Deep-NIR to NIR-II hemicyanine fluorophore scaffolds with dual optically tunable sites for <i>in vivo</i> multiplexed imaging

Dual-locked probes are highly promising for use in medical diagnosis and drug screening due to their enhanced specificity and multiplex detection capabilities. However, most near-infrared (NIR) fluorophores used for dual-locked systems exhibit spectral activity in the blue portion (650-800 nm) of the NIR window, substantially restricting their application in deep-tissue imaging. Herein, for the first time, we report a novel class of deep-NIR to NIR-II hemicyanine fluorophore (Guilin, GL) scaffolds with dual optically tunable sites for dual-locked probe development. These GL dyes, obtained through chloride-substituted cyanine/hemicyanine hybridization, exhibit emission wavelengths spanning 800-950 nm and contain critical <i>meso</i>-Cl and hydroxyl/amino groups, which can be readily converted into two distinct reactive sites for multiplexed imaging analysis. To demonstrate the applicability of GL dyes, we engineered a novel dual-locked probe GL-Cys, which exhibited enhanced emission at 830 nm at low Cys levels and a distinct 765 nm signal elevation at elevated concentrations. More importantly, the GL-Cys probe enables effective differentiation of Cys levels in pancreatic/breast cancer models and corresponding tumor ferroptosis models through <i>in vivo</i> dual-channel ratiometric imaging. Thus, this molecular design paradigm establishes a versatile deep-NIR to NIR-II hemicyanine scaffold that may be generalized for multiplexed imaging of other analytes.