Application of an amperometric detector to the conventional enzyme-immunoassay (EIA)

An amperometric detector is developed which can be directly applied to the conventional enzyme-immunoassay (EIA) employing horseradish peroxidase (POD) as a conjugated enzyme and 3,5,3′,5′-tetramethylbenzidine (TMB) as substrate. In this work, TMB samples with different optical densities (OD) were applied to the cyclic voltammetric analysis and the amperometric measurement at a fixed potential using the detector. The sensitivity of the amperometric analysis was determined to be 24 nA per OD (a.u.) and the detection limit was estimated to be 2.0 nA. The linear correlation between the OD value and the amperometric signal was obtained in the OD range from 0.02 to 2.60 (linearity coefficient = 0.997). The applicability of the amperometric detector to EIA was demonstrated using commercially available EIA kits for the diagnosis of AIDS and hepatitis (HBV and HCV).