A highly sensitive fluorescent probe for monitoring of carboxylesterase activity in drug-induced liver injury and pesticide exposure.

Carboxylesterases play critical roles in drug metabolism and detoxification, and there is an urgent need for tools capable monitoring of CES activity in pathological states. Herein, we developed N-OAC, a novel fluorescent probe based on ICT mechanism, for the highly selective and sensitive detection of CES. Upon CES activation, N-OAC exhibited a significant fluorescence enhancement at 585 nm with a large 145 nm Stokes shift, achieving ultrasensitive detection limit of 3.42 × 10-4 U/mL and excellent linearity (R2 = 0.9986). HRMS and DFT studies collectively confirmed the proposed mechanism wherein CES hydrolyzes the acetoxy group, thereby re-establishing the ICT pathway and leading to fluorescence recovery. N-OAC demonstrated exceptional selectivity over biological interferents and was successfully applied to monitor CES fluctuations in HepG2 cells under DILI and organophosphorus pesticide exposure. Notably, N-OAC visualized CES upregulation by NAC in APAP-injured liver tissues, offering a promising tool for assessing DILI severity and therapeutic efficacy.