Characterisation of horseradish peroxidase immobilisation on an electrochemical biosensor by colorimetric and amperometric techniques

This study presents the use of complementary colorimetric and amperometric techniques to measure the quantity of protein or enzyme immobilised onto a carbon paste electrode modified with a layer of electrodeposited polyaniline. By applying a solution of bovine serum albumin at 0.75 mg/ml, efficient blocking of the electrode from electroactive species in the bulk solution could be achieved. When the horseradish peroxidase was immobilised on the electrode, optimal amperometric responses from hydrogen peroxide reduction were achieved at approximately the same concentration. The mass of enzyme immobilised at this solution concentration was determined by a colorimetric enzyme assay to be equivalent to the formation of a protein monolayer. Under these conditions, amperometric responses from the immobilised layer are maximised and non-specific bulk solution interactions are minimised. At higher immobilised protein concentrations, diminished amperometric responses may be due to inhibited diffusion of hydrogen peroxide to enzyme which is in electronic communication with the electrode surface, or impeded electron transfer.