Dihydroxy-substituted coumarins as fluorescent probes for nanomolar-level detection of the 4-amino-TEMPO spin label

Krzysztof Żamojć , Magdalena Zdrowowicz-Żamojć , Aleksandra Hać , Maciej Witwicki , Paweł Rudnicki-Velasquez , Dariusz Wyrzykowski , Wiesław Wiczk , Lech Chmurzyński


This paper reports on dihydroxycoumarins as fluorescent probes suitable for the detection and determination of the nitroxide radical, namely 4-amino-TEMPO. Since 4-amino-TEMPO is used as a spin label for the detection of various radicals and damage caused by these species, its determination under physiological conditions might help us to understand the mechanism of the oxidative stress. Among different coumarins studied, only dihydroxy-substituted derivatives show high sensitivity, specificity, and selectivity for the nitroxide radical. In this assay, dihydroxy-substituted coumarins under the action of 4-amino-TEMPO show a very fast and significant increase in fluorescence intensity and lifetime. Among them 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement (up to 40 times), with an estimated limit of detection equal to 16.7 nM—a significantly lower value when compared with UV-Vis or electron paramagnetic resonance (EPR) spectroscopy. The method is characterized by an easy procedure of sample preparation and very short time of analysis. The mechanism of the interaction between 6,7-dihydroxycoumarin and 4-amino-TEMPO has been examined with the use of a series of complementary techniques, such as steady-state and time-resolved fluorescence spectroscopy, UV-Vis spectroscopy, electron paramagnetic resonance spectroscopy, potentiometric titration, and high-performance liquid chromatography. It has been proven that the only route of the reaction in the system studied is a proton transfer from the molecule of esculetin to the amino group of the nitroxide. Biological studies performed on prostate cancer cells, breast cancer cells, and normal skin fibroblasts revealed significant anticancer properties of 6,7-dihydroxycoumarin, which caused a considerable decrease in the viability and number of cancer cells, and affected their morphology, contrary to normal fibroblasts. Furthermore, the experiment performed on prostate cancer cells showed that fluorescence emission of esculetin is closely related to intracellular pH—the higher pH, the higher observed fluorescence intensity (in accordance with a chemical experiment). On the other hand, the studies performed in different pH levels revealed that when pH of the solution increases, the observed fluorescence intensity enhancement under the action of 4-amino-TEMPO decreases (better sensing properties of esculetin towards the nitroxide in lower pH).
Author Krzysztof Żamojć (FCh/DGICh/LPCC)
Krzysztof Żamojć,,
- Laboratory of Physicochemistry of Coordination Complexes
, Magdalena Zdrowowicz-Żamojć (FCh/DPCh/LBS)
Magdalena Zdrowowicz-Żamojć,,
- Laboratory of Biological Sensitizers
, Aleksandra Hać (FB/DMBG)
Aleksandra Hać,,
- Katedra Biologii i Genetyki Medycznej
, Maciej Witwicki
Maciej Witwicki,,
, Paweł Rudnicki-Velasquez (FCh/DPCh/LLR)
Paweł Rudnicki-Velasquez,,
- Laboratory of Luminescence Research
, Dariusz Wyrzykowski (FCh/DGICh/PBChN)
Dariusz Wyrzykowski,,
- Pracownia Biologicznej Chemii Nieorganicznej
, Wiesław Wiczk (FCh/DBCh/LP)
Wiesław Wiczk,,
- Laboratory of Photobiophysics
, Lech Chmurzyński (FCh/DGICh/LPCC)
Lech Chmurzyński,,
- Laboratory of Physicochemistry of Coordination Complexes
Journal seriesInternational Journal of Molecular Sciences, ISSN 1422-0067, (N/A 140 pkt)
Issue year2019
Publication size in sheets0.80
Article number3802
Keywords in Englishnitroxide radicals, dihydroxycoumarins, fluorescence, optical chemical sensors
ASJC Classification2700 General Medicine; 1312 Molecular Biology; 1503 Catalysis; 1604 Inorganic Chemistry; 1605 Organic Chemistry; 1606 Physical and Theoretical Chemistry; 1607 Spectroscopy; 1706 Computer Science Applications
URL https://doi.org/10.3390/ijms20153802
Languageen angielski
LicenseJournal (articles only); published final; Uznanie Autorstwa (CC-BY); with publication
Score (nominal)140
Score sourcejournalList
ScoreMinisterial score = 140.0, 08-06-2020, ArticleFromJournal
Publication indicators WoS Citations = 1.000; Scopus SNIP (Source Normalised Impact per Paper): 2018 = 1.224; WoS Impact Factor: 2018 = 4.183 (2) - 2018=4.331 (5)
Citation count*1 (2020-07-03)
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