AMPEROMETRIC DETECTION OF ALKALINE-PHOSPHATASE ACTIVITY AT A HORSERADISH-PEROXIDASE ENZYME ELECTRODE BASED ON ACTIVATED CARBON - POTENTIAL APPLICATION TO ELECTROCHEMICAL IMMUNOASSAY

Amperometric detection of alkaline phosphatase activity has been achie ved using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the enzyme su bstrate. The production of hydrogen peroxide from the dephosphorylatio n of BCIP was measured using an activated carbon electrode with horser adish peroxidase immobilised to its surface by simple passive adsorpti on. This method was easily capable of measuring 10(-12) M alkaline pho sphatase and had a calculated detection limit of 2.2 x 10(-14) M. The horseradish peroxidase electrode system was investigated further as a method for non-competitive electrochemical enzyme immunoassay using th yrotropin (TSH) as the model analyte. This was realised by co-immobili zation 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 subs trate, streptavidin-labelled alkaline phosphatase was added and the cu rrent (generated by enzyme channelling of hydrogen peroxide) measured as a function of TSH concentration. Thus, the activated carbon electro de was used as a combined immunological capture phase and amperometric detection system.