Local Generation and Imaging of Hydrogen Peroxide in Living Cells

Curr Protoc Chem Biol. 2017 Jun 19;9(2):117-127. doi: 10.1002/cpch.20.


Described here is a localized H2 O2 generation-detection system consisting of a yeast D-amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H2 O2 dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H2 O2 generated by the activated DAAO. The system can be easily optimized for various combinations of cell types, conditions and DAAO/sensor subcellular localizations. © 2017 by John Wiley & Sons, Inc.

Keywords: D-amino acid oxidase; HyPer; HyPer2; HyPerRed; biosensors; hydrogen peroxide; metabolic engineering; reactive oxygen species.

MeSH terms

  • Amino Acid Oxidoreductases / metabolism
  • Biocatalysis
  • Biosensing Techniques / methods*
  • Cell Survival
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Hydrogen Peroxide / metabolism*
  • Intracellular Space / metabolism
  • Molecular Imaging / methods*
  • Rhodotorula / enzymology


  • Hydrogen Peroxide
  • Amino Acid Oxidoreductases