Sensitive detection of acetylcholine based on a novel boronate intramolecular charge transfer fluorescence probe

A highly sensitive and selective fluorescence method for the detection of acetylcholine (ACh) based on enzyme-generated hydrogen peroxide (H₂O₂) and a new boronate intramolecular charge transfer (ICT) fluorescence probe, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN), was developed. This strategy involves the reaction of ACh with acetylcholinesterase (AChE) to produce choline, which is further oxidized by choline oxidase (ChOx) to obtain betaine and H₂O₂. The enzyme-generated H₂O₂ reacts with BN and results in hydrolytic deprotection of BN to generate fluorescent product (4-hydroxyl-N-butyl-1,8-naphthalimide, ON). Two consecutive linear response ranges allow determining ACh in a wide concentration range with a low detection limit of 2.7 nM (signal/noise = 3). Compared with other fluorescent probes based on the mechanism of nonspecific oxidation, this reported boronate probe has the advantage of no interference from other biologically relevant reactive oxygen species (ROS) on the detection of ACh. This study provides a new method for the detection of ACh with high selectivity and sensitivity.