Smartphone-integrated NIR fluorescent probe for rapid and sensitive alkaline phosphatase detection in serum and...

Alkaline phosphatase (ALP) is a clinically significant hydrolase enzyme involved in various physiological and pathological processes, making its accurate detection vital for disease diagnosis and monitoring. In this study, we report a new near-infrared (NIR) fluorescent probe based on a triphenylamine-dicyanoisophorone scaffold functionalized with a phosphate group for the rapid and sensitive detection of ALP activity. The probe is designed with a donor-acceptor (D-A) structure, in which triphenylamine serves as a strong electron donor and dicyanoisophorone as an efficient electron acceptor, promoting intramolecular charge transfer (ICT) upon enzymatic activation. Initially, the non-fluorescent probe exhibits a robust "turn-off/on" response upon ALP-mediated dephosphorylation, resulting in a strong NIR emission at 650 nm. The system demonstrates excellent sensitivity with a detection limit of 0.048 U/L and a rapid response time of approximately 8.0 min-outperforming many previously reported ALP sensors. The probe shows high selectivity toward ALP over various biologically relevant metal ions, anions, and amino acids. Furthermore, a smartphone-based RGB analysis enables low-cost and user-friendly quantification of ALP levels. Molecular docking studies support the probe's strong binding affinity to ALP, with a calculated binding energy of -8.2 kcal/mol. Importantly, the probe was successfully applied for the detection of ALP in human serum samples, with results consistent with the standard pNPP assay, confirming its potential for clinical applications. Owing to its superior analytical performance, biocompatibility, and point-of-care potential, this probe represents a promising tool for real-time ALP detection in biomedical and clinical applications.