Mediating <i>in situ</i>-generated fluorescence for Au nanocluster-based multi-color ratiometric detection of alkaline phosphatase

The portable, rapid, and accurate detection methods for alkaline phosphatase (ALP) can significantly improve the efficiency and accuracy of medical testing, especially in early disease screening. Herein, a fluorescent detection was constructed for ALP based on the ALP-mediated <i>in situ</i>-generated fluorescent product for the first time, which employed Au nanoclusters (AuNCs) and ALP-mediated <i>in situ</i> generated fluorescent products as probes by applying 4-methylumbelliferyl phosphate (MeUP) as a substrate. ALP can catalyze the hydrolysis of MeUP to generate 4-methylumbelliferon (MeU), which exhibits blue emission centered at 440 nm. Meanwhile, the AuNCs kept their red fluorescence at 630 nm as a reference signal. Accordingly, a ratiometric fluorescent sensing of ALP was proposed with a consecutive fluorescent color change with a detection limit of 0.0025 U L^-1. The pronounced fluorescence signal variation served as the impetus for the development of a facile, cost-effective portable hydrogel kit, which facilitates equipment-free, visual, and quantitative analysis of ALP activity. Additionally, this system was employed for detecting ALP inhibitors using sodium metavanadate (Na₃VO₄) as a model. Notably, this sensing method and hydrogel kit were applied for ALP detection in human serum samples, yielding promising results.