A Single Fluorescent Probe for Simultaneous Detection of Polarity, Viscosity, and Esterase

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 (λ_ex/em = 490/630 nm) and lipid droplet-anchored green fluorescence (λ_ex/em = 405/525 nm). PQE integrates three functional modules: a quinoline-based polarity sensor exhibiting 48-fold fluorescence attenuation across solvent polarity Δƒ from 0.0205 to 0.3200, a pyrene-derived molecular rotor showing 50-fold fluorescence enhancement over a viscosity range of 0.89 to 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.