Label-free fluorescence turn-on detection of alkaline phosphatase activity using the calcein–Ce³⁺ 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 Ce³⁺ 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 Ce³⁺. 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 Ce³⁺ compared with that between calcein and Ce³⁺. The fluorescence intensity ratio (F-F₀/F₀) exhibited excellent linearity, facilitating sensitive ALP detection. The proposed ALP detection method covers a range from 0 to 1.4 mU/mL (R² = 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.