm2 with a cell voltage of 0.3 V. Thus, chitosan presents a stabilizing effect for the enzyme conformation promoted by the confinement effect in the chitosan solution by electrostatic interactions. Additionally, it facilitated the electron transfer from the enzyme to the electrode due to the presence of embedded chitosan in the enzyme structure acting as an electrical wiring between the electrode and the enzyme (electron transfer rate constant 2.2 s−1). This method involves advantages compared with previously reported chitosan immobilization methods, not only due to good stability of the enzyme, but also to the simplicity of the procedure that can be carried out even for not qualified technicians which enable their easy implementation in industry.