Spin trapping electron paramagnetic resonance (EPR) method is a method that combines the spin-trapping technique with the EPR technique to detect short-lived free radicals.
Why Use Spin Trapping Technology?
Free radicals are atoms or groups with unpaired electrons formed by the covalent bonding of compound molecules under external conditions such as heat and light. They are widely found in nature.
With the development of interdisciplinary disciplines such as biology, chemistry, and medicine, scientists have found that many diseases are associated with free radicals. However, due to their active and reactive nature, the free radicals generated in the reactions are often unstable at room temperature and difficult to be detected directly using conventional EPR spectroscopy methods.
Although short-lived free radicals can be studied by time-resolved EPR techniques or low-temperature fast-freezing techniques, their lower concentrations for most free radicals in biological systems limit the implementation of the above techniques. The spin trapping technique, on the other hand, allows the detection of short-lived free radicals at room temperature through an indirect method.
Fundamentals of Spin Trapping Technology
In a spin-trapping experiment, a spin trap (an unsaturated antimagnetic substance capable of trapping free radicals) is added to the system. After adding the spin trap, the unstable radicals and the trap will form more stable or longer-lived spin adducts. By detecting the EPR spectra of the spin adducts and processing and analyzing the data, we can invert the type of radicals and thus indirectly detect the unstable free radicals.
Figure 1 Principle of spin capture technique (DMPO as an example)
Selection of Spin Trap
The most widely used spin traps are mainly nitrone or nitroso compounds, typical spin traps are MNP (2-methyl-2-nitrosopropane dimer), PBN (N-tert-butyl α-phenyl nitrone), DMPO (5,5-dimethyl-1-pyrroline-N-oxide), and the structures are shown in Figure 2. And an excellent spin trap needs to satisfy three conditions.
1. Spin adducts formed by spin traps with unstable free radicals should be stable in nature and long-lived.
2. The EPR spectra of spin adducts formed by spin traps and various unstable radicals should be easily distinguishable and identifiable.
3. Spin trap is easy to react specifically with a variety of free radicals, and there is no side reaction. Based on the above conditions, the spin trap widely used in various industries is DMPO.
Figure 2 Schematic chemical structure of MNP, PBN, DMPO
Table 1 Comparison of common spin traps
Common Types of Spin-trapping Free Radicals
In spin trapping experiments, the most common ones are O- and N-centered radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), but not all ROS and RNS are free radicals, as shown in Figure 3. Besides, S-center radicals, such as sulfate radicals, are also more common free radicals that can be studied by spin-trapping methods.
Figure 3 Common ROS and RNS
For spin trapping experiments, many factors, such as the time of trapping agent addition, the concentration of trapping agent, solvent type, and pH of the system, can affect the experimental results. Therefore, for different free radicals, it is necessary to choose the spin trap type and concentration, design the experimental scheme, and finally determine the free radical type by EPR spectroscopy, and also eliminate the free radicals by chemical means to verify the results.
Figure 4 EPR spectra of O-(a), N-(b), S-(c) center radicals captured by DMPO.
(Data tested by CIQTEK EPR equipment)
CIQTEK EPR (ESR) Spectrometer
CIQTEK EPR (ESR) spectrometer with in-situ optical stimulation system, high/low-temperature system and the electrochemical system can be used to study various types of paramagnetic molecules, such as free radicals, metal complexes, doped materials, defective materials, metalloproteins, etc., which can meet the demand of multi-scene free radical detection.
X-band continuous wave electron paramagnetic resonance spectrometer EPR200-Plus
Benchtop electronic paramagnetic resonance spectrometer EPR200M
X-band pulsed electron paramagnetic resonance spectrometer EPR100