A Two-Photon Ratiometric Fluorescent Probe for Imaging of Hydrogen Peroxide Levels in Rat Organ Tissues

Hydrogen peroxide (H₂O₂) is important in the regulation of a variety of biological processes and is involved in various diseases. Quantitative measurement of H₂O₂ levels at the subcellular level is important for understanding its positive and negative effects on biological processes. Herein, a two-photon ratiometric fluorescent probe (SHP-Cyto) with a boronate-based carbamate leaving group as the H₂O₂ reactive trigger and 6-(benzo[d]thiazol-2'-yl)-2-(N,N-dimethylamino) naphthalene (BTDAN) as the fluorophore was synthesized and examined for its ability to detect cytosolic H₂O₂ in situ. This probe, based on the specific reaction between boronate and H₂O₂, displayed a fluorescent color change (455 to 528 nm) in response to H₂O₂ in the presence of diverse reactive oxygen species in a physiological medium. In addition, ratiometric two-photon microscopy (TPM) images with SHP-Cyto revealed that H₂O₂ levels gradually increased from brain to kidney, skin, heart, lung, and then liver tissues. SHP-Cyto was successfully applied to the imaging of endogenously produced cytosolic H₂O₂ levels in live cells and various rat organs by using TPM.