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

Hydrogen persulfide/polysulfides (H 2 S 2 /H 2 S n ), as an oxidized derivative of hydrogen sulfide (H 2 S), is capable of directly inducing the S -persulfidation of cysteine residues, thereby modulating the activity of relevant enzymes. Owing to its unique reactive properties, H 2 S 2 /H 2 S n is emerging as a central focus in the study of reactive sulfur species. Therefore, the precise detection of H 2 S 2 /H 2 S 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 H 2 S 2 /H 2 S 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 H 2 S to trace H 2 S 2 /H 2 S n and demonstrated steric hindrance-driven self-disproportionation of persulfides, where less bulky persulfides efficiently yield H 2 S 2 /H 2 S 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 H 2 S 2 /H 2 S n in disease mechanisms and therapeutic development. A bioorthogonal cyclooctyne-ether fluorescent toolkit (Cyne-1‒5) achieves selective, rapid imaging of H 2 S 2 /H 2 S n and protein persulfidation in living systems, resolving long-standing RSS detection challenges.