Ratiometric fluorescence determination of alkaline phosphatase activity based on dual emission of bovine serum albumin-stabilized gold nanoclusters and the inner filter effect

Abstract

A novel and convenient method for the ratiometric fluorescence detection of alkaline phosphatase (ALP) activity was proposed based on dual emission of bovine serum albumin-templated gold nanoclusters (BSA-AuNCs) and the mechanism of the inner filter effect between BSA-AuNCs and p-nitrophenol (PNP). First, ALP catalyzed the hydrolysis of the substrate p-nitrophenyl phosphate (PNPP) to produce PNP. PNP effectively quenched the emission peak of BSA-AuNCs at 410 nm because of the overlap in absorbance feature of PNP and the fluorescence spectrum of BSA-AuNCs, and the peak at 650 nm was almost unaffected. Thus, a sensitive ratiometric method for detection of ALP activity was developed using the fluorescence intensity of BSA-AuNCs at 650 nm as a reference signal. ALP activity versus the ratio of fluorescence intensities at 410 and 650 nm showed good linearity between 0.2 and 5 mU mL⁻¹ (R² = 0.9931) and high sensitivity with a detection limit of 0.03 mU mL⁻¹ (S/N = 3). The developed sensing method was successfully applied to investigate ALP inhibitors and detect ALP in serum samples.