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.