Page 176 - Environmental Nanotechnology Applications and Impacts of Nanomaterials

Reactive Oxygen Species Generation on Nanoparticulate Material 161

Figure 5.2 Reaction of TEMP with singlet oxygen.

The spin-trapping molecules affect the signal of the unpaired electron
on the free radical species that is detected; this effect is known as split-
ting and is most commonly caused by adjacent hydrogen atoms. Often
the splitting leads to a unique pattern for the EPR signals. The combi-
nation of the hyperfine structure (number of lines), line shape, and
hyperfine splitting (the distance between peaks) give a radical its unique
imprint. An ideal spin-trapping molecule will react quickly and specif-
ically with the radical species of interest and produce a characteristic
signal. Two spin traps, which are most frequently used for oxygen rad-
ical detection, are 5,5-dimethyl-1-pyrolline-N-oxide (DMPO) and 2,2,6,6-
tetramethylpiperidine-N-oxyl (TEMP/TEMPO). Figures 5.2, 5.3, and 5.4
give the reaction of DMPO with superoxide and hydroxyl radical and
TEMP with singlet oxygen.
In each case, the product of the reaction is a nitroxide compound,
which is stabilized by charge delocalization between the nitrogen and
oxygen atom. Figures 5.5, 5.6, and 5.7 illustrate typical EPR spectra for
TEMPO, DMPO-OOH, and DMPO-OH.
The TEMPO spectrum is a 1:1:1 hyperfine structure that results from
the interaction of the unpaired electron with the nitrogen nucleus. Both
DMPO-OH and DMPO-OOH have the same interaction, but splitting
occurs due to the presence of adjacent hydrogen and oxygen atoms.
Hydroxyl radical reacts with DMPO about nine orders of magnitude faster
than superoxide, so the DMPO-OH signal will predominate unless
hydroxyl radical is quenched (vide infra). As a result, superoxide detection
with DMPO requires much higher concentrations than hydroxyl detec-
tion would. DMPO-OOH can also decompose to DMPO-OH giving a false
positive for hydroxy radical, but there are ways to avoid this [4, 5]. Many
other spin traps are available, and new ones are developed on a regular
basis.
Another common option for detection of reactive oxygen is chemical
reduction. Two examples are Cytochrome c [6, 7] or nitroblue tetrazolium

Figure 5.3 Reaction of DMPO
with superoxide.