Determination of the activity of alkaline phosphatase based on aggregation-induced quenching of the fluorescence of copper nanoclusters.

A rapid method is described for synthesis of copper nanoclusters (CuNCs) by utilizing L-histidine as the stabilizer and ascorbic acid (AA) as the reductant. The CuNCs display blue-green fluorescence with excitation/emission peaks at 390/485 nm. A sensitive fluorometric assay was worked out for determination of alkaline phosphatase (ALP) activity. If the ALP substrate p-nitrophenylphosphate (PNPP) is enzymatically hydrolyzed, it forms p-nitrophenol (PNP) which reduces the fluorescence of CuNCs because its absorption band at 410 nm overlaps the excitation peak of CuNCs at 390 nm. In addition, the amino groups and imidazole groups on the surface of CuNCs possibly form a complex with the phenol groups of PNP. This induces aggregation-induced quenching of the fluorescence of CuNCs. The fluorescent probe has a linear analytical range that extends from 0.5 mU·mL-1 to 40 mU·mL-1 and a detection limit of 45 μU·mL-1. Graphical abstract Schematic illustration of a fluorometric assay for alkaline phosphatase (ALP) activity that uses L-histidine protected copper nanoclusters (CuNCs), aggregation-induced quenching, and the inner filter effect between PNP and CuNCs.