A dual-locked near-infrared fluorescent probe based on ESIPT and FRET for improved discrimination between normal and cancer cells

Precise imaging identification of tumor tissues from normal tissues is still challenging. In this study, a new dual-locked near-infrared fluorescent probe BBQ650-SS-BTZHC-E was developed for the discrimination between cancer cells and normal cells. The probe consists of a hemicyanine fluorophore conjugated to an esterase substrate and a quencher connected via a disulfide bond. The esterase substrate blocks the excited state intramolecular proton transfer (ESIPT) process, while the quenching agent forms a Förster resonance energy transfer (FRET) pair with the fluorophore. The fluorescence activation is dependent on the presence of both esterase and glutathione (GSH), resulting in the "ESIPT on" and "FRET off" states, which represent an "AND" logic. The characterizations showed that the kinetics of fluorescence activation are dependent on the concentrations of the two activators with a high specificity. Fluorescent imaging experiments using BBQ650-SS-BTZHC-E successfully distinguished cancer cells (HeLa, 4T1, RAW264.7) from normal cells (HFL1) with high contrast and specificity, and imaged 4T1 tumor on live mice. This study provides a simple platform for the development of dual-locked fluorescent probes for bio-imaging applications.