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 F₅₆₆/F₄₃₈ 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.

单原子纳米酶（SAzymes）因其坚固性，出色的催化活性和最大的原子利用特性而引起了生物催化领域的广泛兴趣。在本文中，成功地证明了锚固在N掺杂多孔碳（Fe-SAs / NC）SAzyme上的单原子铁的过氧化物酶活性，并基于Fe-SAs / S构建了用于乙酰胆碱酯酶（AChE）活性的比率荧光法。 NC和聚乙烯吡咯烷酮首次保护了铜纳米簇（PVP-CuNCs）。与H ₂ O ₂存在时，具有过氧化物酶样活性的Fe-SAs / NC催化非荧光邻苯二胺（OPD）氧化为荧光2,3-二氨基吩嗪（DAP），在566 nm处具有最大荧光发射峰。同时，生成的DAP通过荧光共振能量转移（FRET）有效地淬灭了438 nm处的PVP-CuNCs荧光。然而，AChE具有催化乙酰硫胆碱（ATCh）水解生成硫胆碱（TCh）的功能，可以抑制Fe-SAs / NC过氧化物酶样活性来阻止DAP生成并保留PVP-CuNCs荧光。因此，该系统的荧光强度比F ₅₆₆ / F ₄₃₈可以用作AChE活性感测的信号输出，范围为2到70 UL ^-1，检测极限为0.56 UL^-1。此外，基于Fe-SAs / NC和PVP-CuNCs的系统用于测定人血清样品中的AChE。