Fluorescence-based detection of thiols in vitro and in vivo using dithiol probes

Anal Biochem. 2006 May 15;352(2):265-73. doi: 10.1016/j.ab.2006.01.047. Epub 2006 Feb 20.


Thiols play a central role in maintaining biological homeostasis. Their levels can change dramatically in response to oxidative stress associated with toxic insults, bacterial infection, and disease. Therefore, a reagent that can monitor thiol levels both in vitro and in vivo would be useful for assays and as a biomarker. Such a reagent should (i) be selective for thiols, (ii) be able to penetrate cell walls, and (iii) have a low reduction potential so as not to create oxidative stress in a cell. We have developed such a fluorescent reagent (DSSA) based on a dithiol linker: (i) the use of a dithiol linker makes it selective for thiols; (ii) the use of fluorophores that populate neutral states at physiological pH improves cell wall penetration; and (iii) because of the reagent's low reduction potential (-0.60 V), it will not stress cells oxidatively. For example, 5 microM of reagent is responsive to changes in glutathione levels in the physiologically relevant range of 1 to 10mM, yet this would oxidize less than 1% of cellular glutathione. In Escherichia coli, decreased thiol levels were detected in cells deficient in glutathione synthesis. In zebrafish embryos, the DSSA reagent permitted detection of unusually high thiol levels in the zebrafish chorion.

Publication types

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

MeSH terms

  • Animals
  • Cell Proliferation / drug effects
  • Disulfides / chemical synthesis
  • Disulfides / chemistry*
  • Disulfides / pharmacokinetics
  • Escherichia coli / drug effects
  • Fluorescein / chemistry
  • Fluorescence
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / pharmacokinetics
  • Hydrogen-Ion Concentration
  • In Vitro Techniques
  • Molecular Structure
  • Rhodamines / chemistry
  • Sulfhydryl Compounds / analysis*
  • Sulfhydryl Compounds / chemistry*
  • Time Factors
  • Tissue Distribution
  • Zebrafish / embryology
  • Zebrafish / growth & development


  • Disulfides
  • Fluorescent Dyes
  • Rhodamines
  • Sulfhydryl Compounds
  • Fluorescein