Development of a Fluorescence Assay for Rapid Detection of Alkaline Phosphatase Activity in Fresh Milk Based on Inner Filter Effect of Carbon Dots

This work aimed at developing a fluorescence assay for rapid and sensitive determination of alkaline phosphatase (ALP) activity in fresh milk using nitrogen doped carbon dots (N-CDs) as fluorescence probe. Green-emissive N-CDs were synthesized by using ethylenediamine as the nitrogen source and catechol as the carbon source through a hydrothermal method. The obtained N-CDs were characterized by transmission electron microscopy (TEM), UV-vis spectroscopy, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FT-IR) spectra. Control experiments were used to verify the feasibility for constructing the fluorescent assay using the N-CDs. Single-factor experiments were conducted to optimize the pH of working buffer, the concentration of disodium p-nitrophenyl phosphate (PNPP) and enzymatic reaction time for ALP detection. The results indicated that the as-synthesized N-CDs were successfully prepared with all the characterization results being consistent with those in previous works. The optimal wo