st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
Bactericidal efficiency of reactive oxygen radicals
for fungi based on radical density measurement
T. Ohta1, H. Hashizume1, M. Ito1, K. Takeda2, K. Ishikawa2 and M. Hori2
1
Faculty of Science and Technology, Meijo University
1-501 Shiogamaguchi, Tempaku-ku, Nagoya 468-8502
2
Department of Electrical Engineering and Computer Science
Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603
Abstract: We have measured the densities of ground-state atomic oxygen O(3Pj) and singlet
oxygen molecule [O2(1Δg)] produced from the oxygen radical source. The O( 3Pj) density was
between 1.4 x 1014 and 1.5 x 1015 cm-3. O2(1Δg) density was monotonically increased up to
1.2×1015 cm-3 with increasing O2/(Ar+O2) flow rate ratio. O (3Pj) density decreased with increasing exposure distance, while O2(1Δg) density was constant. Based on these results obtained by quantitative analysis of the gas phase, we conclude that O (3Pj) is one of the dominant species responsible for inactivating microorganisms.
Keywords:biological application, reactive oxygen species, inactivation
1. Introduction
Recently, non-equilibrium plasma has been
much attention for applications in biology, medicine,
agriculture and so on. There are many reports that
microorganisms such as fungi, yeast, bacteria, cancer cell, and so on, were inactivated using cold
plasmas. The cold plasma simultaneously produces
various factors, such as vacuum ultraviolet (VUV)
and UV-C emissions, neutral and charged species,
and electric fields, which may synergistically affect
to inactivate microorganisms. Several studies suggested that reactive oxygen species (ROS) may be
the dominant factor on the inactivation of the microorganisms by plasmas. The bactericidal effect of
plasma differs depending on microorganisms owing
to the resistance against the bactericidal factor. It is
essential to study the resistance against the bactericidal factor produced by the plasma based on the
quantitative diagnostics of the plasma. We have focused on inactivating the spores of Penicillium digitatum using non-equilibrium atmospheric pressure
plasma (NEAPP) in gas phase. We reported that the
contributions of UV radiation and ozone were not
dominant for inactivating spores of P. digitatum by
NEAPP.[1] By measuring the radical densities of
ground-state atomic oxygen [O(3Pj=0,1,2)] with an
atmospheric-pressure oxygen radical source, which
only supplies neutral oxygen radicals, we quantitatively elucidated that O(3Pj) is one of the effective
factors responsible for inactivating P. digitatum
spore.[2]
In this study, we have measured the densities of ground-state atomic oxygen O(3Pj) and singlet
oxygen molecule [O2(1Δg)] by ultraviolet absorption
spectroscopy, which are produced from the oxygen
radical source. We have exposed the radicals to P.
digitatum spores to evaluate the bactericidal effects.
2. Experimental
Figure 1 shows a schematic diagram of the
experimental setup containing an oxygen-radical
source employing a non-equilibrium atmospheric
pressure O2/Ar plasma with a vacuum ultraviolet
absorption spectroscopy (VUVAS) optical system.
The radical source was developed based on the
high-density NEAPP, which generates high-density
electrons of about 1016 cm-3.[3, 4] The charged species and optical radiation from the plasma were
blocked by the exit aperture of the radical source, so
that samples are exposed to only neutral species. The
O2+Ar
High AC voltage
Ar purge
lamp
MHCL
O2/He
Height:
10 mm
Radical
source
VUV
Monochromator
MgF2 window
PMT
Absorption length: 6.5 mm
Exhaust
Exhaust
Exhaust
Figure 1. schematic diagram of the experimental
setup containing an oxygen-radical source.
st
chamber containing the radical source was purged
with Ar gas to eliminate the influence of atmospheric gases. Measurements and exposures of radicals to
spores were performed at 10 mm, 15 mm or 20 mm
downstream from the radical head. Flow rate ratio
O2/(O2+Ar) was also varied from 0 to 1.2 % at total
flow rate of 5 slm.
The absolute densities of O(3Pj) and
1
O2( Δg) were measured by VUVAS using a microdischarge hollow-cathode lamp (MHCL) and a
deutrium lamp, respectively.[5, 6] VUV light from
the light source passed though the MgF2 window
and was introduced into the chamber. VUV light
passing through the absorption region was focused
on the slit of a VUV monochromator with the MgF2
lens and detected by a photomultiplier tube.
Number of Survivors (CFU/mL)
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
109
1000
D=20.1 min
100
108
D=16.6 min
D=16.1 min
D=2.4 min
D=7.6 min
D=
5.3 min
107
10
0.2 %
1016
0.6 %
D=0.9 min
1.2 %
0.15
10
-1
00
11
2
33
44
55
2
Exposure Time (min)
66
Figure 2. Number of survivors as a function of
treatment time for three different flow rate ratios
O2/(O2+Ar). Total flow rate was 5 slm.
O(3Pj) density [cm-3]
1E+16
16
10
1E+15
15
10
14
1E+14
10
13
10
1E+13
00
0.2
0.4
0.6
0.8
1
1.2
1.4
0.2
0.4
0.6
0.8
1.0
1.2
1.4
O2/(Ar+O2) [%]
Figure 3. ground-state atomic oxygen O (3Pj) (j=0, 1,
2) density as a function of the O2/(Ar+O2) flow rate
ratio.
[×1014]
15
15
O2(1Δg) density (cm-3)
3. Results and discussion
Figure 2 shows the number of surviving
spores as a function of plasma treatment time for
three different O2 gas mixture ratios. The number of
surviving spores decreased nonlinearly with increasing treatment time. The nonlinearity is assumed
to originate from the stacking of the spores. The D
values at an O2/(Ar+O2) flow rate ratio of 0.6% was
0.9 min. The D values for O2/(Ar+O2) flow rate ratios of 0.2 and 1.2% were larger than that of 0.6%,
namely, the treatment at O2/(Ar+O2) flow rate ratio
of 0.6% was effective.
Figure 3 shows ground-state atomic oxygen O (3Pj) (j=0, 1, 2) density as a function of the
O2/(Ar+O2) flow rate ratio. O2/(Ar+O2) flow rate
ratio of the oxygen radical source was changed between 0 to 1.2 % in the chamber purged with Ar gas
at atmospheric pressure. The O(3Pj) density was estimated to be between 1.4 x 1014 and 1.5 x 1015 cm-3.
The O(3Pj) density increased with increasing O2/(Ar
+O2) mixture flow rate ratio up to 0.6% and it then
decreased with increasing flow rate ratio. Atomic
oxygen is generated through the collision of electrons with O2 gas molecules and its density increases
with the addition of O2 to Ar. However, the further
increase in the amount of O2 leads to the decrease in
electron density and the recombination reaction with
atomic oxygen and oxygen molecule occurred frequently in the remote plasma region.[3] Therefore,
the O(3Pj) density decreased at O2/(Ar+O2) mixing
ratio of greater than 0.6%.
Figure 4 shows O2(1Δg) density as a function of O2/(Ar+O2) flow rate ratio. The density were
measured to be up to 1.2×1015 cm-3 with increasing
O2/(Ar+O2) flow rate ratio. No changes in the density was observed against the exposure distances from
10 mm to 20 mm. Therefore, the results suggested
that O2(1Δg) is long-life time species. Since the D
10
10
55
10mm
15mm
20mm
00
0
0.2 0.4 0.6 0.8
1
1.2 1.4 1.6
0
0.2
0.4 0.6 0.8 1.0
1.2
1.4 1.6
O2/(O2+Ar)(%)
Figure 4. O2(1Δg) density as a function of
O2/(Ar+O2) flow rate ratio.
st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
Radical density (cm-3)
16
1E+16
10
15
1E+15
10
14
1E+14
10
13
1E+13
10
系列1
O(3Pj)
density
系列2
O2(1Δg)
12
1E+12
10
0
0
5
density
10
15
20
5
10
15
20
Exposure distance (mm)
5 µm
25
25
(a)
Figure 5. O (3Pj) and O2(1Δg) densities as a function
of exposure distance.
5 µm
(b)
Figure 6. the number of surviving spores as a func-
Figure 7. SEM image of control spores (A) and rad-
tion of plasma treatment time at three different ex-
ical-treated spores (B).
posure distances, 10 mm, 15 mm and 20 mm.
value is the inverse of the inactivation rate, these
results indicate that the behaviors of the O (3Pj) densities correspond to that of the spore inactivation rate
against O2/(Ar +O2) mixture flow rate ratio.
Figure 5 shows O (3Pj) and O2(1Δg) densities as a function of exposure distance. O (3Pj) density decreased with increasing exposure distance,
while O2(1Δg) density was constant. The life time
of O (3Pj) was estimated to be 0.47 ms from the gradient of the data. The loss processes of O (3Pj) are
reactions with O (3Pj) or O2 via three body reaction
and O (3Pj) density decreased by these loss reactions
with increasing the exposure distance.
Figure 6 shows the number of surviving
spores as a function of plasma treatment time at
three different exposure distances, 10 mm, 15 mm
and 20 mm. At 10 mm distance, number of survivors
rapidly decreased, so that spores completely inactivated for 7 min treatment. D value was estimated to
be 1.2 min. On the other hand, at 15 mm and 20 mm,
D values were 7.9 mim and 19.8 min, and the inactivation efficiencies were much lower than that at 10
mm. From the results of O(3Pj) and O2(1Δg) densities,
the inactivation efficiencies are related to O(3Pj)
densities rather than those of O2(1Δg). We suggested
that O(3Pj) is the main factor in oxygen radicals to
inactivate P. digitatum spores and that in O2(1Δg) is
less effective.
Figure 7 shows the SEM image of control
spores (A) and radical-treated spores (B). The treatment time was 7 min. The radical treatment does not
cause major morphological changes with respect to
the shapes and surface geometries of spores. These
results suggest that oxygen radicals do not damage
surface morphologies of spores, but penetrate into
spores and directly affect various functions of intracellular organelles.
4. Conclusions
We successfully inactivated P. digitatum
spores using an oxygen radical source employing a
non-equilibrium atmospheric-pressure remote O2/Ar
plasma. The ground-state oxygen radical O (3Pj) and
singlet oxygen molecule [O2(1Δg)] densities were
st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
measured using VUV absorption spectroscopy. The
O(3Pj) density was between 1.4 x 1014 and 1.5 x 1015
cm-3. O2(1Δg) density was monotonically increased
up to 1.2×1015 cm-3 with increasing O2/(Ar+O2) flow
rate ratio. O (3Pj) density decreased with increasing
exposure distance, while O2(1Δg) density was constant. Based on these results obtained by quantitative
analysis of the gas phase, we conclude that O (3Pj) is
one of the dominant species responsible for inactivating microorganisms.
5. Acknowledgment
This work was partly supported by a Grant-in-Aid
for Scientific Research on Innovative Areas,
“Frontier Science of Interactions between Plasmas
and Nano-interfaces” (No. 21110006) from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan.
6. References
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[2] S. Iseki, H. Hashizume, F. Jia, K. Takeda, K.
Ishikawa, T. Ohta, M. Ito and M. Hori: Appl.
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[3] M. Iwasaki, H. Inui, Y. Matsudaira, H. Kano, N.
Yoshida, M. Ito and M. Hori: Appl. Phys. Lett.,
92 (2008) 081503.
[4] H. Inui, K. Takeda, H. Kondo, K. Ishikawa, M.
Sekine, H. Kano, N. Yoshida and M. Hori: Appl.
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[5] H. Nagai, M. Hiramatsu, M. Hori, and T. Goto:
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[6] G. Gousset, P. Panafieu, M. Touzeau, and M.
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