Dual-enzyme cascade--an amplified method for the detection of alkaline phosphatase

A method in which a two-enzyme cascade is used for rapid and sensitive detection of alkaline phosphatase is described. A masked inhibitor, 4-(3-oxo-4,4,4-trifluorobutyl)phenyl phosphate, is dephosphorylated by the action of alkaline phosphatase. The resulting compound, 1,1,1-trifluoro-4-(4-hydroxyphenyl)-butan-2-one, acts as a potent inhibitor of the second enzyme, a liver carboxylesterase. A determination of the residual esterase activity provides a highly sensitive indication of the original phosphatase concentration. The sensitivity of this dual-enzyme cascade is approximately 125-fold greater than that observed for the direct detection of phosphatase activity with p-nitrophenyl phosphate.