One probe, two chemistries: an orthogonal fluorescent sensing platform for glutathione and hydrazine in biological...

Simultaneous detection of glutathione (GSH) and hydrazine (N₂H₄) is crucial due to their opposing roles in redox biology, GSH as a key intracellular antioxidant and hydrazine as a toxic nucleophilic pollutant and enzymatic byproduct. We report a pyrimidine-based D-π-A fluorophore (compound 1) that enables selective and differential detection of both analytes through distinct optical and mechanistic pathways. The probe exhibits strong absorption at 570 nm with an emission band at 622 nm, which undergoes significant turn-off fluorescence upon analyte binding (∼5-fold for GSH and ∼22-fold for N₂H₄). These differences arise from unique interaction mechanisms: GSH forms a thioether via rapid Michael addition, while hydrazine forms a hydrazone-type adduct via slower nucleophilic substitution. Distinct absorption shifts (280 nm for GSH vs. 350 nm for N₂H₄), fluorescence lifetimes (τ = 0.00783 ns for GSH vs. 0.01293 ns for N₂H₄), and response kinetics (instantaneous vs. 20 min saturation) further validate orthogonal detection. The limits of detection were 0.87 µM (GSH) and 0.23 µM (N₂H₄). The probe demonstrated practical applicability in diluted urine and serum, and successfully monitored aminoacylase-1 (ACY-1) activity via in situ hydrazine release. Also, the on-field analysis of hydrazine was achieved on a wide range of substrates, including paper strips, glass slides, natural rubber latex etc. Also, the presence of residual hydrazine in food sample wash was determined by a simple dip-stick method. These features establish this system as a robust, ratiometric tool for biomedical diagnostics, and enzymatic activity sensing.