A ratiometric fluorescent probe with dual near infrared emission for in vivo ratio imaging of cysteine.

Accurately detecting cysteine (Cys) in vivo is crucial for diagnosing Cys-related diseases. A novel ratiometric fluorescent probe featuring dual near-infrared emission is developed in this study for the in vivo ratio imaging of Cys. The probe comprises a hemicyanine organic small-molecule dye (HCy-CYS) with specific Cys recognition capabilities covalently coupled with carbon dots (CDs) synthesized using glutathione (GSH) as the carbon source (GCDs), forming a unique composite nanofluorescent probe (GCDs@CYS). The probe undergoes a specific reaction with acrylate upon the addition of Cys, converting HCy-CYS to HCy-OH. Consequently, the GCD fluorescence intensity at 685 nm gradually decreases, whereas that of HCy-OH at 720 nm progressively increases, yielding a ratiometric fluorescence signal. Notably, both emission wavelengths of the probe exceed 650 nm, thereby effectively mitigating the interference from background signals during cellular and in vivo imaging. Furthermore, the probe demonstrates high specificity for Cys, enabling its differentiation from homocysteine and GSH. The Cys concentration and fluorescence ratiometric intensity exhibit a strong linear correlation at 10-150 μM with a detection limit of 0.95 μM. These results indicate that the ratiometric fluorescent probe can serve as a valuable tool for monitoring Cys-related physiological or pathological processes.