Shao_2026_Anal.Methods__

Butyrylcholinesterase (BChE) is closely associated with Alzheimer's disease (AD), with its expression significantly elevated in the brains of AD patients. This enzyme has emerged as a potential biomarker for monitoring AD progression. Therefore, developing a reliable chemical tool to detect BChE both in vitro and in vivo is of considerable interest. In this study, we rationally designed a series of environmentally sensitive fluorescent probes targeting BChE, based on a highly potent, selective, and reversible BChE inhibitor. Among these, the most promising probe ESP4 demonstrated an ultrafast response to BChE with exceptional sensitivity (LOD = 1.81 nM), enabling high-precision detection of BChE. Additionally, ESP4 maintained robust inhibitory activity against BChE in the low nanomolar range (IC(50) = 71.78 +/- 1.90 nM). Notably, ESP4 exhibited excellent selectivity for BChE, showing no interference from other biological species, including acetylcholinesterase (AChE). The probe also accurately measured the IC(50) of tacrine (7.83 nM), a standard BChE inhibitor, demonstrating its reliability in evaluating BChE inhibition. Due to its high sensitivity, rapid response, and superior selectivity, ESP4 enabled real-time imaging of BChE in biological systems such as cells, zebrafish, and AD mouse models. Collectively, these findings highlighted ESP4 as a valuable tool for BChE detection, contributing to a deeper understanding of the physiological role of BChE in health and disease.