A color-tailored fluorogenic sensor for hydrogen peroxide.

HO 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 HO 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 HO. HyPerFLEX enables imaging HO 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 HO concentrations, such as during early glucose-stimulated insulin production, and can measure HO 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.