A fluorescent/colorimetric/electrochemical tri-modal sensing platform for cross-validated dynamic monitoring of endogenous glutathione in plants under stress

Glutathione (GSH) serves as a core cellular redox regulator for plant abiotic-stress responses. Reliable quantification of endogenous plant GSH remains challenging owing to complex matrix interferences, as well as dependencies on specialized instrumentation or transgenic plant materials. Herein, we design and synthesize RFNSP, a resorufin-based molecular probe functionalized with a 2-fluoro-4-nitrobenzenesulfonyl ester recognition moiety, and further construct a fluorescence-colorimetric-electrochemical tri-modal sensing platform for cross-validated GSH quantification in plant samples. Upon reaction with GSH, RFNSP undergoes a two-step sequential activation pathway to generate the conjugated adduct Re-GSH, simultaneously triggering three independent signal outputs; this kinetic feature enables effective discrimination of GSH against cysteine and homocysteine. A graphitic carbon nitride/single-walled carbon nanotube modified carbon-paper electrode (CP/SWCNTs/g-C3N4) is adopted as the electrochemical sensing interface. Benefitting from complementary analytical performances, the platform achieves ratiometric fluorescence detection with a limit of detection (LOD) of 0.5 μM (0.01-1.5 mM), visual colorimetric readout, and electrochemical measurement reaching an LOD of 0.1 μM (2-200 μM). We established chemically tuned Arabidopsis thaliana leaf extracts featuring varied endogenous GSH levels, including cadmium-triggered GSH up-regulation, buthionine-sulfoximine-induced GSH depletion, and N-acetylcysteine-driven GSH restoration. Consistent quantitative results were acquired across all three sensing channels. Free from transgenic materials, confocal microscopy and commercial assay kits, this cross-validated multimodal platform provides a practical analytical tool to advance plant stress-redox biological studies.