Novel ratiometric fluorescent probe for real-time detection of alkaline phosphatase and its application in living cells

A novel ratiometric fluorescent probe has been developed through a simple synthetic route for the detection of alkaline phosphatase(ALP) in aqueous media and for fluorescence imaging in living cells. The introduction of a spontaneous-degradation spacer in the design of the fluorescent probe is beneficial for the ratio detection method and allows the selection of a fluorophore with an amino group. Under catalysis by ALP, the phosphate monoester bond breaks; this is followed by 1,4-elimination, decomposition of the carbamate moiety, and subsequent formation of the 4-amine-1,8-naphthalimide fluorophore. The probe APN shows a significant fluorescence colour change from blue to green in response to ALP, and the fluorescence intensity ratio of the probe solution (F550/F480) has a good linear relationship with the ALP concentration in the range of 0 to 100 U L-1. Our studies have demonstrated that APN exhibits high accuracy in recognising ALP, with a detection limit as low as 0.16 U L-1. Furthermore, the probe shows very good biocompatibility, which is beneficial for its application in biological systems.