A third-generation hydrogen peroxide biosensor based on Horseradish Peroxidase (HRP) enzyme immobilized in a Nafion–Sonogel–Carbon composite

Abstract

A third-generation biosensor based on HRP and a Sonogel–Carbon electrode has been fabricated with the aim of monitoring hydrogen peroxide in aqueous media via a direct electron transfer process. The redox activity of native HRP, typical of thin-layer electrochemistry, was observed. The charge coefficient transfer, α, and the heterogeneous electron transfer rate constant, k s, were calculated to be 0.51 ± 0.04 and 1.29 ± 0.04 s −1, respectively. Topographic study by atomic force microscopy (AFM) shows that the enzyme may have been introduced inside the ionic cluster of the Nafion. The immobilized HRP exhibited excellent electrocatalytical response to the reduction of H 2O 2 and preserved its native state after the immobilization stage. Several important experimental variables were optimized. The resulting biosensor showed a linear response to H 2O 2 over a concentration range from 4 to 100 μM, with a sensitivity of 12.8 nA/μM cm −2 and a detection limit of 1.6 μM, calculated as (3 S.D./sensitivity). The apparent Michaelis–Menten constant K m app was calculated to be 0.295 ± 0.020 mM. The biosensor showed high sensitivity as well as good stability and reproducibility. The performance of the biosensor was evaluated with respect to four possible interferences.