The novel red-fluorescent probe DAR-4M measures reactive nitrogen species rather than NO

Zsombor Lacza, Eszter M. Horváth, Eszter Pankotai, Attila Csordás, Márk Kollai, Csaba Szabó, David W. Busija

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


Introduction: Several fluorescent probes were designed for the measurement of nitric oxide (NO), however, questions arose regarding their specificity and sensitivity in biological samples. In the present study we tested the reaction of a novel rhodamine-based chromophore diaminorhodamine-4M (DAR-4M) with NO and other reactive nitrogen and oxygen species. Methods: We performed fluorometry in 96-well plates in a cell-free buffer with similar ion concentrations as the cytoplasm. Dose-response curves were generated using various NO donors and reactive nitrogen and oxygen species. The effects were compared between the red-fluorescent DAR-4M and its green-fluorescent counterpart 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM). Results: DAR-4M had a markedly higher fluorescence yield to NO donors than DAF-FM, while both probes had a comparable threshold of sensitivity (in the range of 0.1 mM nitroprusside). Both dyes reacted with various NO donors in a dose-dependent manner, while superoxide, hydrogen peroxide, peroxynitrite, or nitroxyl failed to change the fluorescence intensity of the probes. DAR-4M was potentiated in the presence of peroxynitrite to react with low levels of NO donors in a similar manner to DAF-FM. Discussion: We conclude that DAR-4M is a suitable red-fluorescent probe for the qualitative assessment of reactive nitrogen species production, but not specific for NO. Quantitative comparisons among samples is inappropriate since the fluorescent yield is affected by the presence of other oxidants in the sample.

Original languageEnglish (US)
Pages (from-to)335-340
Number of pages6
JournalJournal of Pharmacological and Toxicological Methods
Issue number3
StatePublished - Nov 2005
Externally publishedYes


  • Angeli's salt
  • DAF-2
  • DAF-FM
  • DAR-4M
  • Diaminofluorescein
  • Diaminorhodamine
  • Methods
  • Nitric oxide
  • Peroxynitrite
  • Superoxide

ASJC Scopus subject areas

  • Toxicology
  • Pharmacology


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