Sensitive hydrogen peroxide biosensor based on Tobacco Peroxidase immobilized on p‐phenylenediamine diazonium grafted CNTs. Preventing Fenton‐like inactivation at negative potential

Herein, we present a novel electrode platform for H 2 O 2 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 to enhance the sensitivity, stability and selectivity toward H 2 O 2 detection, as well as preventing the enzyme inactivation due to the electro-Fenton reaction. These results are 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 -NH 2 groups (mainly uncharged at pH 7, considering a pK a 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 H 2 O 2 , a extended linear range between 6 and 900 µM H 2 O 2 , as well as a significant increase in sensitivity (63.1 ± 0.1 µA mM -1 cm -2 ) compared with previous work based on TOP. The developed enzyme electrode showed a great stability, losing only 17.5% of its response along 30 days. Finally, the r-TOP/ p -PD/MWCNTs/G electrode was tested as a screening analytical method for the detection of H 2 O 2 in food samples.