Development of a Xanthene-Based Red-Emissive Fluorescent Probe for Visualizing H₂O₂ in Living Cells, Tissues and Animals

Hydrogen peroxide (H₂O₂) plays important roles in the regulation of many biological processes, and the abnormal level of H₂O₂ has close relation with the initiation and progression of many diseases. Herein, we describe a novel red-emissive fluorescence probe (RhoB) for the visualization of H₂O₂ in living cells, tissues and animals. RhoB was constructed on the basis of a xanthene-based red-emissive dye, and displayed nearly no fluorescence. After the treatment with H₂O₂, RhoB can exhibit red fluorescence with the emission wavelength at 638 nm. RhoB exhibited highly sensitive and selective response to H₂O₂. Density functional theory (DFT) calculations were conducted to shed light on the optical properties of RhoB, and natural bond orbital (NBO) calculations demonstrate that the boron atom shows the highest positive electricity and further support the response mechanism. RhoB was successfully applied for imaging of exogenous and endogenous H₂O₂ in living cells, and also can be utilized for visualizing H₂O₂ in living tissues and animals.