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

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 Km 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.