Nanoprobe-Based Dual-Mode Fluorescent and Colorimetric Assay for Alkaline Phosphatase Activity

Abstract Alkaline phosphatase (ALP) serves as a vital biomarker, and the accurate detection of its activity is essential for clinical diagnosis and biomedical research. In this work, a dual-signal fluorescent probe for an ALP activity assay was developed using blue-emissive carbon dots (B-CDs) and red-emissive resorufin (RS). The sensing mechanism is based on the ALP-catalyzed hydrolysis of p-nitrophenyl phosphate (PNPP) to produce p-nitrophenol (p-NP), which effectively quenches the fluorescence of the B-CDs/RS system and triggers a distinct color change in the solution from pink to orange-yellow. Accordingly, the probe allows the simultaneous detection of ALP activity via fluorescence and RGB colorimetric dual-mode readouts. Experimental results show that B-CDs exhibit a high fluorescence quantum yield (76.5%) and uniform nanoscale size (average diameter of 4.25 nm), demonstrating excellent luminescent properties. The proposed method is rapid and highly sensitive, with a detection limit as low as 0.0172 U/L in the fluorescence mode and 0.256 U/L in the colorimetric mode. Furthermore, the nanoprobe was successfully applied for in vivo dynamic monitoring of ALP activity in zebrafish and for evaluating the inhibitory effect of microcystin LR (MC-LR) on ALP. Overall, this nanoprobe platform provides multifunctional technical support for ALP activity monitoring, enzyme inhibitor screening, and environmental toxin detection, showing great potential for practical applications in clinical diagnosis, bioimaging, and environmental monitoring.