A novel and simple biomolecules immobilization method: Electro-deposition ZrO2 doped with HRP for fabrication of hydrogen peroxide biosensor

For the first time, a very novel and simple immobilization method for fabrication of hydrogen peroxide biosensor was reported in this paper. The biocompatible composite HRP–ZrO2 thin films were synthesized on gold electrode surface based on electro-deposition zirconia doped with horseradish peroxidase (HRP) by cyclic voltammetry scanning in KCl solution containing ZrO2 and HRP. The fabricated process of biosensor was characterized by electrochemical impedance spectroscopy (EIS) and the surface topography of the prepared films was imaged by atomic force microscope (AFM). The HRP in HRP–ZrO2 thin films kept its bioactivity and exhibited excellent electrocatalytical response to the reduction of H2O2. Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP–ZrO2/Au electrode) showed a linear response to H2O2 over a concentration range from 0.02 to 9.45 mM with a detection limit of 2 μM based on a signal-to-noise ratio of 3 under optimized conditions. The apparent Michaelis–Menten constant (KMapp) was evaluated to be 8.01 mM, which indicated the HRP in HRP–ZrO2 thin films kept its native bioactivity and had high affinity for H2O2. Moreover, the proposed biosensor showed high sensitivity, good reproducibility and long-term stability. What is more, this immobilization methodology widened biosensor application in biomolecules immobilization and could further develop for other protein and biomolecules immobilization.