Scope and limitations of the TEMPO/EPR method for singlet oxygen detection: the misleading role of electron transfer |
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| Institution: | 1. Instituto de Tecnología Química UPV-CSIC, Universitat Politècnica de València, 46022 Valencia, Spain;2. Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, 40129 Bologna, Italy;1. Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Department of Dermatology, Venerology and Allergology, Center of Experimental and Applied Cutaneous Physiology, Charitéplatz 1, 10117 Berlin, Germany;2. Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany;3. Skin Barrier Research Group, Faculty of Pharmacy, Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic;4. Department of Physics, Institute of Experimental Physics, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany;5. University of British Columbia, Faculty of Pharmaceutical Sciences, 2405 Wesbrook Mall, V6T1Z3 Vancouver, Canada;1. Key Laboratory for Colloid & Interface Chemistry of Education Ministry, Department of Chemistry, Shandong University, Jinan 250100, PR China;2. Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, PR China;1. Research and Development Division, Iwasaki Electric Co., Ltd., 1-1 Ichiriyma-chyo, Gyoda City, Saitama 361-8505, Japan;2. Faculty of Engineering, Tokai University, Kanagawa, 4-1-1 Kitakaname, Hirastuka City, Kanagawa 259-1292, Japan |
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| Abstract: | For many biological and biomedical studies, it is essential to detect the production of 1O2 and quantify its production yield. Among the available methods, detection of the characteristic 1270-nm phosphorescence of singlet oxygen by time-resolved near-infrared (TRNIR) emission constitutes the most direct and unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in combination with a singlet oxygen probe. This is based on the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6-tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has been widely employed, it can produce misleading data. This is demonstrated by the present study, in which the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the TEMPO/EPR method are compared for a set of well-known photosensitizers. The results reveal that the TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet excited state of the photosensitizer is efficiently quenched by TEMP, acting as electron donor. In such case, generation of the TEMP+ radical cation, followed by deprotonation and reaction with molecular oxygen, gives rise to an EPR-detectable TEMPO signal that is not associated with singlet oxygen production. This knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in chemical, biological, and medical studies. |
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| Keywords: | EPR Photosensitizer Singlet oxygen TEMPO Time-resolved near-infrared emission Free radicals |
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