A real-time fluorescent assay for the detection of alkaline phosphatase activity based on carbon quantum dots

A convenient and real-time fluorometric assay with the assistance of copper ions based on aggregation and disaggregation of carbon quantum dots (CQDs) was developed to achieve highly sensitive detection of alkaline phosphatase activity. CQDs and pyrophosphate anions (PPi) were used as the fluorescent indicator and substrate for ALP activity assessment respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by copper ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, PPi can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to copper ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by re-dispersion of CQDs in the presence of ALP and PPi. Quantitative evaluation of ALP activity in a broad range from 16.7 to 782.6 U/L with the detection limit of 1.1 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility, and provides an example based on disaggregation in optical probe development.