Amperometric detection of alkaline phosphatase activity at a horseradish peroxidase enzyme electrode based on activated carbon: potential application to electrochemical immunoassay

Abstract

Amperometric detection of alkaline phosphatase activity has been achieved using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the enzyme substrate. The production of hydrogen peroxide from the dephosphorylation of BCIP was measured using an activated carbon electrode with horseradish peroxidase immobilised to its surface by simple passive adsorption. This method was easily capable of measuring 10 −12 M alkaline phosphatase and had a calculated detection limit of 2·2 × 10 −14 M. The horseradish peroxidase electrode system was investigated further as a method for non-competitive electrochemical enzyme immunoassay using thyrotropin (TSH) as the model analyte. This was realised by co-immobilization to the electrode surface of both horseradish peroxidase and an anti-thyrotropin monoclonal antibody. After addition of the analyte, a second biotinylated anti-thyrotropin monoclonal antibody and the substrate, streptavidin-labelled alkaline phosphatase was added and the current (generated by enzyme channelling of hydrogen peroxide) measured as a function of TSH concentration. Thus, the activated carbon electrode was used as a combined immunological capture phase and amperometric detection system.