Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells

Hydrogen peroxide (H₂O₂) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H₂O₂ increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H₂O₂ also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H₂O₂ in environmental responses and its role in cellular signaling, there is a need to study the flux of H₂O₂ at high spatio-temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H₂O₂) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H₂O₂, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H₂O₂ demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H₂O₂ was measured in these live cells using the FLIP-H₂O₂ under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H₂O₂. Using this sensor, real-time information of the H₂O₂ level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H₂O₂ on cell physiology and its role in redox signaling.