Cui_2025_Talanta_295_128379

Butyrylcholinesterase (BChE) holds significant physiological functions and is closely associated with various physiological and pathological processes. Endoplasmic reticulum (ER) is the primary subcellular localization for BChE. ER-related events, such as ER stress, autophagy, and disruptions could influence the functional activity of ER-located BChE. Despite its significance, the development of ER-targeting BChE-responsive fluorescent probes remains largely uncharted. Here, we designed and synthesized NIRP, a near-infrared (NIR) fluorescent probe with exceptional ER-targeting capability, enabling highly specific and sensitive imaging of BChE within the ER. NIRP exhibits negligible fluorescence in its native state. However, upon interaction with BChE, NIRP underwent BChE-catalyzed hydrolysis, resulting in a pronounced NIR fluorescence turn-on response. NIRP exhibits high sensitivity toward BChE, with a detection limit of 1.6 mU/mL and a linear response across the concentration range of 0-0.45 U/mL. Notably, NIRP allows monitoring of drug-induced changes in endogenous BChE levels within the ER. Importantly, leveraging NIRP, we revealed for the first time that thapsigargin-induced ER stress reduces BChE level, indicating a critical role for BChE in maintaining ER homeostasis. NIRP represents a robust and powerful molecular tool for visualizing BChE levels in live cells, advancing better understandings of the roles of BChE in ER-related physiology and pathology.