Highly Sensitive Hydrogen Peroxide Biosensor Based on Tobacco Peroxidase Immobilized on p‐Phenylenediamine Diazonium Cation Grafted Carbon Nanotubes: Preventing Fenton‐like Inactivation at Negative Potential

Abstract

AbstractHerein, we present a novel electrode platform for H2O2 detection based on the immobilization of recombinant Tobacco Peroxidase (r‐TOP) onto graphite electrodes (G) modified with p‐phenylenediamine (p‐PD) diazonium cation grafted multi‐walled carbon nanotubes (MWCNTs). The employment of both p‐phenylenediamine moieties and covalent cross‐linking by using glutaraldehyde allowed us to enhance the sensitivity, stability, and selectivity toward H2O2 detection, as well as preventing enzyme inactivation due to the electro‐Fenton reaction. This reaction continuously produces hydroxyl radicals, whose high and unselective reactivity is likely to reduce drastically the operating life of the biosensor. The protection against the electro‐Fenton reaction is mainly ascribed to a beneficial enzyme immobilization leading to a correct orientation achieved through cross‐linking the enzyme in combination with interaction between the uncoupled ‐NH2 groups (mainly uncharged at pH 7, considering a pKa of 4.6) available on the electrode surface and the enzyme. In particular, the electrode based on the r‐TOP/p‐PD/MWCNTs/G platform showed a lower limit of detection of 1.8 μM H2O2, an extended linear range between 6 and 900 μM H2O2, as well as a significant increase in sensitivity (63.1±0.1 μA mM−1 cm−2) compared with previous work based on TOP. Finally, the r‐TOP/p‐PD/MWCNTs/G electrode was tested in several H2O2 spiked food samples as a screening analytical method for the detection of H2O2.