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To detect and distinguish various ROS, we use Electron Spin Resonance (ESR), a very sensitive and the most definitive method to detect short-lived reactive oxygen intermediates. However, ESR itself is not sufficient for detecting short-lived species, such hydroxyl OH● and superoxide anion (O2●-) radicals or singlet oxygen (1Δg). Therefore, the spin-trapping technique has to be used to stabilize highly reactive free radicals.
Figure 1. ESR spectrometer, Bruker EleXsys, Model 500, equipped with 3 microwave bridges operating at 2.4, 9.5 and 34 GHz (left panel). Typical ESR signal of the DMPO-OH spin-adduct resulting from reactive scavenging of hydroxyl OH● radicals by DMPO in water (right panel).
It is well known that nano-crystalline TiO2 and ZnO generate ROS (hydroxyl OH● and superoxide anion O2●- radicals) at high yield in aqueous media under UV light illumination [1,2]. The nitrone spin trap DMPO reacts with hydroxyl (OH●) and superoxide anion (O2●-) radicals, thus yielding two different spin-adducts, DMPO-OH and DMPO-OOH, respectively (FIGURE 1 right panel). In contrast, in the presence of pristine C60 (in organic solvents) or highly water-soluble fullerols (in water solutions), singlet oxygen (1Δg) is usually formed (FIGURE 2) .
Figure 2. The custom-made water-soluble fullerol, C60(OH)19(ONa)17, generates singlet oxygen from the triplet one in aqueous media under illumination with visible light . We apply ESR spin-trapping for detection of ROS both in cell-free experiments in vitro [1-6] as well as in intra-cellular detection of ROS ex vivo .
ESR spin-trapping experiments set also the stage for another type of ex vivo study, where we apply AFM and fluorescence microscopy to monitor the deleterious action of ROS on living cells incubated in the presence nano-engineered materials. It is the combination of these various approaches, which enables us to gain insights in the photo-catalytic properties of various forms of nanoparticles and their potential toxicity .
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