Ultrasensitive bioorthogonal probes for selective discrimination of trace H₂S₂/H₂S _n in sulfide-competitive contexts

Hydrogen persulfide/polysulfides (H₂S₂/H₂S _n ), as an oxidized derivative of hydrogen sulfide (H₂S), is capable of directly inducing the S-persulfidation of cysteine residues, thereby modulating the activity of relevant enzymes. Owing to its unique reactive properties, H₂S₂/H₂S _n is emerging as a central focus in the study of reactive sulfur species. Therefore, the precise detection of H₂S₂/H₂S _n in vivo is critical for elucidating their roles in redox signaling and cellular regulation. However, conventional probes face challenges such as poor sensitivity, cross-reactivity, and instability. Here, we report a bioorthogonal ether linkage fluorescent probe toolkit (Cyne-1‒5) with a cyclooctyne warhead, enabling ultra-sensitive (LOD = 3.3 nmol/L), selective, and real-time tracking of H₂S₂/H₂S _n in living systems. These probes feature rapid activation (>1018-fold fluorescence activation in 5 min), broad spectral coverage (blue to NIR), and exceptional enzymatic stability. Using this toolkit, we uncovered the spontaneous oxidation of H₂S to trace H₂S₂/H₂S _n and demonstrated steric hindrance-driven self-disproportionation of persulfides, where less bulky persulfides efficiently yield H₂S₂/H₂S _n . Furthermore, we achieved the cellular-level visualization of protein S-persulfidation dynamics. This work advances persulfide chemical biology and offers transformative tools for probing H₂S₂/H₂S _n in disease mechanisms and therapeutic development.