Journal of Fluorescence

, Volume 26, Issue 3, pp 919–924 | Cite as

Use and Evaluation of Newly Synthesized Fluorescence Probes to Detect Generated OH• Radicals in Fibroblast Cells

  • Reza Salimi
  • Nilgün YenerEmail author
  • Roghaiyeh Safari


Reactive oxygen species (ROS) are pro-oxidant molecules synthesized in body with various functions and are essential for life. Increasing in reactive oxygen species or decreasing in antioxidants level cause oxidative stress which is very harmful. OH• radical is one of ROS’s, with tendency to bind to lipids, DNA and proteins which cause irreversible damage in cells. The most devastating consequences related to excess OH• radicals occur via direct binding to nucleic acids and proteins. Quantification of this high reactive radical with short life time is difficult. Electron Spin Resonance, Fluorescence, and Luminescence Spectroscopy are commonly used to determine the level of ROS. Fluorescence Probes have higher specificity and sensitivity with their excellent sensors to detect ROS’s compare to the other methods. Also, there are different probes specifically designed for each radical. The purpose of this study was to identify the probe better suiting for detection of OH• radical levels. The two most recommended fluorescence probes, 2-[6-(4 V-Hydroxy) phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and coumarin-3-carboxylic acid (3-CCA) to determine OH• radical levels were compared. Following the formation of OH• radical with Fenton reaction, HPF and 3-CCA probes were added to cells and spectrofluorometric measurements were performed in their respective wavelengths. The mean amplitude of fluorescence for HPF was 32.72 ± 2.37 F.I (n = 40) and for 3-CCA was 52.11 ± 0.5 F.I (n = 40). This difference was statistically significant. 3-CCA also demonstrated more stable measurements at different days compered to HPF.


Fluorescence Probes OH• radicals 2-[6-(4 V-Hydroxy) phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) Coumarin-3-carboxylic acid (3-CCA) 



Project was funded by Dokuz Eylul University Research Support Office with the project number: 2011-KB-SAG-065. We would like to thank Professor Semra Kocturk for her expert advices through this project.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest with the contents of this article.


  1. 1.
    Murrant CL, Reid MB (2001) Detection of reactive oxygen and reactive nitrogen species in skeletal muscle. Microsc Res Tech 55(4):236–248CrossRefPubMedGoogle Scholar
  2. 2.
    Halliwell B, Whiteman M (2004) Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol 142(2):231–255CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Fang YZ, Yang S, Wu G (2002) Free radicals, antioxidants, and nutrition. Nutrition 18(10):872–879CrossRefPubMedGoogle Scholar
  4. 4.
    Gomes A, Fernandes E, Lima JL (2005) Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods 65(2–3):45–80CrossRefPubMedGoogle Scholar
  5. 5.
    Bromme HJ et al (2008) DCFH2 interactions with hydroxyl radicals and other oxidants--influence of organic solvents. Exp Gerontol 43(7):638–644CrossRefPubMedGoogle Scholar
  6. 6.
    Ziech D et al (2010) The role of reactive oxygen species and oxidative stress in environmental carcinogenesis and biomarker development. Chem Biol Interact 188(2):334–339CrossRefPubMedGoogle Scholar
  7. 7.
    Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82(1):47–95CrossRefPubMedGoogle Scholar
  8. 8.
    Dickinson BC, Srikun D, Chang CJ (2010) Mitochondrial-targeted fluorescent probes for reactive oxygen species. Curr Opin Chem Biol 14(1):50–56CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Brandes RP, Janiszewski M (2005) Direct detection of reactive oxygen species ex vivo. Kidney Int 67(5):1662–1664CrossRefPubMedGoogle Scholar
  10. 10.
    Ou B et al (2002) Novel fluorometric assay for hydroxyl radical prevention capacity using fluorescein as the probe. J Agric Food Chem 50(10):2772–2777CrossRefPubMedGoogle Scholar
  11. 11.
    Filaire E, Toumi H (2012) Reactive oxygen species and exercise on bone metabolism: friend or enemy? Joint Bone Spine 79(4):341–346CrossRefPubMedGoogle Scholar
  12. 12.
    Bartosz G (2006) Use of spectroscopic probes for detection of reactive oxygen species. Clin Chim Acta 368(1–2):53–76CrossRefPubMedGoogle Scholar
  13. 13.
    Tai C et al (2002) A new simple and sensitive fluorometric method for the determination of hydroxyl radical and its application. Talanta 58(4):661–667CrossRefPubMedGoogle Scholar
  14. 14.
    Moore J, Yin JJ, Yu LL (2006) Novel fluorometric assay for hydroxyl radical scavenging capacity (HOSC) estimation. J Agric Food Chem 54(3):617–626CrossRefPubMedGoogle Scholar
  15. 15.
    Valko M et al (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39(1):44–84CrossRefPubMedGoogle Scholar
  16. 16.
    Makrigiorgos GM et al (1993) A method for detection of hydroxyl radicals in the vicinity of biomolecules using radiation-induced fluorescence of coumarin. Int J Radiat Biol 63(4):445–458CrossRefPubMedGoogle Scholar
  17. 17.
    Yang XF, Guo XQ (2001) Investigation of the anthracene-nitroxide hybrid molecule as a probe for hydroxyl radicals. Analyst 126(10):1800–1804CrossRefPubMedGoogle Scholar
  18. 18.
    Li P et al (2010) A new highly selective and sensitive assay for fluorescence imaging of *OH in living cells: effectively avoiding the interference of peroxynitrite. Chemistry 16(6):1834–1840CrossRefPubMedGoogle Scholar
  19. 19.
    Mythili Y et al (2004) Protective effect of DL-alpha-lipoic acid on cyclophosphamide induced oxidative cardiac injury. Chem Biol Interact 151(1):13–19CrossRefPubMedGoogle Scholar
  20. 20.
    Xiao H, Parkin KL (2002) Antioxidant functions of selected allium thiosulfinates and S-alk(en)yl-L-cysteine sulfoxides. J Agric Food Chem 50(9):2488–2493CrossRefPubMedGoogle Scholar
  21. 21.
    Soh N (2006) Recent advances in fluorescent probes for the detection of reactive oxygen species. Anal Bioanal Chem 386(3):532–543CrossRefPubMedGoogle Scholar
  22. 22.
    Wardman P (2007) Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radic Biol Med 43(7):995–1022CrossRefPubMedGoogle Scholar
  23. 23.
    Soh N et al (2008) Phospholipid-linked coumarin: a fluorescent probe for sensing hydroxyl radicals in lipid membranes. Anal Sci 24(2):293–296CrossRefPubMedGoogle Scholar
  24. 24.
    Indo HP et al (2007) Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage. Mitochondrion 7(1–2):106–118CrossRefPubMedGoogle Scholar
  25. 25.
    Setsukinai K et al (2003) Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 278(5):3170–3175CrossRefPubMedGoogle Scholar
  26. 26.
    Lemire JA, Harrison JJ, Turner RJ (2013) Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat Rev Microbiol 11(6):371–384CrossRefPubMedGoogle Scholar
  27. 27.
    Grankvist K et al (1979) Superoxide dismutase, catalase and scavengers of hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro. Biochem J 182(1):17–25CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Kaminski CF, Rees EJ, Schierle GS (2014) A quantitative protocol for intensity-based live cell FRET imaging. Methods Mol Biol 1076:445–454CrossRefPubMedGoogle Scholar
  29. 29.
    Manevich Y, Held KD, Biaglow JE (1997) Coumarin-3-carboxylic acid as a detector for hydroxyl radicals generated chemically and by gamma radiation. Radiat Res 148(6):580–591CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Molecular MedicineUniversity of Dokuz EylulIzmirTurkey
  2. 2.Department of Medical Biochemistry, Faculty of MedicineDokuz Eylul UniversityIzmirTurkey
  3. 3.Izmir Biomedicine and Genome centerUniversity of Dokuz EylulIzmirTurkey

Personalised recommendations