Development of a facile and sensitive method for detecting alkaline phosphatase activity in serum with fluorescent gold nanoclusters based on the inner filter effect

In this work, a simple and sensitive method based on the inner filter effect (IFE) of p-nitrophenol (PNP) on the fluorescence of gold nanoclusters (AuNCs) has been developed for detecting Alkaline Phosphatase (ALP) activity. Bright orange fluorescent AuNCs were synthesized by one-pot synthesis and used directly as IFE fluorophores. p-Nitrophenyl phosphate (PNPP) is hydrolyzed by ALP to PNP, which quenches the fluorescence of AuNCs by the IFE. In the presence of ALP, PNPP was converted to PNP, and the absorption band shifted from 310 nm to 405 nm, which resulted in a certain degree of overlap between the absorption of PNP and the excitation of AuNCs. Due to the competitive absorption between AuNCs and PNP, the excitation of AuNCs was clearly diminished, leading to the quenching of the fluorescence of AuNCs. The IFE detection method exhibited a good linear relationship between 0.01 and 7.0 U L^-1 (R² = 0.9990) with the lowest detection limit of 0.003 U L^-1 (the signal-to-noise ratio is 3). The proposed detection method was successfully applied for detecting ALP in serum samples and studying ALP inhibitors.