Sequence-Activated Fluorescent Probe with Heteroatom Modulation Enables Dual Channel Detection of H₂S and ONOO

Hydrogen sulfide (H₂S) and peroxynitrite (ONOO^-) are critical redox biomarkers whose dysregulated fluctuations are closely associated with oxidative stress and related diseases. Because their interplay depends strongly on their relative levels and dynamic changes, the sequential, simultaneous, and distinguishable detection of these two species is highly important, yet it remains challenging. Herein, we employed a heteroatom substitution strategy to modulate the optical properties and sensing behavior of the probe, thereby developing a sequence-activated fluorescent probe, B_SNS, for dual-channel detection of H₂S and ONOO^-. B_SNS exhibits high selectivity and sensitivity toward both analytes, with rapid responses within 5 min, low detection limits of 15 nM for H₂S and 3.2 nM for ONOO^-, and well-separated emission peaks that minimize spectral overlap. These features enable the visualization of exogenous H₂S and ONOO^- in HeLa cells and dynamic monitoring of both species under oxidative stress in a representative cancer model (MC38 cells). Therefore, B_SNS provides a reliable tool for investigating redox biology and holds strong potential for elucidating disease mechanisms and evaluating therapeutic strategies associated with oxidative stress.