WDS'13 Proceedings of Contributed Papers, Part II, 149–153, 2013.
ISBN 978-80-7378-251-1 © MATFYZPRESS
Bioapplication of Plasma Jet Fed by Argon and Argon/O2 Gas Mixture
L. Moravský, M. Klas and Š. Matejčík
Department of Experimental Physics, Comenius University, Mlynská dolina F-2, 842 48 Bratislava, Slovakia.
E. Machová
Institute of Chemistry, Slovak Academy of Sciences, Dubravská cesta 9, 845 38 Bratislava, Slovakia.
Abstract. The atmospheric pressure plasma jet has been widely used for biomedical
applications in recent years. One of the most important applications is the inactivation
of microorganisms and yeast biofilms. This work describes the physical properties of
a self-designed kilohertz-driven plasma jet operated in Ar and Ar-O2 gas mixture at
gas flows as low as 100 sccm. This plasma jet has been applied for inactivation of
cells of Candida albicans which were in suspension in phosphate buffered saline.
Plasma properties have been studied using optical emission spectroscopy (OES).
Yeast viability was evaluated by crafting assay XTT.
Introduction
Plasma can be categorized in high temperature or low temperature plasmas. In low temperature
plasmas of noble gases (such as argon and helium) and chemically active gases (e.g. oxygen and
nitrogen) are commonly used. Non-thermal plasmas may offer new biomedical applications such as
bio-decontamination or sterilization of heat sensitive materials [Hippler et al., 2008; Fricke et al.,
2012]. Inactivation of micro-organisms has been extensively studied over the past decade. Gaseous
nonthermal plasma has unique characteristics because it contains numerous biochemically active
agents like UV photons, OH radicals, O atoms, electronically and vibrationally excited molecules, etc.
[Boudam et al., 2006; Dobrynin et al., 2009; Akishev et al., 2008]. Candidiasis, caused by Candida
species, is the most common fungal infection in humans. Candida albicans is considered as the most
prevalent fungal biofilm-forming pathogen which causes lifethreatening infections by colonizing
polymers used for medical devices [Boudam et al., 2006; Harriott et al., 2009;. Concia et al., 2000;
Arendrup, 2010; Niermann et al., 2010; Wang et al., 2010]. The gas flow rates were predominantly
more than 1000 sccm for biological applications [Fricke et al., 2012; Alkawareek et al., 2012; Zelaya
et al., 2010; Jiang Ch. et al., 2012]. Our work focuses on the application of low cost plasma pen for
decontamination yeast of Candida albicans and to study the parameters of the plasma beam.
Experimental apparatus
A schematic view of the non-equilibrium plasma jet is shown in Figure 1. Electric discharge was
ignited in flowing argon and argon-oxygen mixture in a hollow needle-to-cylinder electrode
configuration. The inner electrode was made of stainless steel and an outer electrode of brass. The kHz
high voltage (HV) power supply consisted of an oscillator, a high voltage transformer and an acoustic
amplifier Behringer EP 2000. The plasma jet was operated with pure argon (purity 99,996%) and a gas
mixture of 1% oxygen (purity 99,995%) and 99% argon, at a total flow rate 100 sccm. Gas flow rate
was set by MKS flow controllers. Electrical discharge parameters were measured using oscilloscope
(Agilent technologies DSO 1012A), connected to high-voltage (Tektronix P6015A) and current
(Rogowski ring) probes. The power supply delivered sinusoidal HV power with amplitudes
of 7 kVpk-pk at operating frequencies around 12 kHz. The discharge was created in a glass capillary
with inner and outer diameter 0.5 mm and 1 mm respectively, and then released to air in a form of
torch-like post-discharge. Optical properties and composition of the plasma jet were studied by optical
emission spectroscopy (using spectrometer Jobin-Yvon H 25 coupled with CCD multichannel detector
Alphalas CCD-S2300-UV). Optical fiber from spectrometer was placed at a fixed distance from the
plasma jet exhaust (1 mm). Samples of the yeast C. albicans were prepared in the form of suspension
in phosphate buffered saline which was delivered in plastic wells shown in Figure 3. The plasma beam
was directed perpendicular to the bottom of the wells. Distance between the beam and surface of
liquid suspension of cells was constant, d = 9 mm. The number of analyzed wells was 196 for both gas
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Figure 1. 1: HV generator, 2: electrode/capillary holder, 3: hollow needle HV electrode, 4: glass
capillary, 5: grounded ring electrode made of copper wire covering the capillary, 6: plasma jet, 7:
bottle of argon and oxygen and MKS flow controllers, 8: quartz optical fiber, 9: OES spectrometer
(Jobin-Yvon H 25) coupled with CCD multichannel detector (Alphalas CCD-S2300-UV).
compositions. The volume of the suspension was 30 μl in each well. Period of exposure of yeast varied
from 10 to 150 seconds for each sample at room temperature (300 K) and atmospheric pressure.
Results and discussion
The optical spectra of the plasma jet are shown in Figures 2a and 2b. The most intensive emission
lines of the postdischarge were the argon (Ar I) emission (4p→4s) lines in the spectral region 690–
860 nm, the 2nd positive system N2 emission bands (C3Πu→B3Πg 300–440 nm) and the transitions of
the OH band (A2Σ+→X2Π Δv = 0) between 306–309 nm (Figures 2a and 2b). The ionic argon lines (Ar
II) between 410–650 nm, the emission bands of N2 first positive system (transition B3Πg→ A3Σu+)
furthermore N2 first negative system (B2Σu+→X2Σg+) and atomic oxygen (O I), which are located at
777.4 and 844 nm, respectively, were not observed. The electron temperature of the plasma jet was not
sufficient to excite N2+(B) excited states.
The detected OH band between 306–309 nm indicates the presence of the water in the afterglow.
We assume that the source of the water is the ambient air, which penetrates by the diffusion into the
working gas.
Preparation of samples
After preparation of the C. albicans samples and their treatment as described in experimental
section, the viability of treated samples was studied using the XTT assay test [Hansen, M.B. et al.,
(1989); Jost L.M. et al., (1992); Roehm, N.W. et al., (1991); Scudiero, P.A. et al., (1988); Tada, H. et
al., (1986); Weislow, O.S. et al., (1989)]. The Figure 3 represents the visible results after exposure and
XTT assay test. Both plastic plates had 6 untreated (1, 2, 5, 6, 9, 10) and 6 treated (3, 4, 7, 8, 11, 12)
columns (Numbering from left to right). The results shown in Figure 4 have been calculated using
calibration curve, which was given by measuring the absorbance at the wavelength 490 nm across the
suspension. The calibration curve was used only for the region between 106 and 108 cells/ml because
under the 106 cells/ml the absorbance of the sample is weak. The values of absorption after the
irradiation with plasma for more than 60 seconds were less than the values in the calibration curve.
The concentration of living cells was less than 106 cells/ml for both measurements. Results (shown in
Figure 5) detected by XTT assay showed that cells in suspension were inactivated by plasma jet.
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Figure 2a. Optical emission spectra of N2 2nd pos. sys. and OH bands in gas mixture Ar + 1 % O2.
The line at 418 nm is an artefact.
Figure 2b. Typical optical emission spectra of argon plasma
Figure 3. Samples of adherent cells in plastic wells. The red-like cells represents the living reference
cells and the other orange and yellow were treated. (The treating time was 10, 30, 60, 90, 120, 150
seconds, in Ar-O2 99:1).
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MORAVSKÝ ET AL.: BIOAPPLICATION OF PLASMA JET FED BY Ar AND Ar/O2
Figure 4. Calibration curve — absorbance of XTT versus number of C. albicans cells, in 30 μl of
suspension.
Figure 5. The number of living cells after the plasma treatment in different gas mixture.
Conclusion
The obtained experimental results show that the application of plasma pen fed by argon and
argon-oxygen gas mixture generated at atmospheric pressure is effective for decontamination of yeast
Candida albicans cells in phosphate buffered saline. If the treating time is 60 s or higher, then the
number of living cells decreased two orders of magnitude. The discharge is full of low-temperature
plasma of excited particles and radicals that cause inactivation of yeast cells of Candida albicans.
Unfortunately, the viability method used in present work, based on XTT assay test, did not allowed to
detect C. albicans bellow 106 cell in 1 ml. The upcoming plans will be changing the gas composition
of operating gas.
Acknowledgments. This work was supported by Slovak Research and Development Agency APVV-0733-11 and UK
grant UK/195/2013.
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