A novel ESIPT-based ratiometric fluorescent probe for selective detection and imaging of hydrazine hydrate in vivo

Hydrazine hydrate (N₂H₄·H₂O), a widely used yet highly toxic industrial reductant, demands rapid and precise detection methods to mitigate environmental and health hazards. Conventional fluorescent probes suffer from small Stokes shifts (<100 nm), causing self-absorption artifacts and limited sensitivity. Herein, a novel ESIPT-based fluorescent probe BBCN was engineered by integrating a cyanomethyl benzoate recognition unit into an HBT scaffold. The α-cyanide-substituted alkene enabled selective N₂H₄ detection through a nucleophilic addition-elimination mechanism. BBCN achieved a remarkable Stokes shift (205 nm) and dual-channel emission (556 nm/645 nm) to circumvent self-absorption and single-channel limitations. BBCN also exhibited ultrafast response (2 min), exceptional selectivity among competing species, and high sensitivity with a detection limit of 0.18 μM. In vitro and in vivo imaging validated BBCN's capability to dynamically visualize hydrazine fluctuations, demonstrating its potential for biomedical applications. This work provided a robust tool for real-time, in situ hydrazine tracking with enhanced accuracy and practicality.