Construction of colorimetric/fluorescent dual-signal probes for the detection of β-galactosidase in senescent cells

The sensitive and selective detection of β-galactosidase (β-Gal) activity and spatial distribution holds critical importance for elucidating the molecular mechanisms underlying cellular senescence and associated pathologies. Herein, we present ratiometric probes (1 and 2) integrating dual signals of colorimetric and red fluorescence for precise β-Gal detection in solution and senescent cells. Strategic introduction of chlorine on a tricyanofuran (TCF)-based fluorophore (1) enabled enzyme-responsive absorption shifts from 420 nm to 575 nm in absorption accompanied by remarkable red fluorescence enhancement (>80-fold at 622 nm). These red-shifted photophysical properties were attributed to the enhanced electron-donating ability from β-Gal-released phenolate residue (1-O), which is stabilized through the incorporation of chlorine into the molecular structure. Probe 1 demonstrated exceptional analytical performance with detection limits of 1.2 × 10⁻⁴ U mL⁻¹ (absorption) and 7.4 × 10⁻⁵ U mL⁻¹ (fluorescence), outperforming conventional probes in sensitivity and selectivity. Therefore, 1 was allowed to track the endogenous β-Gal in senescent A549 cells with a long sensing period (24 h). This work establishes a framework for designing multimodal optical probes through rational structural improvement, offering broad applicability in senescence research, diagnostic assay development, and therapeutic evaluation.