Detection of free radicals
and reactive
oxygen/nitrogen species
Methods
• Direct assay
• Indirect assay
• Chemical and physical method
• Biochemical methods
Electron spin resonance, electron
paramagnetic resonance (ESR, EPR)
• Method for directly measuring of free radicals
• ESR was developed in the late 1940s
• ESR uses paramagnetism of free radicals
• Sample is given in magnetic field generated by magnets and
exposed to microwave radiation
• After absorbing energy, unpaired electrons are excited, they
chang spin and may jump from the ground state to a higher
energy state
ESR spectrum
• Unpaired electron can move between the two energy levels by
either absorbing or emitting of energy
• Absorption and emision is monitored and converted into a
spectrum
Electron spin resonance
spectrometer
sample cavity
Spin trapping
• Spin trapping uses compounds called spin-traps - 5,5-dimethyl-1pyrroline-N-oxide
(DMPO), 2,2,6,6-tetramethylpiperidine-1-oxyl
(TEMPO)
• Spin-trap reacts rapidly with the free radical to form radicaladducts
that are more stable and can be detected
• Spin-traps have been used in experimental animals to
demostrate the production of free radical in vivo
• Toxicity of spin-traps
Plant, Cell and Environment (2004), 27, 1415–1423
Gunera magellanica
DMPO
Ascorbyl
radical
G. magellanica
Lohan B.S. et al.: Determination of the
Antioxidant Status of the Skin by In VivoElectron
Paramagnetic Resonance (EPR)
Spectroscopy. Cosmetics 2015, 2, 286-301
Junwang G. et al.: New Developed Cylindrical TM010 Mode EPR Cavity for Xband
In Vivo Tooth Dosimetry. PLoS ONE 2014, 9, 1-8
Jackson S. K. at al.: In vivo EPR spectroscopy: biomedical and potential diagnostic applications.
Faraday Discuss. 2004, 126, 103-117
Mikuni T. at al.: In vivo Detection of Gastric Cancer in Rats by Electron Paramagnetic Resonance
Imaging. Cancer research 2004, 64, 6495– 6502.
High-Performance Liquid
Chromatographic (HPLC) detection of
free radicals
OH.
OH.
OH.
High Performance Liquid Chromatography (HPLC)
enzym
tyrosine
Electrochemical detection of ROS/RNS
• Electrochemical methods study an analyte by measuring the
potential or current in a solution by electrodes
• Real-time detection using free radical biosensors for measuring
nitric oxide, hydrogen peroxide, hydroxyl radical
https://www.youtube.com/watch?v=6GzNh
0XRfq8
Chemiluminescence measurement for
ROS/RNS detection
• Chemiluminescence is the emission of light as the result of a
chemical reaction of free radical with specific compounds
• This emitted light can be measured with the help of a
luminometer
• Luminol is widely used in free radical detection
ROS/RNS
endoperoxide
Fluorescence measurement for free radical
detection
fluorescence microscopyluminometer
• Fluorescence is the emission of light by a substance that absorbed
light
• In most cases, the emitted light has a longer wavelength, and
therefore lower energy, than the absorbed radiation
Free Rad Biol Med (2010), 48, 983-1001
dihydroethidium 2-hydroxyethidium
Fluorescence measurement for free radical
detection
fluorescence microscopy
control cells cells with oxidative stress
Dichlorofluorescin diacetate
Dichlorofluorescin
Dichlorofluorescein
Indirect methods of ROS/RNS detection
• Monitoring of the presence of lipid peroxidation products
– Detection of malondialdehyde by thiobarbituric acid using
spectrophotometer (532 nm wavelength)
thiobarbituric acid
Indirect methods of ROS/RNS detection
• Monitoring of the presence of lipid peroxidation products
– Detection of malondialdehyde by thiobarbituric acid using
spectrophotometer (532 nm wavelength)
thiobarbituric acid
- Detection of conjugated dienes using spectrophotometer
(220-300 nm wavelength)
alkanes
- Breath alkanes (pentane, ethane) as an index of lipid
peroxidation (detection by gas chromatography)
• Monitoring of markers for oxidative damage of proteins
- Detection damaged proteins by 2,4-dinitrophenylhydrazine
using spectrophotometer (360–390 nm wavelength)
• Monitoring of markers for oxidative damage of NA
- Detection of 8-hydroxy-2-deoxyguanosine by HPLC with
electrochemical detection as a marker of damaged DNA