Bio Applications and Plasma Medicine by the Atmospheric Microplasma
Kazuo Shimizu1, Shigeki Tatematsu2, Hodaka Fukunaga2 and Marius Blajan1
Organization for Innovation and Social Collaboration, Shizuoka University, Japan
2
Graduate School of Engineering, Shizuoka University, Japan
1
Abstract: Atmospheric microplasma has been intensively studied for various application fields, since this
technology has features shown here: generated around only 1 kV under atmospheric pressure, discharge gap of only
10 to 100um and dielectric barrier discharge. Low discharge voltage atmospheric plasma process is an economical
and effective solution for various applications such as sterilization, odor removal, surface modification and so on.
New possibility of the bio application of microplasma, so called “Plasma Medicine” will be introduced here.
Keywords:
1. Introduction
Microplasma studied in our group is atmospheric
pressure non thermal plasma which requires
relatively low discharge voltage, has possibility for
application in various fields e. g. sterilization, odor
removal, surface treatment [1]. Atmospheric
microplasma is a type of dielectric barrier discharge
thus does not require costly vacuum enclosures. The
discharge gap is set to an order of micrometers which
is extremely low, enabling the plasma to start at
discharge voltage of around 1 kV.
Most of the plasma sterilization methods are using
direct plasma method [2] and a number of studies
have been conducted on plasma jet [3]. Few attempts
have been made to use remote plasma sterilization
methods. Active species are generated between the
electrodes and exposed to bacteria. In this paper, the
sterilization effect by remote microplasma methods
with air was experimentally investigated.
2. Experimental Setup
Figure 1 shows the microplasma electrodes of
surface treatment. The electrodes used in this study
were stainless steel electrodes (thickness 1 mm,
aperture ratio 30%, discharge gap 100 µm) covered
with a dielectric material and faced together with a
spacer (thickness 100 µm, aperture area 100 µm) in
between. Due to small discharge gaps (0~100) µm, a
high intensity electric field (107~108 V/m) could be
obtained with relatively low discharge voltages
around 1 kV.
A neon transformer was used as an AC high voltage
power supply. The microplasma was generated by
applying the high voltage on electrodes. Process
gases were flowed into the reactor from the top and
flown through the holes of electrodes. The electrodes
were attached to a glass tube and inserted in to an
acrylic tube in order to keep a controlled gas
composition around the electrodes and target to be
sterilized. The radicals and ions generated by
microplasma collided with the object, which was E.
coli DH5α in this study.
An image of a streamer generated in the discharge
is shown in Figure 2 [4]. It was observed that the
streamer diameter was about 10 µm for 100 µm
discharge gap using as discharge gas a N 2/Ar mixture.
Discharge voltage was 1 kV and the streamer was
thinner than previously reported [5]. Streamers were
generated between the electrodes, which generate
various radicals and ions that could affect the bacteria
as a target. Emission spectrum of microplasma was
measured with an ICCD camera and a spectrometer.
To examine the effect of microplasma for
bacteriologic shape, the shape was observed by fieldemission-type SEM (scanning electron microscope).
The ESR (electron spin resonance analyzer) was used
to analyze the radicals dissolved in water by
microplasma discharge. By-products derived from
microplasma discharge was analyzed by FT-IR
(fourier transform infrared spectroscopy). Surface
potential of PEN (polyethylene naphthalate) film was
measured by the electrostatic potential monitor with
measuring probe.
Fig. 1. The image of microplasma electrodes for
sterilization. The targets were the various kinds of
bacteria.
Figure 4. Marx generator was used for microplasma
generation and discharge voltage was fixed to 1.1 kV.
Fig. 2. An image of light emission of Ar microplasma
discharge. Discharge gas: 1% N2 in Ar; discharge
voltage: 1 kV; discharge gap: 100 μm [4].
Gram-negative bacteria Escherichia coli DH5α were
the target to be sterilized in this study. E. coli was
cultured at 37°C, for 16 hours in the liquid medium
by incubator and applied on the agar medium. Then
the agar medium was put on the Z-axis stage in the
reactor. The distance between electrodes and the Petri
dish could be adjusted by changing the height of the
stage. Microplasma was generated between the
electrodes by the applied voltage thus active species
and radicals were produced according to each gas
composition. These active species and radicals
reached E. coli on the agar medium by process gas
flow. Finally, the agar medium were cultured again
thus it could be observed the results of the
sterilization process. This plasma process is called
remote plasma process.
3. Results and Discussion
Figure 3 shows the images of the sterilization of E.
coli by using atmospheric air microplasma.
(a) Air: Vd= 1.3 kV.
(b) Air:Vd= 1.5 kV.
Fig. 3. The sterilization result of E. coli by using air
microplasma. Treatment time; 60 s, distance from
electrodes; 2 mm.
Discharge voltage was adjusted between, 1.3 ~ 1.5
kV(Air). Gas flow rate was 10 L/min and the agar
medium was exposed at 2 mm distance from
electrodes. Exposure time was 60 s.
Sterilization area expanded with the increase of
applied voltage. Sterilization effect was observed
near the center of Petri dish by Ar microplasma
exposure. OH radical from Ar microplasma discharge
was observed by emission spectroscopy as shown in
Fig 4. An example of the emission spectrum of
excited OH radical by atmospheric microplasma. Gas
flow rate; 10 L/min, applied voltage; negative pulse
1.1 kV, frequency; 1 kHz [6].
Figure 5 shows the detected OH radical peaks
dissolved in the water which analyzed by the ESR
after the air microplasma exposure. Discharge
voltage was fixed to 1.3 kV, gas flow rate was 10
L/min and the water was exposed at 5 mm distance
from electrodes. Exposure time was fixed to 90 s. In
this analysis, spin trap agent 5,5-dymethyl-1pyrroline N-oxide(DMPO) was used due to the shortlived radical of OH radical in liquid phase. The four
peaks shown in figure 6 correspond to the OH radical
peaks generated by microplasma and detected by the
spin trap agent.
Fig. 6. OH signal detected by an ESR measurement.
It is considered that OH radical sterilized E. coli by
remote microplasma process.
Figure 7 shows the change of sterilization
performance by air microplasma treatment with the
exposure time. Discharge voltage was fixed at 1.4 kV.
Gas flow rate was 10 L/min and the agar medium
was exposed at 2.3 cm distance from electrodes.
Exposure time was varied from 0 to 60 s.
Sterilization effect was calculated by colony counting
method. The colony number was defined as 1.0
without any microplasma treatment and the change of
colony number was plotted in the graph after
microplasma treatment. To increase sterilization
effect, ethanol gas was added in air as the process
gasses. Ethanol concentration in air was 1.3%
measured by the gas indicator tube.
Fig. 7. Sterilization effect versus treatment time by
air microplasma. Distance from electrodes: 2.3 cm;
discharge voltage: 1.4 kV.
Ethanol is generally known as sterilization agent.
Thus about 40% of E. coli was sterilized by air with
ethanol gas exposure. About double-digits decreasing
of bacterial number was confirmed by air
microplasma treatment. On the other hand more than
six-digits decreasing of bacteria number was
achieved by air and vaporized ethanol remote
microplasma. To analyze the sterilization mechanism,
ozone concentration in the reactor was measured by
ozone monitor. 25 ppm (air microplasma) and 11
ppm (air + 1.3% ethanol microplasma) were
measured by microplasma generation. Therefore,
sterilization effect is not positively correlated with
ozone concentration. Thus it could be considered that
active species or chemical substances derived from
ethanol contributed to the bacterial sterilization.
Figure 8 shows the sterilization rate by air and
vaporized ethanol remote microplasma.
Fig. 8. The effect of the distance of microplasma
exposure at various discharge voltage. Distance from
electrodes; 2.3 -20 cm; discharge voltage;1.2-1.4 kV,
gas flow rate; 10 L/min.
Discharge voltage was adjusted 1.2 ~ 1.4 kV and the
ethanol concentration in air was 1.3%. Gas flow rate
was 10 L/min and the agar medium was exposed at
2.3 ~ 20 cm distance from electrodes. Exposure time
was fixed to 60 s. Sterilization effect was calculated
by colony counting method. Sterilization effect was
advanced with the increase of applied voltage by air
and vaporized ethanol remote microplasma. More
than six-digits decreasing of bacteria number was
achieved at the distance of 15 cm from the electrodes
by air and vaporized ethanol remote microplasma.
Sterilization effect was observed even at the distance
of 50 cm from electrodes. Thus it could not be
considered that E. coli was sterilized only by radicals
derived from microplasma due to its extremely-short
life time of radicals.
Figure 9 shows the gas composition of analysis after
the microplasma process with the addition of ethanol
by the FT- IR spectroscopy.
Fig. 9. The gas composition of air and vaporized
ethanol microplasma analyzed by the FT-IR.
Discharge voltage was fixed to 1.4 kV and the
ethanol concentration in air was 1.3%. Gas flow rate
was 10 L/min. C2H5OH was identified due to air and
ethanol vaporized microplasma. In addition, C2H4O
peak was observed by FT-IR spectroscopy. It could
be considered that C2H4O plays the important role of
bacterial sterilization.
To observe the effect of microplasma on the shape
of bacteria, a field-emission-type SEM was used.
(a) Before treatment
(b) Tr= 60s, Vd= 1.4 kV.
(x 6,000)
(x 6,000)
Figure 10 Image of E. coli before and after Ar and air
microplasma treatment.
Figure 10 shows the image of E. coli before and
after air and vaporized ethanol microplasma
treatment. Ethanol concentration in the air was 1.3%
and distance between electrodes and E. coli was fixed
at 2.3 cm. Similarly, shape change of E. coli was
observed after air and vaporized ethanol microplasma.
In general it is considered that various radicals
derived from O2 contributed to the sterilization
effects.
(a) Surface potential versus discharge voltage in
remote microplasma process.
(b) Surface potential versus discharge voltage in Ar
plasma jet process.
Fig. 11. Surface potential of PEN film after Ar
microplasma treatment (a) and Ar plasma jet (b).
Surface charge potential measurement for the
plasma process could be important factor when
plasma is used to treat a particular surface. Especially,
when the target is the soft matter or cells, animals, or
human being since electroporation could be strongely
suggested after plasma process on the cells [7]. It
could cause mutations of DNA in cells.
PEN film was used instead of human body to
measure the increase of surface potential in this
study. Figure 11 (a) shows the surface potential of
PEN film after Ar microplasma treatment. Discharge
voltage was adjusted 0.8 ~ 1.0 kV and PEN film was
exposed at 1-2 mm distance from electrodes.
Exposure time was fixed to 15 s. Figure 11 (b) shows
the surface potential of PEN film after Ar plasma jet.
Discharge voltage was adjusted 2.4 ~ 6.0 kV and
PEN film was exposed at 15-30 mm distance from
electrodes. Exposure time was fixed to 15 s.
Maximum surface potential was measured to be
about 45 V at 1 mm from electrodes by Ar
microplasma exposure. It is almost same level as
using a static charge eliminator. On the other hand,
about 650 V of surface potential was measured at 15
mm by applying the Ar plasma jet. It could be
considered to be high for the actual use for the
medical fields or semiconductor device.
Thus only a small increase in surface potential was
measured after microplasma treatment and it could be
considered that microplasma treatment is electricallysafe as a medical device.
4. Conclusion
Microplasma generated at atmospheric pressure and
low discharge voltage was used for sterilization of E.
coli and surface treatment of the thin polymer film.
1) Air microplasma sterilized a wider area and a sixdigits decrease of bacterial counts was achieved by
air and ethanol vaporized microplasma treatment.
C2H4O was detected as by-product by air and
vaporized ethanol microplasma.
2) The photos taken by the SEM indicated that
microplasma sterilization acted as an etching
process.
3) The increase of surface potential was measured to
be below 50 V after Ar microplasma exposure.
Plasma process for medical field application must be
safe, when the target is a soft matter, especially for
human being.
Microplasma is now on the stage for the medical
field trial. For example, sterilization, cell activation,
cancer treatment, tooth care and so on.
5. References
[1] K. Shimizu, Y. Komuro, S. Tatematsu, M.
Blajan, Pharmaceutica Analytica Acta, Special
issue title: PK/PD: Antifungal and Antibacterial
1, 2153-2435-S1-001 (2011).
[2] M. Laroussi, F. Leipold, Int. J. Mass Spectrom.
233, 81 (2004).
[3] M. A. Malik, A. Ghaffar, S. A. Malik, Plasma
Sources Sci. Technol. 10, 82 (2001).
[4] M. Blajan, and K. Shimizu , IEEE Trans. PS,
40, 1730(2012).
[5] U. Kogelschatz, IEEE Trans. Plasma Sci.
30,1400 (2002).
[6] K. Shimizu, M. Blajan, S. Tatematsu, IEEE
Trans. on IAS 48, 1182 (2012).
[7] N. Y. Babaeva, M. Kushner, J. Phys. D: App.
Phys. 43, 185206 (2010).