Liu_2026_Anal.Methods__

Accurate detection of acetylcholinesterase (AChE) activity and screening of its inhibitor drugs are crucial for detecting and intervening in neurodegenerative or aging-associated diseases. Herein, a sensitive and reliable ratiometric fluorescence platform is developed for sensing AChE activity and its inhibitor drugs. This strategy is based on an AZM hybrid, which is constructed by self-assembling a MOF@MOF architecture (ZIF8@MIL101(Al)) with AgAu nanoclusters (AgAuNCs) via electrostatic interactions. ZIF8 serves to enhance the fluorescence of AgAuNCs, while MIL101(Al) acts as a stable reference probe. Upon introduction of AChE and the substrate acetylthiocholine (ATCh), the generated thiocholine (TCh) quenches the AgAuNC fluorescence, whereas the MIL101(Al) signal stays constant, leading to a concentration-dependent change in the fluorescence intensity ratio. The ratiometric response enables sensitive detection of AChE activity with a limit of detection (LOD) of 0.032 U L(-1). The platform is applied to AChE determination in human serum samples. The system demonstrates the feasibility of inhibitor screening with tacrine as a model inhibitor. This work provides a simple ratiometric platform that holds promise for AChE-related clinical diagnosis and drug discovery.