Inner filter effect-induced ratiometric fluorescence detection of alkaline phosphatase using a Zn-MOF/AuNCs...

Alkaline phosphatase (ALP) is a crucial biomarker for the diagnosis and monitoring of hepatobiliary and bone disorders, necessitating the development of reliable and convenient methods for its activity assay. Herein, a ratiometric fluorescence sensing platform for ALP activity detection was constructed based on a dual-emission probe composed of a zinc-based metal-organic framework (Zn-MOF) and gold nanoclusters (AuNCs) via simple physical mixing. At a single excitation wavelength of 370 nm, Zn-MOF and AuNCs exhibited two well-resolved emission peaks at 430 nm and 650 nm, respectively. ALP catalyzed the hydrolysis of p-nitrophenyl phosphate (p-NPP) to produce p-nitrophenol (p-NP), which simultaneously quenched the fluorescence of both Zn-MOF and AuNCs through the inner filter effect (IFE). As a result, the ratiometric fluorescence value F₄₃₀/F₆₅₀ decreased progressively with increasing ALP activity. The proposed method exhibited a linear response in the range of 3-600 U L^-1 with a detection limit of 0.65 U L^-1. The quenching mechanism was confirmed to be the IFE rather than dynamic quenching based on spectral overlap analysis and fluorescence lifetime measurements. Moreover, the sensing system demonstrated excellent selectivity, good tolerance to salt and oxidative interference, and satisfactory applicability in human serum samples with recoveries of 90.4-108.6%. This work provides a facile and reliable ratiometric fluorescence strategy for ALP detection, which avoids complex material synthesis and offers built-in self-calibration through dual-signal synchronous response.