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An exonuclease-assisted fluorescence sensor for assaying alkaline phosphatase based on SYBR Green I

Wang, Huiyu, Ma, Changbei, Li, Zekun, Wu, Kefeng
Molecular and cellular probes 2019 v.45 pp. 26-30
DNA, alkaline phosphatase, blood serum, catalytic activity, detection limit, fluorescence, humans
In this report, we propose a fast, reliable and convenient approach to determine the alkaline phosphatase (ALP) activity based on a label-free fluorescence strategy. Upon catalysis of ALP, dephosphorylated dsDNA hampers the λ exonuclease (λexo) cleavage, shows high affinity to SYBR Green I (SG I), resulting in a strong fluorescence emission peak at 520 nm. In the absence of ALP, the dsDNA with 5′-phosphoryl-termini could be employed as a substrate of λexo. After cleavage, a weak fluorescence emission peaks at 520 nm could be observed. The assay was both selective and sensitive, and the detection limit was found to be as low as 3 U/L. This method was utilized to evaluate Na3VO4 as ALP inhibitor. The method was successfully applied to the determination of the activity of ALP in spiked human serum samples.