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A High Precision Method for Quantitative Measurements of Reactive Oxygen Species in Frozen Biopsies
Norwegian University of Science and Technology, Trondheim, Norway.
Umeå University, Sweden.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Norwegian University of Science and Technology, Trondheim, Norway.
Linköping University, Department of Medical and Health Sciences, Division of Radiological Sciences. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Biomedical Engineering.
2014 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 3Article in journal (Refereed) Published
Abstract [en]


An electron paramagnetic resonance (EPR) technique using the spin probe cyclic hydroxylamine 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetr​amethylpyrrolidine(CMH) was introduced as a versatile method for high precision quantification of reactive oxygen species, including the superoxide radical in frozen biological samples such as cell suspensions, blood or biopsies.

Materials and Methods

Loss of measurement precision and accuracy due to variations in sample size and shape were minimized by assembling the sample in a well-defined volume. Measurement was carried out at low temperature (150 K) using a nitrogen flow Dewar. The signal intensity was measured from the EPR 1st derivative amplitude, and related to a sample, 3-carboxy-proxyl (CP•) with known spin concentration.


The absolute spin concentration could be quantified with a precision and accuracy better than ±10 µM (k = 1). The spin concentration of samples stored at −80°C could be reproduced after 6 months of storage well within the same error estimate.


The absolute spin concentration in wet biological samples such as biopsies, water solutions and cell cultures could be quantified with higher precision and accuracy than normally achievable using common techniques such as flat cells, tissue cells and various capillary tubes. In addition; biological samples could be collected and stored for future incubation with spin probe, and also further stored up to at least six months before EPR analysis, without loss of signal intensity. This opens for the possibility to store and transport incubated biological samples with known accuracy of the spin concentration over time.

Place, publisher, year, edition, pages
San Francisco: Public Library of Science , 2014. Vol. 9, no 3
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-105262DOI: 10.1371/journal.pone.0090964ISI: 000332483600116PubMedID: 24603936OAI: diva2:705058
Available from: 2014-03-14 Created: 2014-03-14 Last updated: 2015-06-01Bibliographically approved

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Lindgren, MikaelGustafsson, Håkan
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ChemistryThe Institute of TechnologyDivision of Radiological SciencesFaculty of Health SciencesDepartment of Biomedical Engineering
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