Han_2025_Anal.Chim.Acta_1372_344463

BACKGROUND: The dynamic interplay between esterase activity and physicochemical microenvironments-such as polarity and viscosity-is critical for decoding early cellular dysfunction in processes like apoptosis, ferroptosis, and drug-induced toxicity. However, conventional probes typically report only a single parameter, obscuring interdependent changes in enzyme activity and membrane properties. This technological gap limits our ability to capture real-time, spatially resolved fluctuations within subcellular compartments. Developing a multifunctional, dual-emission probe that concurrently monitors esterase activity, polarity, and viscosity would thus provide unprecedented insight into complex pathophysiological mechanisms and enhance diagnostic precision. RESULTS: We propose a dual-targeted fluorescent probe PQE, which achieves simultaneous monitoring of esterase activity, polarity and viscosity in living cells through mitochondria-localized red fluorescence (lambda(ex/em) = 490/630 nm) and lipid droplet-anchored green fluorescence (lambda(ex/em) = 405/525 nm). PQE integrates three functional modules: a quinoline-based polarity sensor exhibiting 48-fold fluorescence attenuation across solvent polarity delta from 0.0205 to 0.3200, a pyrene-derived molecular rotor showing 50-fold fluorescence enhancement over a viscosity range of 0.89-965 cP, and an acetoxymethyl (AcOM) esterase-activatable group demonstrating 39-fold fluorescence increase after hydrolysis. Using PQE, we distinguished live cells, early apoptotic cells, and fixed cells, and observed that ferroptosis-induced esterase inactivation coincided with increased mitochondrial viscosity and decreased lipid droplet polarity. In addition, PQE also dynamically visualized esterase inhibition and viscosity-polarity imbalance in acetaminophen and LPS-induced hepatotoxicity models. SIGNIFICANCE: Compared to single-parameter detection, the simultaneous monitoring of multiple markers in a single probe enables comprehensive profiling of enzymatic activity, viscosity, and polarity within the same cellular context. This real-time, organelle-specific approach enhances sensitivity and accuracy in detecting ferroptotic, hepatotoxic events and may accelerate applications in diagnostics and drug screening.