Genetically encoded fluorescent indicator for intracellular hydrogen peroxide

Nat Methods. 2006 Apr;3(4):281-6. doi: 10.1038/nmeth866.


We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins / pharmacology
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Biosensing Techniques / methods*
  • Cytoplasm / metabolism
  • DNA-Binding Proteins / metabolism
  • Escherichia coli Proteins / metabolism
  • Genetic Code
  • HeLa Cells / metabolism
  • HeLa Cells / ultrastructure
  • Humans
  • Hydrogen Peroxide / analysis*
  • Hydrogen Peroxide / metabolism
  • Intracellular Membranes / metabolism*
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics
  • Mitochondria / metabolism
  • Nerve Growth Factors / pharmacology
  • PC12 Cells / metabolism
  • PC12 Cells / ultrastructure
  • Prokaryotic Cells / metabolism
  • Rats
  • Repressor Proteins / metabolism
  • Sensitivity and Specificity
  • Time Factors
  • Transcription Factors / metabolism


  • Apoptosis Regulatory Proteins
  • Bacterial Proteins
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Luminescent Proteins
  • Nerve Growth Factors
  • Repressor Proteins
  • Transcription Factors
  • oxyR protein, E coli
  • yellow fluorescent protein, Bacteria
  • Hydrogen Peroxide