Enhanced oxidase-like activity of g-C3N4 nanosheets supported Pd nanosheets for ratiometric fluorescence detection of acetylcholinesterase activity and its inhibitor

The undesirable enzymatic activity of nanozymes under near neutral pH condition and the traditional single signal output always restrict the analytical application of nanozyme-based biosensors. Herein, graphitic carbon nitride nanosheets supported palladium nanosheets composite (Pd/g-C₃N₄) with both oxidase-like activity and fluorescent property is synthesized. Notably, Pd/g-C₃N₄ exhibits enhanced oxidase-like activity compared to Pd NSs under pH 7.4. By combining Pd/g-C₃N₄ with o-phenylenediamine (OPD), a ratiometric fluorescence assay for acetylcholinesterase (AChE) activity detection is developed. Pd/g-C₃N₄ can catalyze oxidation of nonfluorescent OPD to fluorescent oxidized OPD (oxOPD, Em = 565 nm), which can quench fluorescence of g-C₃N₄ supporter (Em = 441 nm) through fluorescence resonance energy transfer (FRET). However, in presence of AChE, acetylthiocholine can be hydrolyzed into thiocholine, which will block the oxidase-like activity of Pd/g-C₃N₄ and then hamper the FRET process. This ratiometric fluorescence assay is also viable to screen AChE inhibitor. This work will guide design of ratiometric fluorescence assay based on nanozymes with improved enzymatic activity.