ZEPTOMOLE DETECTION LIMIT FOR ALKALINE-PHOSPHATASE USING 4-AMINOPHENYLPHOSPHATE, AMPEROMETRIC DETECTION, AND AN OPTIMAL BUFFER SYSTEM

Trace detection of bovine alkaline phosphatase was studied using the substrate, 4-aminophenylphosphate, and amperometric detection of the product, 4-aminophenol. Six aminoethanol buffers were compared with respect to their abilities to stabilize the product and to promote enzyme activity. In ethanolamine and 2-(methylamino)ethanol buffers, progress curves were linear from zero time to at least 60 min, while progress curves in the other buffers became non-linear in a short time due to product decomposition. The highest reaction rate at pH 10.0 was found in the 2-(methylamino)ethanol buffer, while the highest pseudo-rate constant, V(max)/K(m), was found in tris(hydroxymethyl)aminomethane. Higher buffer concentration and the addition of magnesium ion enhanced enzyme activity, while the addition of zinc ion decreased enzyme activity. In 1.0 F, pH 10.0, 2-(methylamino)ethanol buffer and in the presence of 1 mg ml-1 MgCl2, the detection limit for aqueous mouse IgG-alkaline phosphatase conjugate was 25 amol ml-1 or 500 zeptomoles (10(-21) mol). The reaction time was 60 min. Under similar conditions, the detection limit for IgG-alkaline phosphatase adsorbed onto a microtiter plate was 4 attomoles.