Optimizing the synthesis of red-emissive nitrogen-doped carbon dots for use in bioimaging

Synthesizing highly efficient red-emissive carbon dots (CDs) is a challenge that still impedes widespread applications of CDs in bioimaging. Herein, we demonstrate a facile, isolation-free synthesis of deep red (600-700 nm) emissive nitrogen-doped CDs (nCDs) based on microwave-assisted pyrolysis of citric acid and ethylenediamine. The duration of pyrolysis, the molar ratio of acid to amine, and the concentration of reactants were optimized using Central Composite Design and Response Surface Methodology to yield deep red fluorescence. We demonstrated their applicability on three different cell lines (retinal epithelial, lens epithelial, and Chinese hamster ovary cells). We measured the viability, the generation of reactive oxygen species, and percentage of apoptotic cells to determine their level of toxicity in cell culture. Confocal images showed that the nCDs fluoresced at different wavelengths depending upon the excitation wavelength and were excitable up to 635 nm. Furthermore, the ex vivo imaging of porcine ocular globes and post-mortem imaging of a whole mouse exemplified the utility of nCDs.