Nitrogen doped carbon dots for turn-off fluorescent detection of alkaline phosphatase activity based on inner filter effect

Abstract

The abnormal expression level of alkaline phosphatase (ALP) will lead to serious diseases. Therefore, a sensitive and rapid assay for ALP activity monitoring is of vital importance. In this work, a fluorescence turn-off approach for the detection of ALP is designed on the basis of nitrogen doped carbon dots (N-CDs), which were synthesized by one-step hydrothermal method and applied as signal readout. p-Nitrophenylphosphate (PNPP) can be hydrolyzed into p-nitrophenol (PNP) by ALP and their absorption peaks are different under alkaline conditions, so it was chosen as the ALP substrate. The absorption spectrum of PNP has good overlap with the excitation and emission spectra of N-CDs, thus the fluorescence of N-CDs can be effectively quenched by PNP via the inner filter effect (IFE). Consequently, quantitative detection of ALP is realized because the relative fluorescence intensity is linearly with the ALP activity in a wide range from 0.05 to 40 U L^-1. The detection limit is 0.02 U L^-1 (S/N = 3), which is much lower than the normal level of serum ALP in adults (about 40-190 U L^-1). Moreover, the assay was successfully applied to evaluate ALP inhibitor efficiency and screen ALP inhibitors in drug discovery. It is also demonstrated that N-CDs possesses low cytotoxicity, excellent biocompatibility and photostability, and can be successfully applied in vivo fluorescence imaging, showing great potential in clinical applications.

Keywords: Alkaline phosphatase; Fluorescence imaging; Inner filter effect; Nitrogen doped carbon dots; Quench.