Fluorescent probes for in vitro and in vivo quantification of hydrogen peroxide

Hydrogen peroxide (H₂O₂) plays essential roles in redox signaling and oxidative stress, and its dynamic concentration is critical to human health and diseases. Here we report the design, syntheses, and biological applications of HKPerox-Red and HKPerox-Ratio for quantitative measurement of H₂O₂. Both probes were successfully applied to detect endogenous H₂O₂ fluxes in living cells or zebrafish, and biological effects of multiple stress inducers including rotenone, arsenic trioxide, and starvation were investigated. As H₂O₂ is a common by-product for oxidase oxidation, a general assay was developed for ultrasensitive detection of various metabolites (glucose, uric acid, and sarcosine). Moreover, cellular H₂O₂ measurements were achieved for the first time by combining flow cytometry with live cell calibration. This study provides a pair of unique molecular tools for advanced H₂O₂ bio-imaging and assay development.