Novel Ratiometric Fluorescent Probe based on Internal Reference of Lanthanide/nucleotide for Alkaline Phosphatase Detection

Based on the specific hydrolytic ability of alkaline phosphatase (ALP), a novel biocompatible ratiometric lanthanide fluorescent probe based on internal reference (CIP@SiO₂-Ce/ATP-Tris) was constructed with Ce³⁺ as the central ion, adenosine triphosphate (ATP) as the ligand, Tris as the auxiliary ligand and ciprofloxacin (CIP) encapsulated into SiO₂ nanoparticles as the reference signal. The fluorescent probe emits characteristic fluorescence at 363 nm belonging to Ce³⁺ as the working signal and at 435 nm belonging to CIP as the reference signal. Dephosphorylation disrupted the coordination of Ce/ATP-Tris with the enzymatic reaction of ALP, which resulted in a fluorescence quenching of Ce³⁺. The reference fluorescence was kept stable because of protective effect by encapsulated SiO₂. The biosensor analysis method was achieved by comparing the relationship between I₄₃₅/I₃₆₃ and ALP concentration. The detection limit is 0.0025 U/L, and the linear range of detection is 0.1-20 U/L. It was subsequently used to detect ALP in samples of fetal bovine serum and human serum, and promising results were obtained.