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.
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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.
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