Abstract: Highly sensitive and stable acetylcholinesterase detection is critical for diagnosing and treating various neurotransmission-related diseases. In this study, a novel colorimetric-fluorescent dual-mode biosen-sor based on highly dispersive trimetal-modified graphite-phase carbon nitride nanocomposites for acetylcholinesterase detection was designed and synthesized by phosphorus doping and a mixed-metal MOF strategy. The specific surface area of trimetal-modified graphite-phase carbon nitride nanocom-posites increased from 15.81 to 96.69 g m^-2, and its thermal stability, interfacial charge transfer, and oxidation-reduction capability were enhanced compared with those of graphite-phase carbon ni-tride. First-principles density functional theory calculations and steady-state kinetic analysis are ap-plied to investigate the electronic structures and efficient peroxidase-mimicking properties of trimetal-modified graphite-phase carbon nitride nanocomposites. The oxidation of 3,3’,5,5’-tetramethylbenzidin was inhibited by thiocholine, which originates from the decomposition of thiocholine iodide by Acetyl-cholinesterase (AChE), resulting in changes in fluorescence and absorbance intensity. Due to the indepen-dence and complementarity of the signals, a highly precise colorimetric-fluorescent dual-mode biosensor with a linear range for detecting AChE of 4-20 μU mL-1 and detection limits of 0.13 μU mL^-1 (colori-metric) and 0.04 μU mL^-1 (fluorescence) was developed. The spiking recovery of AChE in actual samples was 99.0 %-100.4 %. Therefore, a highly accurate, specific, and stable dual-mode biosensor is available for AChE detection, and this biosensor has the potential for the analysis of other biomarkers.

Key words: Graphitic carbon nitride, Zeolitic imidazolate framework-8, Colorimetric-fluorescent dual-mode sensor, Density functional theory calculations, Acetylcholinesterase