# Deep-NIR to NIR-II hemicyanine fluorophore scaffolds with dual optically tunable sites for in vivo multiplexed imaging

## TL;DR

Researchers developed new deep-NIR to NIR-II fluorophores with dual optical sites for improved in vivo multiplexed imaging in cancer models.

## Contribution

The first deep-NIR to NIR-II hemicyanine fluorophore scaffolds with dual optically tunable sites for dual-locked probe development are introduced.

## Key findings

- GL dyes emit wavelengths between 800–950 nm with meso-Cl and hydroxyl/amino groups for reactive site conversion.
- GL-Cys probe enables in vivo dual-channel imaging to differentiate cysteine levels in cancer and tumor ferroptosis models.
- The GL scaffold offers a versatile design for multiplexed imaging of various analytes.

## Abstract

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 meso-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 in vivo 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.

We have described a library of innovative deep-NIR to NIR-II hemicyanine fluorophore scaffolds (Guilin, GL) with dual optically tunable sites for dual-locked probe development.