Hydroxyl-Masking-Induced Excited-State Molecular Twisting for the Design of Highly Sensitive Ratiometric Fluorescent Probes

Abstract Ratiometric fluorescent probes enable quantitative biomarker detection by reducing concentration-dependent errors of intensity-based measurements. However, spectral crosstalk between dual emissions and environmental interference remain major limitations for accurate imaging. Here, we developed a single-excitation fluorescent platform (CP-EG) for constructing crosstalk-free and environmentally robust ratiometric probes by introducing a tunable hydroxyl group into a coumarin−benzopyrylium scaffold and incorporating a hydrophilic ethylene glycol chain. The resulting probes exhibited the “disappeared” long-wavelength emission when the hydroxyl group was masked. Density functional theory calculations revealed that hydroxyl masking induces pronounced excited-state twisting, with the dihedral angle of the etherified dye reaching 89.78°, thereby resulting in the quenching fluorescence. Biomarker-triggered demasking restored the plane-like excited-state structure and activated strong long-wavelength fluorescence, enabling highly sensitive ratiometric responses. Notably, CP-EG and its derived probes exhibit minimal variation in emission ratios under changes in polarity, viscosity, and pH, ensuring reliable fluorescence analysis in complex biological environments. Leveraging this platform-based probe (CP-EG-CE), we simultaneously monitored oxidative stress- and drug-induced liver injury-associated changes in esterase activity across two distinct organelles. This hydroxyl-masking-induced excited-state molecular twisting strategy provides a versatile approach for developing high-fidelity ratiometric fluorescent probes, enabling the accurate biomarker imaging and molecular diagnostics.