Determination of Acetylcholinesterase Activity Based on Ratiometric Fluorescence Signal Sensing

Acetylcholinesterase (AChE) plays an important role in the treatment of human diseases, environmental security and global food supply. In this study, the simple fluorescent indicators and MnO₂ nanosheets were developed and integrated to establish a ratiometric fluorescence sensing system for the detection of AChE activity. Two fluorescence signals could be recorded independently at the same excitation wavelength, which extended the detection range and enhanced the visibility of results. Fluorescence of F-PDA was quenched by MnO₂ nanosheets on account of inner filtering effect. Meanwhile, the nonfluorescent OPD was catalytically oxidized to 2,3-diaminophenazine by MnO₂ nanosheets. The acetylcholine (ATCh) was catalytically hydrolyzed by AChE to enzymatic thiocholine, which decomposed MnO₂ to Mn²⁺, recovered the fluorescence of F-PDA and reduced the emission of ox-OPD. Utilizing the fluorescence intensity ratio F₄₆₈/F₅₅₈ as the signal readout, the ratiometric fluorescence method was established to detect AChE activity. Under the excitation wavelength of 410 nm, the ratio F₄₆₀/F₅₅₈ against the AChE concentration demonstrated two linear relationships in the range 0.05 -1.0 and 1.0-50 U·L^- 1 with a limit of detection (LOD) of 0.073 U·L^- 1. The method was applied to the detection of AChE activity and the analysis of the inhibitor Huperzine-A. Due to the advantages of high sensitivity and favorable selectivity, the method possesses an application prospect in the activity deteceion of AChE and the screening of inhibitors.