Fluorescent\nIron-Doped Polymer Dot Nanozyme-Based\nCascade System for Dual-Mode Detection of Acetylcholinesterase Activity\nand Its Inhibitors

The\nadvancement of acetylcholinesterase (AChE) activity\nand its\ninhibitor assays is crucial for clinical diagnosis, drug screening,\nand environmental monitoring. A nanozyme-mediated cascade reaction\nsystem could offer promising prospects for a wide range of applications\nin such biosensing; however, the creation of nanozyme catalysts with\ndiverse functionalities remains a significant challenge. Herein, we\nhave proposed a multifunctional iron-doped polymer dots (Fe-PDs) nanozyme\npossessing excellent fluorescence and peroxidase (POD)-mimicking activity.\nNotably, the Fe-PDs nanozyme is capable of catalyzing H<sub>2</sub>O<sub>2</sub> to produce a series of reactive oxygen species, which\ncan simultaneously quench the fluorescence of Fe-PDs and induce a\nchromogenic reaction of 3,3′,5,5′-tetramethylbenzidine\n(TMB), enabling the dual-mode detection of H<sub>2</sub>O<sub>2</sub> through both fluorescence turn-off and absorbance turn-on signals.\nFurthermore, by integrating acetylcholine (ACh) and choline oxidase\n(ChOx), we have developed a three-enzyme (AChE–ChOx–POD)\ncascade-based fluorometric and colorimetric dual-mode sensing platform\nfor monitoring AChE activity and its inhibitors. The sensitive and\nconvenient dual-mode sensor has achieved low limits of detection with\n0.5 mU/mL (fluorometry) and 0.014 mU/mL (colorimetry) for AChE, respectively,\nwhich are superior to the traditional Ellman’s assay. More\nsignificantly, this sensor can also be extended to detect the reversible\nand irreversible inhibitors of AChE, such as tacrine (IC<sub>50</sub> = 23.3 nM) and carbaryl (LOD = 0.8 nM). We firmly believe that this\ninnovative dual-mode nanozyme-involved multienzyme cascade system-based\nsensing strategy will stimulate further exploration and serve as a\nversatile and practical tool for biochemical sensing applications.