COMPARISON OF METHODS FOR FOLLOWING ALKALINE-PHOSPHATASE CATALYSIS - SPECTROPHOTOMETRIC VERSUS AMPEROMETRIC DETECTION

An amperometric method for alkaline phosphatase is described and compared to the most widely used spectrophotometric method. Catalytic hydrogenation of 4-nitrophenylphosphate (the substrate in the spectrophotometric method) gives 4-aminophenylphosphate (the substrate in the amperometric method). The latter substrate has the formula C6H6NO4PNa2·5H2O and a Mr of 323. The Michaelis constant for 4-aminophenylphosphate in 0.10 m, pH 9.0. Tris buffer is 56 μm, while it is 82 μm for 4-nitrophenyl phosphate. The amperometric method has a detection limit of 7 nm for the product of the enzyme reaction, which is almost 20 times better than the spectrophotometric method. Similarly, with a 15-min reaction at room temperature and in a reaction volume of 1.1 ml, 0.05 μg/l alkaline phosphatase can be detected by electrochemistry, almost an order of magnitude better than by absorption spectrophotometry. Amperometric detection is ideally suited for small-volume and trace immunoassay. © 1991.