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