Sensitive fluorometric detection of alkaline phosphatase using a water-soluble conjugated polymer

Abstract

In this paper, we developed a new fluorescence method for highly sensitive and selective detection of the alkaline phosphatase (ALP) activity with phenyl phosphate (PP) as the enzyme substrate and water-soluble conjugated polymer PPESO₃ as a fluorescent probe. Phenyl phosphate could be hydrolyzed to phenol via the ALP-catalyzed reaction. In the presence of horse radish peroxidase (HRP) and H₂O₂, phenol was rapidly oxidized to quinone which could efficiently quench the fluorescence of the conjugated polymer PPESO₃. The fluorescence intensity change of PPESO₃ is proportional to the concentration of ALP. Thus, we established a fluorescence method for ALP activity detection based on a water-soluble fluorescent conjugated polymer–enzyme hybrid system. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I₀ (I₀ and I were the fluorescence intensity of the sensing system in the absence and presence of ALP, respectively) and the concentration of ALP in the range of 0–30 U L⁻¹, and the detection limit for ALP was 0.5 U L⁻¹. The present method was applied to the determination of ALP in spiked human serum samples with satisfactory results.