Highly sensitive amperometric enzyme immunoassay for alpha-fetoprotein in human serum

A sandwich amperometric enzyme immunoassay with flow injection for alpha-fetoprotein in human serum has been developed with alkaline phosphatase as the enzyme label. p-Hydroxyphenyl phosphate was used as the substrate for alkaline phosphatase. The hydrolysis product, hydroquinone, was detected by oxidative amperometry in a flow injection system. The amperometric wall jet detector was fitted with a glassy carbon working electrode held at 350 mV vs. Ag/AgCl. The detection limit of hydroquinone in 30 mM borate buffer pH 9.5 was 1.2 x 10(-10) M (linearity range: 10(-9)-5.12 x 10(-6 M). A detection limit for free alkaline phosphatase of 1.2 x 10(-15) M (linearity range: 10(-15)-10(-13) M), or about 36 000 molecules, was observed (same borate buffer and incubations of 10 min at 25 degrees C). These conditions were maintained for the amperometric alpha-fetoprotein immunoassay. For comparison purposes, a photometric detection system was set up, with p-nitrophenyl phosphate as enzyme substrate and the same pair of antibodies and incubation conditions. The detection limit for alpha-fetoprotein obtained by amperometry, 0.07 ng/ml (linearity range = 5-500 ng/ml), was 14 times lower than by photometry. The amperometric enzyme immunoassay correlates well with a commercial colorimetric immunoassay (r = 0.986, slope = 0.967, n = 240).