Mediatorless biosensor for H 2O 2 based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold

Abstract

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H 2O 2 due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4–1.5 A M −1 cm −2 at −50 mV versus Ag∣AgCl. The response to H 2O 2 in the concentration range 0.1–40 μM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H 2O 2 detection was 10 nM at the electrodes modified with recombinant HRPs.