Reagentless biosensor for hydrogen peroxide based on immobilization of protein in zirconia nanoparticles enhanced grafted collagen matrix

A novel matrix, zirconia nanoparticles enhanced grafted collagen (ZrO2-grafted collagen) hybrid composite, for immobilization of protein and biosensing was developed. The scanning electron microscopy, UV–vis and Fourier transform infrared spectra, and electrochemical measurements showed that the matrix was well biocompatible and could retain the bioactivity of immobilized protein to a large extent. The direct electron transfer of the immobilized myoglobin (Mb) exhibited a couple of stable and well-defined redox peaks with the formal potential of −336 mV (versus SCE) in 0.1 M pH 7.0 PBS. This matrix could accelerate the electron transfer between Mb and the electrode with a surface-controlled process and an electron transfer rate constant of 3.58 ± 0.35 s−1 at 10–500 mV s−1. The Mb immobilized in the matrix showed a high thermal stability up to 70 °C and an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) without the help of an electron mediator. The linear response range of the biosensor to H2O2 concentration was from 1.0 to 85.0 μM with the limit of detection of 0.63 μM at a signal-to-noise ratio of 3σ. The biosensor exhibited high sensitivity, acceptable stability and reproducibility. This work opened a way for the further study on the direct electron transfer and biosensing application of the immobilized protein in collagen-related matrices.