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

SIMPLE SUMMARY: Spatially and temporally defined H(2)O(2) signatures are essential parts of various signaling pathways. Therefore, monitoring H(2)O(2) dynamics with high spatio–temporal resolution is significantly important to understand how this ubiquitous signaling molecule can control diverse cellular responses. In this study, we designed and characterized a Fluorescence Resonance Energy Transfer (FRET)-based genetically encoded H(2)O(2) sensor that provides a powerful tool to monitor the spatio–temporal dynamics of H(2)O(2) fluxes. We have used this sensor to monitor the flux of H(2)O(2) in live cells under stress conditions. Using this sensor, real-time information of the H(2)O(2) level can be obtained non-invasively and would help to understand the adverse effect of H(2)O(2) on cell physiology and its role in redox signaling. ABSTRACT: Hydrogen peroxide (H(2)O(2)) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H(2)O(2) increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H(2)O(2) also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H(2)O(2) in environmental responses and its role in cellular signaling, there is a need to study the flux of H(2)O(2) at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H(2)O(2)) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H(2)O(2), and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H(2)O(2) 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 H(2)O(2) was measured in these live cells using the FLIP-H(2)O(2) under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H(2)O(2). Using this sensor, real-time information of the H(2)O(2) level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H(2)O(2) on cell physiology and its role in redox signaling.