Early diagnosis and therapeutic intervention for Alzheimer's disease (AD) necessitate advanced tools for detecting acetylcholinesterase (AChE) activity and screening AChE inhibitors (AChEIs). This study developed a novel bimetallic MOF nanozyme, Cu–NH2–88B(Fe), exhibiting significant peroxidase-like activity. This nanozyme was integrated with AChE to construct a dual-enzyme cascade biosensing platform, which achieved highly sensitive AChE detection, with a detection limit of 0.01 mU/mL, and demonstrated excellent accuracy in fetal bovine serum (spiked recoveries: 92.07–111.09%). Additionally, the platform also enabled quantitative assessment of synthetic AChEIs, determining IC50 values for donepezil (9.85 nM), neostigmine (1.41 μM), huperzine A (6.21 μM), and galantamine (611.31 μM), all of which exhibited broad linear ranges and high sensitivity. All four compounds exhibited a broad linear range and excellent sensitivity. Innovatively, the platform was applied to screen AChE inhibitory activity in seven traditional Chinese medicines (TCMs). Crucially, when comparing four extraction methods, ultrasound-assisted extraction (UAE) proved most effective in liberating active compounds. The UAE-obtained extract of Poria cocos (Yunnan) showed the strongest inhibition, achieving an AChE inhibition rate of 39.03% at 0.01 mg/mL. Notably, the screening results across different species, origins, and extraction methods (including the superior UAE) showed high consistency with the classical Ellman method, validating the platform's reliability. This study not only provides a low-cost, easy-to-operate, sensitive, and reliable analytical strategy for AChE activity detection and AChE inhibitor development, but more importantly, it successfully applies nanozyme technology to the complex system of TCMs, offering strong technical support for the screening and preliminary identification of potential anti-AD active components from TCM resources.
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