Sensitive and selective label-free alkaline phosphatase detection based on DNA hairpin probe

Abstract

Here, we present a label-free, simple and signal-on architecture for fluorescent alkaline phosphatase (ALP) biosensor utilizing an SYBR Green I (SG) assisted fluorescence amplification method. The strategy relies on the fact that ALP provides a significant barrier to lambda exonuclease (λ exo) activity by a dephosphorylating DNA hairpin probe (HP), and SG shows a considerable fluorescence intensity enhancement upon binding to double-stranded DNA (dsDNA) than single-stranded DNA (ssDNA). Our method is simple, sensitive and selective, which can also successfully detect the activity of ALP in complex biological fluids. The results have revealed that the method allows a specific and quantitative assay of the target ALP with a wide linear response range from 0.4 to 200 U/mL and a detection limit of 0.05 U/mL. © 2012 The Japan Society for Analytical Chemistry.