Monitoring alkaline phosphatase activity using blue- and red-emissive biocompatible fluorescent probes in human serum...

Alkaline phosphatase (ALP) is a clinically significant biomarker, requiring the development of sensitive and selective detection methods. Here, two new ESIPT-based fluorescent probes, HCN-P and HIP-P, were thoughtfully designed and synthesized by combining a benzothiazole fluorophore with tunable intramolecular charge transfer (ICT) properties. Both probes demonstrated outstanding analytical performance, including low detection limits (0.060 U/L for HCN-P and 0.032 U/L for HIP-P), broad linear ranges (0.05-0.65 U/L and 0.025-0.2 U/L, respectively), and quick response times (around 5-6 min). A marked increase in fluorescence quantum yields (from 0.00117 to 0.0784 for HCN-P and from 0.0079 to 0.0771 for HIP-P) further confirmed the activation mechanism. Kinetic studies showed favourable enzyme-substrate interactions, with K_m values of 11.3 μM (HCN-P) and 5.82 μM (HIP-P), and Vmax values of 6.39 and 7.49, respectively. Both probes exhibited excellent selectivity for ALP even in the presence of various interfering species, and inhibition experiments using sodium orthovanadate (Na₃VO₄) confirmed enzyme-specific activation. Molecular docking studies indicated favourable binding affinities, with free energies of -7.1 kcal/mol (HCN-P) and -7.9 kcal/mol (HIP-P). The probes were successfully used in human serum samples, showing good agreement with the standard pNPP method, with relative standard deviations (RSDs) from 2.3% to 3.9%. Additionally, biocompatibility (∼85% cell viability at 10 μM) and effective fluorescence imaging in HeLa cells demonstrated their potential for biological applications. Therefore, this work introduces a versatile ESIPT-ICT synergistic strategy for creating fluorescent probes with tunable emission, high sensitivity, and strong applicability in biological systems.