Reagentless biosensor for hydrogen peroxide based on self-assembled films of horseradish peroxidase/laponite/chitosan and the primary investigation on the inhibitory effect by sulfide.

Horseradish peroxidase (HRP) was successfully incorporated into the laponite/chitosan (Chit)-modified glassy carbon electrode (GCE) using step-by-step self-assembly. The self-assembly processes were monitored by X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectra (EIS). Direct electrochemistry and electrocatalysis of the self-assembled HRP were investigated. Cyclic voltammetry of HRP/laponite/Chit/GCE, in anaerobic phosphate buffer solution (0.025 M PBS, pH 7.0), displayed a pair of stable and quasi-reversible peaks at potentials Epa=0.024 V and Epc=-0.132 V vs. SCE, attributed to the HRP-Fe(III)/HRP-Fe(II) redox couple. The electrochemical reaction of the HRP/laponite/Chit/GCE exhibited a surface-controlled electrode process. The electron transfer rate constant was estimated to be 1.82 s(-1). The self-assembled HRP maintained its biological activity and exhibited an excellent electrocatalytic performance for the reduction of H2O2 with fast amperometric response (10 s), broad linear range (2.9×10(-5) to 1.4×10(-3) M), good sensitivity (19.7±0.5 mA M(-1) cm(-2)) and low detection limit (5×10(-6) M) at a signal-to-noise ratio of 3. Moreover, the inhibitory of sulfide to the self-assembled HRP was also investigated.