Characterization of the direct electron transfer and bioelectrocatalysis of horseradish peroxidase in DNA film at pyrolytic graphite electrode

Abstract

Direct electron transfer between horseradish peroxidase (HRP) in a DNA film and pyrolytic graphite (PG) electrode was observed at the formal potential of −0.208 V (versus Ag/AgCl (sat. KCl)) involving the Fe III/Fe II redox couple. DNA played the role of charge carrier and facilitated the electron transfer between HRP and the PG electrode. The peak current of the redox process increased linearly with scan rate from 0.05 to 2 V/s, which is typical in thin layer electrochemistry. The number of electrons and the average apparent heterogeneous electron transfer rate constant k s involved in the redox reaction were calculated to be 0.94 and 1.130 s −1, respectively. The formal potential dependence on pH with 54 mV per pH indicated a one-proton-transfer coupled with a one-electron-transfer reaction. Scanning electron microscopy (SEM) showed the different morphologies for isolated DNA film and the complex film of HRP and DNA. Visible absorption and reflectance–absorption infrared (RAIR) spectra proved the heme environment of HRP in DNA film to be in its native status. The embedded HRP molecules in DNA film retained the electrocatalytic activity for hydrogen peroxide. The sensitivity of the HRP-DNA-PG biocomposite was 115.6 μA/cm 2 for 2.5×10 −4 mol/l H 2O 2. This HRP-DNA film modified PG electrode represented a valid example of mediator-free amperometric biosensors.