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New noncellular fluorescence microplate screening assay for scavenging activity against singlet oxygen

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Abstract

In the present study, a new fluorescence microplate screening assay for evaluating scavenging activity against singlet oxygen (1O2) was implemented. The chemical generation of 1O2 was promoted using the thermodissociable endoperoxide of disodium 3,3′-(1,4-naphthalene)bispropionate (NDPO2). The detection of 1O2 was achieved using dihydrorhodamine 123 (DHR), a nonfluorescent molecule that is oxidizable to the fluorescent form rhodamine 123 (RH). The combined use of a 1O2-selective generator and a highly sensitive probe (DHR) was then successfully applied to perform a screening assay of the 1O2 scavenging activities of ascorbic acid, penicillamine, cysteine, N-acetylcysteine (NAC), methionine, reduced glutathione (GSH), dihydrolipoic acid, lipoic acid, and sodium azide. All of these antioxidants exhibited concentration-dependent 1O2 scavenging capacities. They could be ranked according to observed activity: ascorbic acid> cysteine> penicillamine> dihydrolipoic acid > GSH> NAC> sodium azide> lipoic acid (IC50 values of 3.0 ± 0.2, 8.0 ± 0.7, 10.9 ± 0.8, 25.2 ± 4.5, 57.4 ± 5.9, 138 ± 13, 1124 ± 128, 2775 ± 359 μM, mean±SEM, respectively) > methionine (35% of scavenging effect at 10 mM). In conclusion, the use of NDPO2 as a selective generator for 1O2 and its fluorescence detection by the highly sensitive probe DHR is shown to be a reliable and resourceful analytical alternative means to implement a microplate screening assay for scavenging activity against 1O2.

Generation and detection of singlet oxygen

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Acknowledgements

The authors acknowledge the financial support provided by FCT and FEDER in the ambit of the project POCI/QUI/59284/2004. David Costa acknowledges FCT and FSE his PhD grant (SFRH/BD/10483/2002).

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Authors and Affiliations

  1. REQUIMTE, Departamento de Química-Física, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030, Porto, Portugal

    David Costa, Eduarda Fernandes, João L. M. Santos & José L. F. C. Lima

  2. Departamento de Química, Universidade de Aveiro, 380-193, Aveiro, Portugal

    Diana C. G. A. Pinto & Artur M. S. Silva

Authors
  1. David Costa
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  2. Eduarda Fernandes
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  3. João L. M. Santos
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  4. Diana C. G. A. Pinto
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  5. Artur M. S. Silva
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  6. José L. F. C. Lima
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Correspondence to Eduarda Fernandes.

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Costa, D., Fernandes, E., Santos, J.L.M. et al. New noncellular fluorescence microplate screening assay for scavenging activity against singlet oxygen. Anal Bioanal Chem 387, 2071–2081 (2007). https://doi.org/10.1007/s00216-006-0998-9

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  • DOI: https://doi.org/10.1007/s00216-006-0998-9

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