st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
Quantitative detection of plasma-generated free radicals in liquids by electron
paramagnetic resonance spectroscopy
Helena Tresp1,2, Malte U. Hammer1,2, Klaus-Dieter Weltmann2, Stephan Reuter1,2
1
2
Centre for Innovation Competence Plasmatis, Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany
2Leibniz Institute for Plasma Science and Technology INP Greifswald e.V., Felix-Hausdorff-Str. 2, 17489 Greifswald,
Germany
Abstract: This work focused on qualitative and quantitative detection of oxygen free radicals
in liquids after plasma treatment with an atmospheric pressure argon plasma jet by electron
paramagnetic resonance spectroscopy (EPR). For the treatment, a shielded plasma jet, where
the active effluent zone is surrounded by a protective gas curtain was used.
Keywords: plasma medicine, EPR, ESR, radicals, plasma-liquid-interaction, ROS, •OH, O2•-
1. Introduction
In the field of plasma medicine the important role of
liquid analysis was revealed in the last couple of years
[1, 2]. Especially the plasma generated free radicals have
a large effect on the chemical formation of reactive
species, which have themselves a large impact on
mammalian systems [3]. For instance nitric oxide (•NO) is
a major signaling molecule in biology [4], hydroxyl
radicals (•OH) and superoxide anion radicals (O2•-) are
well known for triggering oxidative stress response of
cells [5]. In the present work, these oxygen radicals were
investigated by the complex technique of electron
paramagnetic resonance (EPR) spectroscopy, also called
electron spin resonance (ESR) spectroscopy. To detect
these radicals in liquids at room temperature with EPR,
the radicals need to be stabilized by the use of chemical
agents called spin trap. This technique was developed
1968 by Janzen and Blackbur [6]. The spin trap forms a
more stable adduct with the radical which remains its
radical character so that it can be detected by EPR.
2. Methods and Materials
A shielded atmospheric pressure argon plasma jet
(kinpen, neoplas GmbH, Germany) with a feed gas flow
rate of 3 standard liters per minute (slm) argon (purity
99.999%) and as shielding gas pure oxygen (purity
99.995%) with a gas flow rate of 5 slm were used to treat
a volume of 5 mL liquid placed in a 60 mm Petri dish in
an meandered pattern (Fig. 1) for 3 minutes [7]. The used
liquids were Dulbecco’s phosphate buffered solution
(DPBS), Rosewell Park Memorial Institute (RPMI) cell
culture media, and sodium chloride solution (0.85%
NaCl). All were treated using the same parameter set
meaning same treatment time, same volume, same time
gap between treatment and measurement.
The used spin trap 5,5,-dimethyl-1-pyrroline-N-oxide
(Dojindo Laboratoire Kumamoto, Japan) was dissolved
directly in the investigated solution (100 mM) and
measured once before treatment. For the radical detection
an X-band EPR (EMXmicro, Bruker BioSpin GmbH,
Germany) was used. Detailed information about the
working procedure is described in a previous publication
[8].
Fig. 1 Experimental setup for the shielded plasma jet treatment
of liquid in a 60 mm diameter Petri dish.
3. Results and Discussion
In Fig. 2 the EPR spectra of plasma treated DPBS is
shown. The Landé factor and the hyperfine coupling were
determined for the peaks of the measured spectra,
compared with database to identify to the radicals. These
parameters were utilized to simulate the spectra for each
detected radical respectively and for the combination of
the radicals for an absolute measurement of its
concentration in the liquid. The peak assignment of the
measured peaks of plasma treated DPBS is shown in
Fig.2.
The four larger peaks with an intensity ratio of 1:2:2:1
marked with a purple star are related to the hydroxyl
radical, the small peaks near to the ones of •OH marked
with a blue triangle represent the superoxide anion
radical.
st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
measured spectra
simulated spectra of DMPO•/OH
simulated spectra of DMPO•/OOH
simulated spectra
0.002 Tesla
Fig. 2 EPR spectra of plasma treated Dulbecco’s phosphate
buffered solution. The measured spectra, the simulated
DMPO•/OH, the simulated DMPO•/OOH, and the simulation of
the measured spectra, which consists of the combination of the
DMPO•/OH and DMPO•/OOH spectra, are shown.
The use of DMPO as spin trap has some disadvantages,
one is the fact that the reaction rate of 5,5-dimethyl-1pyrroline-N-oxide with superoxide anion radicals (Eq.1)
is much shorter than its reaction rate with hydroxyl
radicals (Eq.2) [9].
kO2•- < 102 M-1s-1
k•OH > 109 M-1s-1
(Eq.1)
(Eq.2)
Another drawback is the short half-life of the
DMPO•/OOH of around one minute and that the spin
adduct of the superoxide anion radical can react to the
more stable spin trap adduct of DMPO•/OH [9]. So the
determined hydroxyl radical concentration could originate
also from the superoxide radical, not only from the •OH.
Fig. 3 EPR spectra after 3 minutes treatment time of different
biological relevant solutions, a) 0.85% sodium chloride solution,
b) Dulbecco’s phosphate buffered solution, and c) Rosewell
Park Memorial Institute cell culture medium.
As can be seen in Fig. 3 the complexity of the liquid
play a non-negligible role for the radicals which are
generated by plasma treatment of the liquid.
Sodium chloride solution shows only the hydroxyl
radical peaks (Fig. 3a). In the spectra of 3 minutes plasma
treated Dulbecco’s phosphate buffered solution absorption
peaks of the hydroxyl radical and the super oxide anion
radical can be detected (Fig. 3b). The EPR spectrum of
plasma treated RPMI (Fig. 3c) shows like NaCl only the
peaks of the •OH radical.
By comparison of the determined radical concentrations
it becomes clear that the highest amount of radicals were
produced in DPBS, less in RPMI and the smallest amount
in NaCl solution (Eq.3,4).
[radical]DPBS > [radical]RPMI > [radical]NaCl
5.8µM > 3.6 µM > 2.3 µM
(Eq.3)
(Eq.4)
A comparison of the ingredients of the investigated
solutions shows that NaCl solution is the less complex
liquid because it contains only mainly sodium chloride
and water, DPBS has two additional possible sources of
oxygen or hydroxyl (the sodium phosphate dibasic
(Na2HPO4) and monopotassium phosphate (KH2PO4)).
RPMI also is comprised of sodium phosphate dibasic
(Na2HPO4) in about the same amount as DPBS. RPMI
also has radical scavenging ingredients such as amino
acids and vitamins. This explains the differences in
detected radicals after plasma treatment, because the
treatment itself was the same for all treatments and only
the type of liquid changes. Similarly it can be assumed for
the presence of the superoxide anion radical in treated
DPBS. As was pointed out above it can be also the case
that detected DMPO•/OH concentration originated partly
or completely from superoxide anion radicals. A not
visible DMPO•/OOH peak pattern does not directly
indicate that no O2•- in the liquid.
4. Conclusion
Biological relevant liquids, which are not only the
complex ones like cell culture media, also easier ones for
instance phosphate buffered solution and sodium chloride
solution were investigated concerning their behavior in
radical generation by plasma treatment with an
atmospheric pressure argon plasma jet. The outcome of
this study is that the complexity is important. The formed
radical concentration is dependent from the ingredients
but it could not said neither that as easier the solution as
bigger is the amount of produced radicals nor that a more
complex a solution formed higher concentrations of
radicals caused by the possible sources and scavengers for
the radicals.
st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
5. References
[1] Oehmigen K, Hahnel M, Brandenburg R, et al.
Plasma Processes and Polymers 2010 7 250-257
[2] Oehmigen K, Winter J, Hähnel M, et al. Plasma
Processes and Polymers 8 904-913
[3] Reuter S, Tresp H, Wende K, et al. IEEE Transactions
on Plasma Science, Special Issue on Plasma
Medicine 2012
[4] Beckman J S and Koppenol W H American Journal
of Physiology 1996 271 C1424-37
[5] Aruoma O I Journal of the American Oil Chemists
Society 1998 75 199-212
[6] Janzen E G and Blackbur.Bj Journal of the American
Chemical Society 1968 90 5909-&
[7] Reuter S, Winter J, Schmidt-Bleker A, et al. IEEE
Transactions on Plasma Science; Special Issue on
Atmospheric Pressure Plasma Jet Applications 2012
[8] Tresp H, Hammer M U, Weltmann K-D, et al. Journal
of Physics D: Applied Physics 2013
[9] Halliwell B and Gutteridge J M C Free Radicals in
Biology and Medicine: Oxford University Press,
2007
6. Acknowledgment
This work is funded by German Federal Ministry of
Education a Research (BMBF) (grant number
03Z2DN12).