Fluorimetric determination of alkaline phosphatase activity in human serum by use of a flow-through biosensor

A fluorimetric method for the determination of alkaline phosphatase activity based on the use of a flow-through biosensor is reported. The biochemical basis of the method is the hydrolysis of 4-methyl-umbelliferone-phosphate catalyzed by the analyte with fluorimetric monitoring of the 4-methyl-umbelliferone formed (lambda ex = 365 nm, lambda em = 445 nm). The enhancement of sensitivity achieved when the reaction product is retained on the support packed in the flow-cell makes the method suitable for determination of the analytes in serum samples after 1:50 dilution. The linear range is found to be between 0.1 and 20 U l-1, with relative standard deviation less than 2.2%. The use of this biosensor was tested by the determination of the analyte in human serum from healthy and sick individuals with excellent recoveries (94-103%).