3,3',5,5'-TETRAMETHYLBENZIDINE AS ELECTROCHEMICAL SUBSTRATE FOR HORSERADISH-PEROXIDASE BASED ENZYME IMMUNOASSAYS - A COMPARATIVE-STUDY

The use of 3,3',5,5'-tetramethylbenzidine (TMB) as an electrochemical substrate for horseradish peroxidase (HRP) was investigated. HRP activ ity has been detected using flow injection analysis at a glassy carbon working electrode polarised at +100 mV versus Ag/AgCl in 0.1 mol(-1) citrate-phosphate buffer (pH 5.0). The optimum concentrations were 2 x 10(-4) mol l(-1) TMB and 10(-3) mol l(-1) H2O2. The detection limit o btained after 15 min of incubation was 8.5 x 10(-14) mol l(-1) HRP wit h the amperometric method. This limit was lower than that obtained usi ng hydroquinone as HRP substrate and comparable to that with the p-ami nophenyl phosphate-alkaline phosphatase system. Better performance was achieved with amperometric than spectrophotometric detection using TM B in a competitive ELISA for rabbit immunoglobulin G as a model analyt e.