Novel Protocol for Covalent Immobilization of Horseradish Peroxidase on Gold Electrode Surface

A novel protocol for immobilization of horseradish peroxidase (HRP) onto diazonium functionalized screen-printed gold electrode (SPGE) has been successfully developed. This protocol involved 1) electrochemical reduction of p-nitrophenyl diazonium salts synthesized in situ in acidic aqueous solution to graft a layer of p-nitrophenyl on SPGE, 2) electrochemical reduction of the nitro groups to convert to amines, 3) chemical reaction with nitrous acid to transform the amine to diazonium derivative and 4) chemical coupling of the enzyme with the diazonium group to form a covalent diazo bond. The fabricated biosensor showed the direct electrochemistry of HRP and displayed electrocatalytic activity towards the reduction of hydrogen peroxide (H2O2) without any mediator. The biosensor exhibited fast amperometric response to H2O2. The catalytic current increased with increasing H2O2 concentration from 5 mu M to 30 mu M and the detection limit of the biosensor was 2 mu M. The biosensor exhibited acceptable sensitivity, good reproducibility and long-term stability.