A fluorescent assay for alkaline phosphatase activity based on inner filter effect by in-situ formation of fluorescent azamonardine

Abstract

A simple and sensitive fluorescent assay for determination of alkaline phosphatase (ALP) activity has been developed based on the inner filter effect (IFE) of p-nitrophenol (PNP) through the in-situ formation of azamonardine. Herein, p-nitrophenylphosphate (PNPP) was used as ALP substrate, which could be hydrolyzed to PNP with an absorption band centered at 405 nm. Under the alkaline condition, the introduction of resorcinol into the oxygen-containing dopamine solution would trigger the formation of fluorescent azamonardine with an intense emission band centering at 460 nm when excited at 415 nm. As a result, the fluorescence of azamonardine could remarkably decrease in the presence of PNP via IFE due to the absorption spectrum of PNP overlaps with the excitation spectrum of azamoardine. An IFE-based fluorescent assay has been thereby proposed for the ALP activity detection showing a linear range from 0.1 to 6.0 mU mL−1 with the detection limit down to 0.07 mU mL−1. Moreover, the developed fluorescent assay was successfully applied to probe ALP in human serum samples with satisfactory results. This IFE-based detection strategy exhibits several merits such as high sensitivity, good anti-interference ability, time-saving and easy operation simply by mixing the commercial reagents together. Therefore, it may promote the development of simple and sensitive fluorescent assays for the enzyme activity detections.