Single Fluorescent Probe Responds to H₂O₂, NO, and H₂O₂/NO with Three Different Sets of Fluorescence Signals

Hydrogen peroxide (H2O2) acts as a signaling molecule in a wide variety of signaling transduction processes and an oxidative stress marker in aging and disease. However, excessive H2O2 production is implicated with various diseases. Nitric oxide (NO) serves as a secondary messenger inducing vascular smooth muscle relaxation. However, mis-regulation of NO production is associated with various disorders. To disentangle the complicated inter-relationship between H2O2 and NO in the signal transduction and oxidative pathways, fluorescent reporters that are able to display distinct signals to H2O2, NO, and H2O2/NO are highly valuable. Herein, we present the rational design, synthesis, spectral properties, and living cell imaging studies of FP-H2O2-NO, the first single-fluorescent molecule, that can respond to H2O2, NO, and H2O2/NO with three different sets of fluorescence signals. FP-H2O2-NO senses H2O2, NO, and H2O2/NO with a fluorescence signal pattern of blue–black–black, black–black–red, and black–red–red, respectively. Significantly, we have further demonstrated that FP-H2O2-NO, a single fluorescent probe, is capable of simultaneously monitoring endogenously produced NO and H2O2 in living macrophage cells in multicolor imaging. We envision that FP-H2O2-NO will be a unique molecular tool to investigate the interplaying roles of H2O2 and NO in the complex interaction networks of the signal transduction and oxidative pathways. In addition, this work establishes a robust strategy for monitoring the multiple ROS and RNS species (H2O2, NO, and H2O2/NO) using a single fluorescent probe, and the modularity of the strategy may allow it to be extended for other types of biomolecules.