Carbon-dot-based ratiometric fluorescent probe for imaging and biosensing of superoxide anion in live cells

In this article, a ratiometric fluorescent biosensor for O₂ ^•- was developed, by employing carbon dots (C-Dots) as the reference fluorophore and hydroethidine (HE), a specific organic molecule toward O₂^•-, playing the role as both specific recognition element and response signal. The hybrid fluorescent probe CD-HE only emitted at 525 nm is ascribed to C-Dots, while HE was almost nonfluorescent, upon excitation at 488 nm. However, after reaction with O₂ ^•-, a new emission peak ascribed to the reaction products of HE and O₂^•- was clearly observed at 610 nm. Meanwhile, this peak gradually increased with the increasing concentration of O ₂^•- but the emission peak at 525 nm stayed constant, leading to a ratiometric detection of O₂^•-. The inorganic-organic fluorescent sensor exhibited high sensitivity, a broad dynamic linear range of ∼5 × 10^-7-1.4 × 10^-4 M, and low detection limit down to 100 nM. The present probe also showed high accuracy and excellent selectivity for O₂^•- over other reactive oxygen species (ROS), metal ions, and so on. Moreover, the C-Dot-based inorganic-organic probe demonstrated long-term stability against pH changes and continuous light illumination, good cell-permeability, and low cytotoxicity. Accordingly, the developed fluorescent biosensor was eventually applied for intracellular bioimaging and biosensing of O₂ ^•- changes upon oxidative stress.