Nitrogen-doped carbon dots as a ratiometric fluorescent probe for determination of the activity of acid phosphatase, for inhibitor screening, and for intracellular imaging.

The author describe a method for preparation of green fluorescent nitrogen-doped carbon dots (N-CDs) through hydrothermal treatment of a mixture of lotus leaf juice and ethylenediamine (EDA). The N-CDs have uniform size, good dispersibility and water solubility. Under 316 and 366 nm photoexcitation, they show dual fluorescence with emission peaks at 415 and 509 nm, respectively. They are positively charge and display low cytotoxicity. This makes them an excellent choice for fluorometric assays and for bioimaging. A ratiometric assay was developed for the determination of the activity of acid phosphatase (ACP). It is based on the aggregation- induced quenching (AIQ) of the fluorescence of the N-CDs by sodium hexametaphosphate (NaPO). Enzymatic hydrolysis of (NaPO) by ACP leads to the disintegration of (NaPO) and to the restoration of fluorescence. The measurement of the ratio of fluorescence at two wavelengths (415 and 509 nm), background interference and fluctuating signals can be widely eliminated. The method works in the 1-50 U·L ACP activity range and has a detection limit of 0.43 U·L. It was successfully applied (a) to the determination of ACP in spiked serum samples, (b) to ACP inhibitor screening, and (c) to imaging of ACP in HePG2 cells. Graphical abstract Schematic presentation of the synthesis of nitrogen-doped carbon dots (N-CDs), and their application to the ratiometric fluorometric determination of acid phosphatase (ACP) based on the aggregation-induced quenching and enzymatic hydrolysis.