Horseradish peroxidase immobilized in TiO 2 nanoparticle films on pyrolytic graphite electrodes: direct electrochemistry and bioelectrocatalysis

Abstract

Horseradish peroxidase (HRP)–TiO 2 film electrodes were fabricated by casting the mixture of HRP solution and aqueous titania nanoparticle dispersion onto pyrolytic graphite (PG) electrodes and letting the solvent evaporate. The HRP incorporated in TiO 2 films exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks at about −0.35 V versus saturated calomel electrode (SCE) in pH 7.0 buffers, characteristic of HRP–Fe(III)/Fe(II) redox couple. The electron exchange between the enzyme and PG electrodes was greatly enhanced in the TiO 2 nanoparticle film microenvironment. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant ( k s) and formal potential ( E°′) were estimated by fitting the data of square wave voltammetry with nonlinear regression analysis. The HRP–TiO 2 film electrodes were quite stable and amenable to long-time voltammetric experiments. The UV-Vis spectroscopy showed that the position and shape of Soret absorption band of HRP in TiO 2 films kept nearly unchanged and were different from those of hemin or hemin–TiO 2 films, suggesting that HRP retains its native-like tertiary structure in TiO 2 films. The electrocatalytic activity of HRP embedded in TiO 2 films toward O 2 and H 2O 2 was studied. Possible mechanism of catalytic reduction of H 2O 2 with HRP–TiO 2 films was discussed. The HRP–TiO 2 films may have a potential perspective in fabricating the third-generation biosensors based on direct electrochemistry of enzymes.