Label-free fluorescence turn-on detection of alkaline phosphatase activity using the calcein-Ce complex.

This study introduces an innovative approach for the real-time and efficient detection of alkaline phosphatase (ALP) activity, using a calcein fluorescence probe and leveraging the static quenching properties of calcein fluorescence by Ce metal ions. In this method, calcein serves as the signal element, with its fluorescence effectively preserved through energy transfer or charge transfer when coordinated with Ce. Conversely, ALP catalyzes the phosphopeptide substrate to generate a substantial amount of Pi, preventing calcein fluorescence quenching due to the higher affinity between Pi and Ce compared with that between calcein and Ce. The fluorescence intensity ratio (F-F/F) exhibited excellent linearity, facilitating sensitive ALP detection. The proposed ALP detection method covers a range from 0 to 1.4 mU/mL (R = 0.9942), with the limit of detection at 0.069 mU/mL (S/N = 3). Additionally, this method was successfully applied for detecting ALP in serum samples and studying its inhibitors. This research introduces a novel clinical diagnosis approach for ALP sensing while broadening the potential applications of calcein.