Rational Design of ICT‐Based Ratiometric Fluorescent Probe for Real‐Time Detection of Esterase Enzyme in Living Cells

ABSTRACT Esterases are a major class of mammalian hydrolase enzymes involved in diverse biological processes, and their dysregulation is implicated in several metabolic disorders. Activatable in situ fluorescence techniques offer high‐resolution spatiotemporal analysis, enabling precise investigation of their biological roles. Considering this, we have designed and synthesized a ratiometric fluorescence probe, PTDB‐OAc, based on an intramolecular charge transfer (ICT) for the esterase enzyme activity detection. The mechanism was supported by ESI‐MS, 1 H‐NMR spectra, DFT, VMD analysis and MD simulation studies. Owing to its excellent ratiometric response (<30 s), low detection limit (5.17 × 10 −4 U/mL), and excellent selectivity, the probe effectively monitored time‐dependent esterase activity in RAW 264.7 macrophage cells, enabling real‐time imaging of endogenous esterase variations and demonstrating its strong potential as a reliable ratiometric tool for studying esterase function in living biological systems.