Ratiometric naphthalimide-based fluorescent probes for highly selective detection of carboxylesterase activity

Carboxylesterase (CES) is a key Phase I metabolic enzyme, and the accurate assessment of its activity is essential for studies in drug metabolism, toxicology, and disease diagnosis. However, many existing fluorescent probes suffer from cross-reactivity with homologous enzymes such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In this study, we designed carbamate as a CES-specific hydrolyzable moiety and conjugated it to the highly photostable naphthalimide fluorophore NIOH via either chlorinated or non-chlorinated self-immolative linkers, thereby developing two ratiometric fluorescent probes, NI-Cl and NI-W. Both probes exhibited negligible responses to AChE and BChE while enabling selective and sensitive ratiometric detection of CES. NI-Cl demonstrated an exceptionally low detection limit of 0.038 U/mL, and NI-W also showed strong performance with a detection limit of 0.048 U/mL. Importantly, NI-Cl successfully visualized endogenous CES activity in different live cells, providing a robust tool for elucidating the physiological and pathological roles of CES.