Dual-emissive carbonized polymer dots for the ratiometric fluorescence imaging of singlet oxygen in living cells

Singlet oxygen (1O2) is a type of reactive oxygen species (ROS), playing a vital role in the physiological and pathophysiological processes. Specific probes for monitoring intracellular 1O2 still remain challenging. In this study, we develop a ratiometric fluorescent probe for the real-time intracellular detection of 1O2 using o-phenylenediamine-derived carbonized polymer dots (o-PD CPDs). The o-PD CPDs possessing dual-excitation-emission properties (blue and yellow fluorescence) were successfully synthesized in a two-phase system (water/acetonitrile) using an ionic liquid tetrabutylammonium hexafluorophosphate as a supporting electrolyte through the electrolysis of o-PD. The o-PD CPDs can act as a photosensitizer to produce 1O2 upon white LED irradiation, in turn, the generated 1O2 selectively quenches the yellow emission of the o-PD CPDs. This quenching behavior is ascribed to the specific cycloaddition reaction between 1O2 and alkene groups in the polymer scaffolds on o-PD CPDs. The interior carbon core can be a reliable internal standard since its blue fluorescence intensity remains unchanged in the presence of 1O2. The ratiometric response of o-PD CPDs is selective toward 1O2 against other ROS species. The developed o-PD CPDs have been successfully applied to monitor the 1O2 level in the intracellular environment. Furthermore, in the inflammatory neutrophil cell model, o-PD CPDs can also detect the 1O2 and other ROS species such as hypochlorous acid after phorbol 12-myristate 13-acetate (PMA)-induced inflammation. Through the dual-channel fluorescence imaging, the ratiometric response of o-PD CPDs shows great potential for detecting endogenous and stimulating 1O2 in vivo.