Horseradish peroxidase immobilized in TiO2 nanoparticle films on pyrolytic graphite electrodes: direct electrochemistry and bioelectrocatalysis

Horseradish peroxidase (HRP)-TiO₂ 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 TiO₂ 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 TiO₂ 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-TiO₂ 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 TiO₂ films kept nearly unchanged and were different from those of hemin or hemin-TiO₂ films, suggesting that HRP retains its native-like tertiary structure in TiO₂ films. The electrocatalytic activity of HRP embedded in TiO₂ films toward O₂ and H₂O₂ was studied. Possible mechanism of catalytic reduction of H₂O₂ with HRP-TiO₂ films was discussed. The HRP-TiO₂ films may have a potential perspective in fabricating the third-generation biosensors based on direct electrochemistry of enzymes.