Rational Design of Hemicyanine Platform for Engineering High-Contrast Activatable NIR-II Probes

Activatable NIR-II hemicyanine probes are crucial tools for high-contrast deep-tissue biomarker imaging. However, their development is constrained by an inherent dilemma: extending π-conjugation to narrow the energy gap inevitably compromises the fluorescence brightness in the activated state (dim on), while the strong intramolecular charge transfer (ICT) effect makes effective quenching in the locked state difficult to achieve (leaky off), leading to poor response sensitivity and a low on/off ratio. Here, we rationally designed a xanthene-cyanine hybrid NIR-II activatable platform (RC-OH), wherein activated-state brightness was enhanced by strengthening the steric hindrance to suppress intermolecular aggregation (bright on), while locked-state quenching was promoted by amplifying nonradiative decay via the inductive effect of an ortho-chlorine atom on the phenolic hydroxyl group (tight off). Leveraging the dual-optimized RC3-OH platform, we constructed RC3-Cys, RC3-ALP, and RC3-ONOO probes and validated their high on/off ratio (up to 16-fold) and specific detection of cysteine (Cys), alkaline phosphatase (ALP) and peroxynitrite (ONOO-), respectively. These results highlight RC3-OH as a versatile NIR-II platform for constructing high-sensitivity, biomarker-activatable NIR-II probes.