Fluorescent assay for alkaline phosphatase by integrating strand displacement amplification with DNAzyme-catalytic recycling cleavage of molecular beacons

Abstract

A fluorescence sensing method for the quantification of alkaline phosphatase (ALP) was developed by integrating the strand displacement amplification with DNAzyme-catalytic recycling cleavage of molecular beacons. ALP can hydrolyze a 3′-phosphoralated primer into a 3′-hydroxy primer which can initiate the strand displacement amplification to produce the Mg2+-dependent DNAzyme. The DNAzyme can then catalyze the cleavage of the DNA molecular beacon labeled with FAM fluorophore at its 5′-end and BHQ1 quencher at its 3′-end, turning on the fluorescence of FAM fluorophore. The content of ALP in a sample can be deduced from the measured fluorescence intensity. Due to the cascading nature of its amplification strategy, the proposed method achieved sensitive and specific ALP detection in human serum samples. Its results were in good consistent with the corresponding values obtained by a commercial ALP detection kit. The limit of detection of the proposed method for ALP is about 0.015 U/L, lower than some methods recently reported in literature, demonstrating its potential for ALP detection in biomedical research and clinical diagnosis.