Synthesis and application of a ratiometric probe for hydrogen peroxide

Ruwen Yin, Yuan Fang, Xinqi Zhou, Cliff I. Stains

Research output: Chapter in Book/Report/Conference proceedingChapter


Molecular imaging of biological analytes provides detailed insights into signaling processes. Ratiometric probes are particularly attractive due to the ability to quantify analyte production. However, design strategies for ratiometric probes can be hindered by spectral overlap of the product and reactant species. In this chapter, we provide protocols for the synthesis and application of RF620, a ratiometric probe for H2O2 displaying dramatic changes in both excitation and emission wavelengths, designed using an approach we term chemoselective alteration of fluorophore scaffolds. The probe contains a chemoselective functional group within a fluorescent xanthene scaffold, resulting in the in situ synthesis of a new fluorophore upon reaction with H2O2. Under physiological conditions, RF620 exhibits far-red to near-infrared excitation and emission, and upon reaction with H2O2, RF620 is chemically converted into tetramethylrhodamine, producing a significant (~ 66 nm) blue-shift in excitation and emission. RF620 can be used for ratiometric, molecular imaging of endogenous H2O2 production in living cells.

Original languageEnglish (US)
Title of host publicationChemical Tools for Imaging, Manipulating, and Tracking Biological Systems
Subtitle of host publicationDiverse Methods for Optical Imaging and Conjugation
PublisherAcademic Press Inc.
Number of pages14
StatePublished - 2020

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988


  • Cell signaling
  • Fluorescent probe
  • Hydrogen peroxide
  • Ratiometric probe
  • Reactive oxygen species

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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