Polyacrylic Acid-Ca2+(Ce3+) Nanoclusters as Fluorescent "Off-On-Off" Probes for Quantitative Detection of Pyrophosphate and Alkaline Phosphatase

Alkaline phosphatase (ALP) is an essential enzyme widely distributed in the human body, and it can hydrolyze pyrophosphate (PPi) under alkaline conditions. Therefore, ALP and PPi activities are associated with many diseases and it is crucial to develop sensitive and selective methods for detecting ALP and PPi. Herein, poly(acrylic acid) (PAA)-Ca2+(Ce3+) nanoclusters fluorescent probe was synthesized aiming to detect PPi and ALP quantitatively. The nanoclusters present monodisperse, and their particle size is 0.9-2.4 nm. The probe exhibits no fluorescence due to electron transfer; whereas it can combine with PPi to yield fluorescence increase, arising from the enhanced electric-dipole transition of Ce3+ under the ligand effect of PPi. Moreover, the probe displays good selectivity to PPi, and the fluorescence changes display a linear relationship with PPi concentration of 3-20 mM (y = 11.29x - 23.47, R-2 = 0.994). Furthermore, the probe was mixed with PPi to form PAA-Ca(Ce):PPi nanoparticle (Monodisperse, average size: 1.94 nm) and showed high fluorescence selectivity and responsiveness to ALP due to the ALP hydrolyzing effect and the fluorescence difference between PAA-Ca(Ce):PPi and PAA-Ca(Ce):Pi. Based on the relationship between the fluorescence reduction rate of the probe and the logarithm of ALP concentration (y = 0.0594x - 0.00111, 5-200 U/L, R-2 = 0.996), ALP was successfully detected quantitatively, and the detection limit is calculated to be 0.62 U/L. Through this approach, we developed a fluorescence "turn-on" for PPi detection and "turn-off" for ALP detection. This study provides a promising strategy for the diagnosis of diseases related to PPi and ALP.