Ratiometric detection and imaging of endogenous alkaline phosphatase activity by fluorescein-coumarin-based fluorescence probe

Alkaline phosphatase (ALP) is a type of enzyme that widely exists in various tissues of the human body; it plays an important role in regulating many cell functions. The development of a sensitive and accurate tool to detect the changes of ALP activity in organisms can contribute to research in the fields of biochemistry, cytology, clinical medicine, etc. In this paper, a small organic molecule-based ratiometric fluorescent probe (FCP) was designed based on the hydroxyl electron-donating group in fluorescein-coumarin protected by the phosphate group. ALP can trigger the fluorescence change through the enzyme-catalyzed cleavage of phosphoryl ester groups, and the ratio of ALP can be measured at wavelengths of 465 nm and 530 nm. The probe had high selectivity and sensitivity to ALP, and the detection limit measured under the optimal conditions in an aqueous medium reached 0.006 mU/mL. The ALP activity of human serum samples was determined using the probe and found to be in good agreement with that measured using commercial ALP kits. Finally, the probe was also successfully applied to image ALP in living hepatocytes with good selectivity and sensitivity.