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 O₂^•– 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