A New Ratiometric SERS Sensing Strategy Based on Molecular Dissociation Using Dual-Alkyne Reporters

Abstract Traditional responsive SERS probes rely on direct modification of Raman reporters, which suffer from poor specificity and weak anti-interference capability. Most existing designs are tailored for single analytes and lack general applicability. To address these methodological drawbacks, we propose a new ratiometric SERS sensing strategy based on molecular dissociation using dual-alkyne reporters. Two carbon−carbon triple bonds with distinct Raman shifts serve as signal units, delivering high signal-to-background ratios in the biological Raman silent region. A set of model compounds were used to explore the effects of electronic properties and steric hindrance on self-immolative elimination. This strategy realizes signal transduction via self-immolative-elimination-based molecular dissociation. Analyte-induced dissociation releases one alkyne-labelled fragment from the Au-bound molecular scaffold, resulting in differential spatial confinement of the two alkyne reporters within the SERS-active environment and consequently differential attenuation of their Raman signals. Quantitative detection based on signal intensity ratios minimizes signal fluctuations arising from probe concentration and nanoparticle aggregation. Hypochlorous acid was selected as a model analyte to verify the performance of the probe HYP. The sensor exhibits a limit of detection of 0.83 μM, and excellent selectivity, pH stability and biocompatibility for in situ detection and imaging of HClO in living cells. This modular design can be readily adapted to various analytes by replacing the recognition moiety. It establishes a generalizable paradigm for oxidation-responsive SERS analysis and provides a universal route for developing advanced alkyne-based Raman probes.