Cascade-activated fluorescent probe for temporal imaging of H₂S and microviscosity dynamics under inflammatory stress

Inflammation is a critical pathological process closely associated with redox imbalance, metabolic dysregulation, and microenvironment changes. Hydrogen sulfide (H₂S) and microviscosity are both involved in inflammation stress, but their temporal relationship remains unclear. Existing H₂S-viscosity dual-responsive probes mainly provide parallel readouts or are often limited by insufficient spectral separation. Herein, we developed a cascade-activated fluorescent probe PPAC-SNO₂ for sequential detection of H₂S and viscosity with a large spectral separation of 178 nm. Upon reaction with H₂S, PPAC-SNO₂ undergoes sulfonate cleavage to release PPAC-OH, producing red emission at 630 nm. Under high-viscosity conditions, the excited-state conformational evolution of PPAC-OH is restricted, generating blue-green emission at 452 nm. The large spectral separation minimizes channel crosstalk and enables accurate dual-channel imaging. In the lipopolysaccharide/aminooxyacetic acid group, where endogenous H₂S biosynthesis was inhibited, delayed viscosity elevation was still observed, indicating that viscosity elevation is associated with inflammatory stress. In summary, PPAC-SNO₂ enables dual-channel visualization of H₂S and viscosity with large spectral separation and low crosstalk, providing a useful tool for probing time-dependent chemical signaling and microenvironment change during inflammatory stress.