Single-atom iron containing nanozyme with peroxidase-like activity and copper nanoclusters based ratio fluorescent strategy for acetylcholinesterase activity sensing

Single-atom nanozymes (SAzymes) have attracted extensive interests in bio-catalysis filed because of their robustness, excellent catalytic activity and unique feature of maximum atomic utilization. Herein, the peroxidase-like activity of single-atom iron anchored on N-doped porous carbon (Fe-SAs/NC) SAzyme was successfully demonstrated and a ratio fluorescence method for acetylcholinesterase (AChE) activity sensing was constructed based on Fe-SAs/NC and polyvinylpyrrolidone protected copper nanoclusters (PVP-CuNCs) for the first time. With H2O2 presence, Fe-SAs/NC with peroxidase-like activity catalyzed oxidation of nonfluorescent o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP) with maximum fluorescence emission peak at 566 nm. Meanwhile, the generated DAP efficiently quenched PVP-CuNCs fluorescence at 438 nm through fluorescence resonance energy transfer (FRET). However, AChE possessed function of catalyzing the hydrolysis of acetylthiocholine (ATCh) to generate thiocholine (TCh), which can inhibit the Fe-SAs/NC peroxidase-like activity to block DAP generation and preserve PVP-CuNCs fluorescence. Therefore, the fluorescence intensity ratio F-566/F-438 of the system can serve as signal output for AChE activity sensing ranging from 2 to 70 U L-1 with a detection limit of 0.56 U L-1. Moreover, Fe-SAs/NC and PVP-CuNCs based system was utilized for AChE determination in human serum sample.