In situ measurement of superoxide and hydroxyl radicals by frequency mixing detection technique.

Frequency mixing magnetic detection (FMMD) was used to detect superoxide from hypoxanthine and xanthine reaction and to detect hydroxyl radical from the Fenton reaction. FMMD was also applied to measure the reactive oxygen species (ROS) level released from microglial cells. We could assess the formation and extinction of the free radicals without a spin trap reagent. The FMMD signal amplitude scaled with the concentration of the radicals. It was verified that no signals are obtained from the substrates and reagents. Based on the observations and on previous research, we suggest that the FMMD signals originate from superoxide and hydroxyl radicals, indicating that FMMD can be used to detect O-centered radicals. Subsequent analysis of free radicals generated from living microglial cells showed that there were significant differences between the activated microglial cells and resting ones. The results of this research are promising regarding the applications of FMMD for in situ measurement of free radicals from various sources, including the cell.