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Alkaline phosphatase is a common enzyme used in electrochemical enzyme immunoassays, with phenyl phosphate as a substrate. The phenol compound produced by the enzyme-catalyzed hydrolysis of phenyl phosphate is measured by oxidative amperometry. Recently, the use of 4-aminophenyl phosphate as a substrate in these assays has been advocated and extensively studied by several authors.l-6 In this note we report that 4-aminophenol, the hydrolysis product of 4-aminophenyl phosphate, can be amperometrically detected at concentration levels as low as 10-10 M (M=mot dm 3) by the principle of polarographic catalytic current''8, in which enzymatic recycling9 with a redox enzyme (that is, diaphorase) is involved; the sensitivity of the electrochemical assay of alkaline phosphatase is greatly improved by this method. Alkaline phosphatase (EC 3.1.3.1, Lot No. 06290) was purchased from Toyobo Co., Ltd. The enzyme was dissolved with a 1% bovine serum albumin (BSA) solution containing 0.25 mM MgCl2 and stored in a refrigerator. Diaphorase (EC 1.6.99.-, Type I) from Bacillus stearothermophillus was a gift from Unitika Ltd. The enzyme was dissolved with a 1% BSA solution. 4Aminophenyl phosphate (PAPP, Aldrich Chemical Co., Inc.), 4-aminophenol (PAP, Wako Pure Chemical Industries, Ltd.) and NADH (reduced form of nicotinamide adenine dinucleotide, Oriental Yeast Co., Ltd.) were used without further purification. PAPP was dissolved in a 0.1 M carbonate buffer (pH 10.3) containing 0.25 mM MgC12, immediately before use. PAP was dissolved in a 0.02 M acetate buffer (pH 4.5). Other chemicals were of analytical-reagent grade. The working electrode was a glassy carbon disk electrode (4=0.3 cm). The electrode was polished to a mirror-like finish with 0.05 μm alumina, followed by through rinse with deionized water in an ultrasonic bath. The counter electrode was a platinum electrode. The potentials were measured against an Ag/ AgCI (sat. KCl) reference electrode. Electrochemical measurements were carried out in an H-type plexiglass cell with a sample volume of 0.4 ml, thermostated at 37±0.3 ° C, with a potentiostat (Yanaco polarographic analyzer P-1000) connected with an X Y recorder (Yokogawa Modal 3025). Figure 1 shows cyclic voltammograms of 10.4 M PAP in a 0.1 M tris-buffer solution of pH 8.5 without (a) and with (b) 3.3X10.8 M diaphorase and 10-3 M NADH. The results clearly show that the anodic current due to the oxidation of PAP to 4-quinone imine (PQI),

analytical sciences

Sensitive Amperometry of 4-Aminophenol Based on Catalytic Current Involving Enzymatic Recycling with Diaphorase and Its Application to Alkaline Phosphatase Assay