A color-tailored fluorogenic sensor for hydrogen peroxide

H₂O₂ is an important signaling molecule and redox regulator of normal cellular metabolism and a major element of oxidative stress. Here we report HyPerFLEX (HyPer with flexible fluorogen excitation), a sensor from the HyPer family designed for high-precision H₂O₂ monitoring in living cells. HyPerFLEX combines the redox-sensitive OxyR domain from Neisseria meningitidis and circularly permuted fluorogenic protein Y-FAST, yielding oxygen-independent fluorescence upon oxidation of OxyR by H₂O₂. HyPerFLEX enables imaging H₂O₂ dynamics in living cells, with tunable spectra from green to far red for multicompartment imaging, even under prolonged hypoxia. It surpasses HyPer7 in detecting ultralow H₂O₂ concentrations, such as during early glucose-stimulated insulin production, and can measure H₂O₂ levels in the highly oxidizing endoplasmic reticulum lumen. These advanced features and broad compatibility make HyPerFLEX a powerful tool for studying oxidative stress and cellular signaling.