Characterization of an atmospheric pressure plasma jet array and its application to

cancer cell treatment using plasma activated medium

Dong Yeong Kim, Sun Ja Kim, Hea Min Joh, and T. H. Chung

Citation: Physics of Plasmas 25, 073505 (2018); doi: 10.1063/1.5037249

View online: https://doi.org/10.1063/1.5037249
View Table of Contents: http://aip.scitation.org/toc/php/25/7
Published by the American Institute of Physics

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 PHYSICS OF PLASMAS 25, 073505 (2018)

Characterization of an atmospheric pressure plasma jet array and its

application to cancer cell treatment using plasma activated medium
Dong Yeong Kim, Sun Ja Kim, Hea Min Joh, and T. H. Chunga)
Department of Physics, Dong-A University, Busan 49315, South Korea
(Received 23 April 2018; accepted 21 June 2018; published online 9 July 2018)
An atmospheric pressure plasma jet array source driven by a pulsed bipolar voltage of several tens
of kilohertz was characterized and utilized for cancer cell treatment. Electrical and optical emission
characteristics of plasma jet array were obtained as functions of the applied voltage, gas flow rate,
and pulse frequency, and the optimal operating parameters were obtained. The emission intensities
from reactive oxygen and nitrogen species (RONS) in a gas phase, such as OH, NO, H, and O,
were measured by optical emission spectroscopy. In the plasma-liquid interaction experiment, the
OH concentration was measured using the TA solution. The nitrite concentration in the plasma
treated media (DW, HBSS, and DMEM) was measured using Griess reaction assay. Human skin
cancer cells injected with plasma treated liquid were observed for changes in cell viability using
the MTS assay. The results demonstrate that the plasma jet array source can be a good candidate
for delivering RONS to liquid for plasma activated medium (PAM). Published by AIP Publishing.
https://doi.org/10.1063/1.5037249

I. INTRODUCTION effects as that of direct plasma exposure but facilitate flexi-

bility and precision of delivery with potentially gentler con-
Cold atmospheric pressure plasmas have drawn tremen-
ditions as may be demanded with in-vivo conditions.19,20
dous attention due to their widespread potentials for biomedical
Furthermore, it was found that there is an optimum dose of
applications including bio-decontamination, sterilization, and
PAM to induce significant cancer cell apoptosis while keep-
cancer therapy.1–5 Atmospheric pressure plasma jet (APPJ)
ing minimum damage to normal cells.21 APPJs’ anticancer
generates a stable plasma plume and transports reactive species
activity is primarily due to the delivery of reactive oxygen
away from the plasma generating electrode using gas flow.6
and nitrogen species (RONS). Many studies demonstrate that
However, the treatment area of a single plasma jet is less than
the concentration of H2O2, NO2–, and NO3– can fully
1 cm2, which is too small to adapt to the large-scale treatment.6
account for the majority of RONS produced in PAM.16–18
When multiple plasma jets are arranged in close proximity,
This study aims to explore the possibility of APPJ array
their interaction may result in an intense and energetic APPJ.7
to generate PAM for cancer therapy. For that purpose, the
With the plasma jet array, large area plasma treatment is possi-
effect of different parameters (applied voltage, gas flow rate,
ble in many fields, including biological and medical fields, and
and pulse repetition frequency) on the plasma properties of
the area can be adjusted by changing the number of plasma jet.
the plasma jet array driven by a pulsed bipolar wave of sev-
Some recent reports have proposed various types of
eral tens of kilohertz was studied to obtain homogenous
APPJ array to enhance the treatment area.6–12 With a rising
plasma jets. Then, a chemical probe method was used to
interest in the enlarged scale of plasma jets, their spatial uni-
measure the reactive species such as OH and NO2– generated
formity and stability have been a central issue due to the
in the APPJ array treated liquids. Finally, the PAM treated
interactions between individual jets and between jets and tar-
by the APPJ array was applied to cancer cells in-vitro to
gets.7,8,13 Up to now, the effect of the operating parameters
assess its applicability in cancer therapy.
on the behavior of the plasma jet array has been studied to
obtain homogeneous plasma jets.13,14 The properties of the
II. EXPERIMENTAL SETUP
plasma plume and the generation of reactive species can be
controlled by changing the APPJ configuration, applied volt- Figure 1 shows a schematic of the jet array source
age, pulse frequency, and gas flow rate.15 driven by a pulsed bipolar high voltage and the photograph
There are numerous studies showing that non-thermal of the plasma plume contacting a medium in a liquid con-
atmospheric pressure plasmas are effective against cancer tainer. The plasma jet array consists of 16 tungsten pin wire
cells both in-vitro and in-vivo. Living cells and tissues were electrodes and quartz tubes with pencil-shaped nozzle. The
directly exposed to plasma treatment, or the cell culture details of the jet array are found in our previous paper.22 The
medium or buffer solution was first exposed to plasma treat- helium was used as a plasma-forming gas and fed through
ment [so-called plasma-activated medium (PAM)] and then the gas inlet. The plasma plume propagated into ambient air.
added to medium-free cells.16–20 Indirect treatments with The voltage and the current were measured using high volt-
PAM demonstrate similar bactericidal and or cytotoxic age probe (PPE 20 kV LeCroy) and current probe (3972
Pearson). The optical emission spectra were obtained using a
a)
Author to whom correspondence should be addressed: thchung@dau.ac.kr fiber optic spectrometer (USB-2000 þ XR1-ES OceanOptics).

1070-664X/2018/25(7)/073505/9/$30.00 25, 073505-1 Published by AIP Publishing.

073505-2 Kim et al. Phys. Plasmas 25, 073505 (2018)

circuit. The plume temperature which was measured using a

fiber optic temperature sensor (Luxtron, M601-DM&STF)
remained less than 40  C at the applied voltage of 4.5 kVpp.
With an increase in the applied voltage (to 6.38 kVpp), the
plume temperature was increased up to 45.2  C. The current
and voltage measurements are utilized to detect the discharge
power and energy consumption, and discharge uniformity is
investigated by using a photo sensor. As shown in the previ-
ous work,22 the spatial distributions of the gas temperature
and wavelength-integrated intensity were quite uniform
along the jet array irrespective of applied voltage, gas flow
rate, and pulse frequency.
Figure 2 exhibits the time evolutions of the applied volt-
age [Va(t)], discharge current [Id(t)], discharge power
[P(t) ¼ Id(t)*Va(t)], and wavelength-integrated intensity from
FIG. 1. (a) Photograph of the plasma plumes from the jet array contacting a
the plasma plume at the operating condition: 6.38 kVpp, 8 l/
medium in a liquid container and (b) the schematic of the experimental min, and 50 kHz. The discharge current is obtained by sub-
setup. tracting the displacement current (with plasma off) from the
measured (total) current (with plasma on). It was observed
A photo sensor amplifier (Hamamatsu C6386-01) was used to that the discharge produced two peaks of the discharge cur-
observe the temporal evolution of the wavelength-integrated rent and optical emission every cycle of the applied voltage,
plasma emission. the main peak (positive streamer) occurred at the positive
RONS in gas phase spread into liquid phase and react with half cycle of the voltage waveform, and the weak peak (neg-
water to produce various biologically active reactive species in ative streamer) occurred at the negative half cycle. Peak
the liquid phase. The presence of H2O2 accelerates the decom- discharge current during the primary discharge (positive
position of ozone and increases the OH radical concentration in
water.23 As a method of OH radical detection, we utilized the
hydroxylation of terephthalic acid (TA), which is a typical pho-
tocatalytic reaction that specifically oxidizes TA. That is, the
OH radical reacts with TA to form hydroxyterephthalic acid
(HTA) which fluoresces at k ¼ 425 nm.24 Fluorescence inten-
sity was observed to increase after plasma treatment, indicating
the total amount of OH radicals trapped by TA. From the fluo-
rescence intensity emitted from the plasma treated solution, the
concentration of OH radicals in the solution could be estimated.
Reactive nitrogen species are also formed in the liquid phase.
Nitrite concentration was determined using the Griess reagent
(Molecular Probes). In liquids, nitric oxide is converted into
nitrite and nitrate ions.25 The Griess reaction can be used to
analyze nitrite and can provide a good measure of the concen-
trations of these nitrogen oxides.25
To investigate the effect of plasma-induced ROS on cells,
cell viability was examined using melanoma (Hs 895.T) cells.
Cells were maintained and seeded in Dulbecco modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum
and 1% penicillin-streptomycin at 37  C in a humidified incu-
bator supplemented with 5% CO2. Cell viability was assessed
by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay with
the use of a kit (Promega) according to the manufacturer’s
instructions. Assays are performed by adding a small amount
of the solution reagent directly to culture wells, incubating for
2–4 h, and then recording the absorbance at 490 nm with the
VersaMax Microplate Reader (Molecular Devices).

III. RESULTS AND DISCUSSION FIG. 2. (a) Waveforms of the applied voltage [Va(t)] and (b) time evolutions
of the discharge current [Id(t)], (c) discharge power [P(t) ¼ Id(t)*Va(t)], and
Plasma plumes were generated and propagated to (d) wavelength-integrated emission intensity at the operating condition: 6.38
grounded-electrode relatively uniformly without an auxiliary kVpp, 8 l/min, and 50 kHz.
073505-3 Kim et al.
streamer) exceeds that during the negative streamer, 47.5 mA
versus 11.6 mA. The amplitudes of the discharge power for
the positive and negative pulses were 108.6 W and 37.4 W,
respectively. The average power was 4.27 W.
Figure 3(a) shows the waveforms of the discharge power
for different applied voltages. The current amplitude signifi-
cantly increased with the applied voltage. The temporal dif-
ference between the initiation of the voltage pulse and the
discharge current peak maximum, which is due to the propa-
gation processes of the plasma plume, was reduced with an
increase in the applied voltage. As the applied voltage is
increased from 4.41 kVpp to 7.38 kVpp, the peak power
deposited into jet array increases from 51.4 W to 110.1 W.
Figures 3(b) and 3(c) show the dependence of discharge
power on the gas flow rate and on the pulse frequency, respec-
tively. When the gas flow rate was raised from 2 l/min to 12 l/
min, the current peaks rose and reached a plateau at 12 l/min.
When the pulse frequency was raised from 30 kHz to 40 kHz,
the peak power increased. However, the peak power remained
not much changed above 40 kHz. The decrease in the pulse
frequency is corresponding to an increase in the interval time
between each discharge. Long interval time may result in a
decrease in the seed electron density, thus the discharge cur-
rent (or power) may decrease.7 However, in the range of the
pulse frequency of 40 kHz–60 kHz (employed in this study),
this effect does not make a significant influence.
FIG. 3. Time evolutions of the discharge power for different (a) applied vol-
tages, (b) gas flow rates, and (c) pulse frequencies. The insets show the peak
power as functions of applied voltage, gas flow rate, and pulse frequency.
Phys. Plasmas 25, 073505 (2018)
Figure 4 exhibits the time evolution of the wavelength-
integrated emission intensity as functions of (a) applied volt-
age, (b) gas flow rate, and (c) pulse frequency. It should be
noted that these time evolutions resemble those of the dis-
charge power (Fig. 3). This indicates that the dissipated
power correlates well with light emission. Similar to the tem-
poral profiles of the discharge power, the temporal difference
between the initiation of the voltage pulse and the onset of
light pulse differs depending on the applied voltage, gas flow
rate, and pulse frequency. The plasma bullet speed deter-
mines the temporal difference. With increasing applied volt-
age, the bullet speed increases due to enhanced electric field
[Fig. 4(a)], whereas an increase in the gas flow rate deters
bullet speed [Fig. 4(b)]. The effect of the gas flow speed is
the stretching of the electric field profile in space close to jet
nozzle—the higher the flow, the longer and less steep the
electric field profile, which is caused by differences in gas
mixing as a function of gas flow speed.26 As shown in Fig.
4(c), the temporal difference shortens with increasing pulse
frequency. For the plasma propagation outside the tube in
surrounding air, higher repetition rates lead to the higher
propagation velocity, which in turn leads to an increase in
the plasma plume length.27
The treatment efficiency depends on the electrical
energy per pulse. The electrical energy per pulse was
obtained through integration of the power impulse over time,
FIG. 4. Time evolutions of the wavelength-integrated emission intensity
(photodetector signal) for different (a) applied voltages, (b) gas flow rates,
and (c) pulse frequencies.
073505-4 Kim et al.
while the wavelength-integrated light emission per pulse was
obtained through integration of the wavelength-integrated
intensity impulse over time. As shown in Fig. 5, the electri-
cal energy per pulse and the wavelength-integrated light
emission per pulse correlate well with each other and
increase with applied voltage and gas flow rate, but remain
not much changed with pulse frequency. Although, in their
dependences on the operating parameters, the electrical
energy per pulse exhibits a little difference from those of the
peak power (the insets of Fig. 3), the discrepancy lies within
a tolerable limit. Especially, the change in the wavelength-
integrated light emission per pulse with the operating param-
eters is observed to behave in a similar fashion with those of
the peak wavelength-integrated intensity.
Figure 6(a) illustrates the optical emission spectrum
from the jet array. Ambient air flows into the plasma jet and
reacts with the plasma to produce many kinds of active spe-
cies. The peak of nitric oxide (NO) at 283 nm is due to the
chemical reaction of N and O2 (or N and O). The hydroxyl
(OH) radical at 309 nm is produced via dissociative electron
attachment to the water vapor molecules. The N2þ first nega-
2P þ 2P þ
tive system at 391 nm (B u ! X g ) is attributed to
Penning ionization (He*þ N2 ! He þ N2þþ e–) and charge
transfer (Heþ þ N2 ! He þ N2þ) followed by direct
electron-impact excitation (e– þ N2þ ! N2þ*þ e–). The N2*
second positive system (C3 Pu ! B3 Pg) are clearly
observed at 315, 337, 357, 375, and 380 nm. The emission
FIG. 5. Electrical energy per pulse and the wavelength-integrated light emis-
sion per pulse for different (a) applied voltage, (b) gas flow rates, and (c)
pulse frequencies.
Phys. Plasmas 25, 073505 (2018)
line at 656 nm corresponds to the Ha line, which is generated
by the collision between water vapor molecule and electrons
(H2O þ e ! H þ OH þ e). The He 706 nm emission [3s 3S
! 2p 3P] indicates the presence of energetic energy elec-
trons above 20.96 eV. The O: 777 and 844 nm lines suggest
the presence of highly reactive excited O atoms such as O
[3p 5P] and O [3s 5S].28 Metastable state He can dissociate
the oxygen molecules and excite atomic oxygen to the
excited state.13 The optical spectra reveal that the APPJ array
of this work exhibit significantly enhanced intensity levels of
OH (309 nm) band and N2þ band (391 and 427 nm), and rela-
tively high levels of reactive radicals such as NO, O, and Ha.
Especially, it is well known that the OH radical can induce
an important biological response.16–21,23,24 In this respect,
the high level of OH generated in our jet array can provide a
significant merit in biomedical applications including cancer
therapy.
Figures 6(b)–6(d) represent the change in the intensity
peaks with varying applied voltage, gas flow rate, and pulse
frequency. The effect of increasing the applied voltage is
that the faster, more intense ionization wave penetrates fur-
ther into the plume, and this leads to enhanced discharge cur-
rent creating more reactive species [Fig. 6(b)]. As shown in
Fig. 6(c), as the gas flow rate is increased, the intensity peaks
increase, reaching the maximum at 8 l/min, then decrease
slightly afterwards. This behavior was also observed in our
previous experiment on a single APPJ (at much lower flow
rate than 8 l/min).29 It is also observed that as the pulse fre-
quency is raised from 30 kHz to 60 kHz, the emission inten-
sity of most of the peaks (except N2þ 391 nm peak) increases
slightly [Fig. 6(d)]. This trend is in an overall agreement
with other experiment.30,31 In atmospheric-pressure non-
equilibrium plasma, the plasma is generated intermittently.
The intensity at each wavelength observed using a spectrom-
eter is the time-averaged effective intensity. For a fixed
exposure time of the spectrometer, the time-averaged effec-
tive intensity at each wavelength is expected to increase line-
arly with the pulse frequency.32 However, it should be noted
that the increase in the emission intensities at each wave-
length with the pulse frequency is not significant in the range
of 40–60 kHz.
The intensity ratio of two atomic lines of He, 587 nm (3d
3
D ! 2p 3P) and 706 nm (3s 3S ! 2p 3P), is equivalent to the
ratio of upper state’s density, which depends on the electric
field or the electron energy.33 If we assume that the excitation
coefficient ratio of He at 706 nm and O at 777 nm remains
constant with the control parameters, we can estimate the
effect of the control parameters on O density by taking the
emission intensity ratio of O/He. Figures 7(a)–7(c) illustrate
the intensity ratios He(587 nm)/He(706 nm) and O(777 nm)/
He(706 nm) as functions of applied voltage, gas flow rate, and
pulse frequency, respectively. It is observed that these ratios
exhibit similar trend of change with the control parameters.
This indicates that oxygen atom density correlates with the
electron energy and is enhanced with increasing applied volt-
age, and at specific values of gas flow rate (8 l/min) and pulse
frequency (40 kHz).
The relation between the observed spectral-line intensi-
ties and the excitation levels for He I is shown in Fig. 8(a).
073505-5 Kim et al.
The characteristic spectral lines of helium 587, 667, 706, and
728 nm were chosen to determine the electron excitation
temperature (Texc) under a Boltzmann approximation.32,34
When electron collisions dominate the excitation process,
the electron excitation temperature is closely related to the
electron temperature. However, it should be noted that Texc
will only provide an approximate measure of the electron
temperature. In order to provide more accurate information
on the electron temperature, more elaborate collisional-
radiative modelling should be coupled with optical emission
spectroscopy.35–37 Figures 8(b)–8(d) represent the electron
excitation temperature of the jet array as functions of applied
voltage, gas flow rate, and pulse frequency, respectively. The
excitation temperature rises with applied voltage. With an
increase in gas flow rate, the Texc rises and reaches a maxi-
mum at 8 l/min, and then declines slightly. With varying
pulse frequency from 30 kHz to 60 kHz, the Texc exhibits a
maximum at 40 kHz, and then decreases slightly after that
point. It is worth noting that the changes in the Texc resemble
those of the intensity ratios shown in Fig. 7. This may sup-
port that the excitation temperature can be a measure of
the electron energy. Especially, a slight increase in Texc at
Phys. Plasmas 25, 073505 (2018)
FIG. 6. (a) Typical optical emission
spectrum from the jet array. The peak
intensities of important lines as func-
tions of (b) applied voltage, (c) gas
flow rate, and (d) pulse frequency.
40 kHz may correlate with the changes in the discharge
power [Fig. 3(c)], the wavelength-integrated intensity [Fig.
4(c)], the electrical energy, and the wavelength-integrated
light emission per pulse [Fig. 5(c)], and the line intensity
ratio [Fig. 7(c)] with pulse frequency.
Plasma-liquid interaction experiments are carried out by
plasma treatment with aqueous TA solution (2 ml) in a rectan-
gular quartz container. To investigate the response under differ-
ent discharge conditions, in every case, the fixed distance
between the open end of electrodes and surface of liquids was
1 mm. Fluorescence intensity increased after reaction with
plasma; the change was used to estimate the quantity of OH
radicals trapped by TA. The fluorescence intensity corresponds
to a time-integrated OH radical concentration in the liquid.
Aqueous solution of TA was prepared by dissolving TA
(Sigma) in distilled water containing NaOH (Wako). A series
of 2HTA dilutions in NaOH solution were made to generate a
calibration curve over the range of 0–50 lM. Figures 9(a)–9(c)
show the fluorescence spectra of HTA as functions of treatment
time (0, 1, 2, and 3 min), applied voltage (4.41, 5.38. 6.38, and
7.40 kVpp), and gas flow rate (4, 6, and 8 l/min). As treatment
time elapsed, the fluorescence intensity increased [Fig. 9(a)];
073505-6 Kim et al. Phys. Plasmas 25, 073505 (2018)

FIG. 7. The intensity ratios of two atomic lines He(587 nm)/He(706 nm) and
O(777 nm)/He(706 nm) as functions of (a) applied voltage, (b) gas flow rate,
and (c) pulse frequency.

this change indicates an increase in the total amount of OH rad-

icals trapped by TA.24 The 2HTA concentration increased
nearly linearly with treatment time [Fig. 9(d)] and with applied
voltage [Fig. 9(e)]. The calculated OH radical density in the
solution was comparable to that obtained by high-voltage (up
to 20 kVpp) pulsed discharge.24 The results indicate that OH
concentration increased with treatment time and applied volt-
age, in agreement with the previous measurement of the OH
density.24 Concerning the gas flow rate dependence, the gas
flow rate of 6 l/min has the highest fluorescence followed by
8 and 4 l/min [Fig. 9(f)]. This indicates that adequate gas flow
promotes the transport of RONS into liquid.38
FIG. 8. (a) Boltzmann plot method to estimate the excitation temperature.
The electron excitation temperature as functions of (b) applied voltage, (c)
gas flow rate, and (d) pulse frequency.
Reactive nitrogen species are also formed in the liquid
phase. Figure 10 shows the results of the nitrite (NO2–) quan-
titation assay performed on three different solutions [deion-
ized water (DW), DMEM, and HBSS (serum-free Hanks’
balanced salt solution)] after plasma treatment. For the quan-
tification of the nitrite concentrations, a calibration curve
073505-7 Kim et al. Phys. Plasmas 25, 073505 (2018)

FIG. 9. Fluorescence spectra of aqueous TA solutions exposed to the APPJ array for different (a) treatment time (0, 1, 2, and 3 min), (b) applied voltage (4.41,
5.38. 6.38, and 7.40 kVpp), and (c) gas flow rate (4, 6, 8 l/min). 2HTA concentration obtained by plasma treatment to a TA solution as functions of (d) treatment
time (applied voltage: 6.38 kVpp and gas flow rate: 6 l/min), (e) applied voltage (treatment time: 2 min and gas flow rate: 6 l/min), and (f) gas flow rate (treat-
ment time: 2 min and applied voltage: 6.38 kVpp).

was prepared using the standard sodium nitrite solutions

(Molecular Probes). No significant difference is observed in
gas-treated control liquids. On the other hand, the nitrite con-
centration increased after plasma exposure with increasing
gas flow rate (2, 4, and 6 l/min) and applied voltage (4.3, 6.3,
and 8.3 kVpp) and becomes higher in order of DMEM,
HBSS, and DW. DMEM contains a higher concentration of
amino acids and vitamins, as well as additional supplemen-
tary components. The buffer solution (HBSS) is simply com-
posed of various salts. Plasma can interact more with
components in complex media (like DMEM) compared to
water and buffer solution. Especially, the nitrite concentra-
tions dramatically increased up to 97 lM in plasma-treated
DMEM within 30 s of treatment. It takes more than 2 min to
produce relatively high concentrations of nitrite when using
a single jet.25,29,31,39 Considering a short plasma treatment
time of 30 s, the obtained nitrite concentration is higher than
that obtained by single jets.25,29,31,39
We tested whether plasma-activated HBSS caused a
change in cell viability. Two milliliters of HBSS in a rectan-
gular quartz container was treated with plasma for 30 s. Cells
were incubated with plasma-activated HBSS for 90 min at
37  C, and cells were then washed with PBS (phosphate-
buffered saline) and incubated for 24 h in fresh medium with
0.1% FBS. Figure 11(a) shows bright-field images of cells
obtained after the 24-h incubation. It was shown that cell
numbers significantly reduced with increasing applied volt-
age (4.3 kVpp ! 8.3 kVpp) compared with the gas-control
cells. RONS and their derived species are generally consid-
ered to be the main bioactive species in plasma-activated
liquids which affect cell response.40 The results suggested
FIG. 10. Nitrite concentrations in different media (DW, HBSS, and DMEM)
that our newly developed jet array source can rapidly gener- as functions of (a) gas flow rate (2, 4, and 6 l/min) at Va ¼ 6.38 kVpp and (b)
ate high levels of RONS which is a primary cause of cellular applied voltage at a gas flow rate of 6 l/min. Treatment time was 30 s.
073505-8 Kim et al.
FIG. 11. (a) Bright-field images of cells (upper row: gas control and plasma
treatment—4.3 KVpp and bottom row: plasma treatment—6.3 kVpp and 8.3
kVpp) and (b) measurement of cell viability by MTS assay at 24 h after
plasma-activated HBSS treatment (gas-treated, 4.3 kVpp, 6.3 kVpp, and 8.3
kVpp). Each point represents mean 6 SD of three replicates, and the scale
bar indicates 100 lm.
damage that leads to cell death. According to the results
(Figs. 9 and 10), the ability of our jet array to generate
RONS such as OH and NO2– is much more effective than
that of single jet.29,31 MTS assay was used to quantitatively
determine viability of cells treated with plasma-activated
HBSS. As shown in Fig. 11(b), viability dramatically
decreased with a rise in applied voltage, which indicates a
fatal damage of the cell by plasma-activated liquids. Plasma-
activated liquids as a clinical application in cancer therapy
could be an efficient tool when direct plasma treatment is not
convenient to apply.21,41 Our source could potentially be
used for the applications with the rapid and abundant produc-
tion of RONS.
IV. CONCLUSION
An atmospheric-pressure plasma jet array source driven
by pulsed bipolar high voltages of several tens of kilohertz
was characterized. The jet array device exhibits plasma sta-
bility and uniformity while remaining low gas temperature.
As the applied voltage and gas flow rate increase, the
Phys. Plasmas 25, 073505 (2018)
discharge current and power and the light emission intensity
increase. The high level of RONS in our jet array can pro-
vide a significant merit in large-area plasma treatments of
biological samples. It is observed that the production of reac-
tive species in the gas phase can be controlled with the oper-
ating parameters such as applied voltage, gas flow rate, and
pulse frequency. The production of reactive species in the
plasma-treated liquid exhibits a similar trend to that in the
gas phase with respect to the operating parameters. The
nitrite concentration increases with increasing gas flow rate
and applied voltage and becomes higher in order of DMEM,
HBSS, and DW. It is found that the plasma jet array provides
abundant reactive species to the target and the power dissipa-
tion and the production of reactive species depend on plasma
properties, which can be controlled by operating parameters
such as applied voltage, gas flow rate, and pulse frequency.
The cell viability decreases with increasing RONS. Although
the quantification of RONS and cell viability test were not
performed in a wide range of the control parameters, this
work demonstrates that the plasma jet array can be a good
candidate for delivering RONS to liquid for plasma activated
medium (PAM). Therefore, it would be a next challenge to
control the quantities of each RONS existed in PAM by
adjusting the parameters of APPJ array and to correlate their
effects on cell death precisely.
ACKNOWLEDGMENTS
This work was supported by the National Research
Foundation of Korea under Contract Nos.
2015R1D1A1A09056870 and 2015R1C1A1A02036615.
1
M. Keidar, “Plasma for cancer treatment,” Plasma Sources Sci. Technol.
24, 033001 (2015).
2
K. Gazeli, C. Noel, F. Clement, C. Dauge, P. Svarnas, and T. Belmonte,
“A study of helium atmospheric-pressure guided streamers for potential
biological applications,” Plasma Sources Sci. Technol. 22, 025020 (2013).
3
G. Isbary, T. Shimizu, Y. F. Li, W. Stolz, H. M. Thomas, G. E. Morfill,
and J. L. Zimmermann, “Cold atmospheric plasma devices for medical
issues,” Expert Rev. Med. Devices 10, 367 (2013).
4
G. Nayak, H. A. Aboubakr, S. M. Goyal, and P. J. Bruggeman, “Reactive
species responsible for the inactivation of Feline calicivirus by a two-
dimensional array of integrated coaxial microhollow dielectric barrier dis-
charges in air,” Plasma Process Polym. 15, e1700119 (2018).
5
M. Vandamme, E. Robert, S. Lerondel, V. Sarron, D. Ries, S. Dozias, J.
Sobilo, D. Gosset, C. Kieda, B. Legrain, J. M. Pouvesle, and A. L. Pape,
“ROS implication in a new antitumor strategy based on non-thermal
plasma,” Int. J. Cancer 130, 2185 (2012).
6
F. Liu, B. Zhang, Z. Fang, M. Wan, H. Wan, and K. (Ken) Ostrikov, “Jet-
to-jet interactions in atmospheric-pressure plasma jet arrays for surface
processing,” Plasma Process Polym. 15, e1700114 (2018).
7
Y. Xia, W. Wang, D. Liu, Y. Peng, Y. Song, L. Ji, Y. Zhao, Z. Qi, X.
Wang, and B. Li, “An atmospheric-pressure microplasma array produced
by using graphite coating electrodes,” Plasma Process Polym. 14,
e1600132 (2017).
8
M. Ghasemi, P. Olszewski, J. W. Bradley, and J. L. Walsh, “Interaction of
multiple plasma plumes in an atmospheric pressure plasma jet array,”
J. Phys. D: Appl. Phys. 46, 052001 (2013).
9
Z. Cao, Q. Nie, D. L. Bayliss, J. L. Walsh, C. S. Ren, D. Z. Wang, and M.
G. Kong, “Spatially extended atmospheric plasma arrays,” Plasma Sources
Sci. Technol. 19, 025003 (2010).
10
Q. Y. Nie, Z. Cao, C. S. Ren, D. Z. Wang, and M. G. Kong, “A two-
dimensional cold atmospheric plasma jet array for uniform treatment of
large-area surfaces for plasma medicine,” New J. Phys. 11, 115015 (2009).
073505-9 Kim et al.
11
P. P. Sun, J. H. Cho, C. H. Park, S. J. Park, and J. G. Eden, “Close-packed
arrays of plasma jets emanating from microchannels in a transparent poly-
mer,” IEEE Trans. Plasma Sci. 40, 2946 (2012).
12
Z. Cao, J. L. Walsh, and M. G. Kong, “Atmospheric plasma jet array in
parallel electric and gas flow fields for three-dimensional surface
treatment,” Appl. Phys. Lett. 94, 021501 (2009).
13
R. Wang, H. Sun, W. Zhu, C. Zhang, S. Zhang, and T. Shao, “Uniformity
optimization and dynamic studies of plasma jet array interaction in argon,”
Phys. Plasmas 24, 093507 (2017).
14
C. Zhang, T. Shao, Y. Zhou, Z. Fang, P. Yan, and W. Yang, “Effect of O2
additive on spatial uniformity of atmospheric-pressure helium plasma jet
array driven by microsecond-duration pulses,” Appl. Phys. Lett. 105,
044102 (2014).
15
M. Wan, F. Liu, Z. Fang, B. Zhang, and H. Wan, “Influence of gas flow
and applied voltage on interaction of jets in a cross-field helium plasma jet
array,” Phys. Plasmas 24, 093514 (2017).
16
P.-M. Girard, A. Arbabian, M. Fleury, G. Bauville, V. Puech, M. Dutreix,
and J. Santos Sousa, “Synergistic effect of H2O2 and NO2 in cell death
induced by cold atmospheric He plasma,” Sci. Rep. 6, 29098 (2016).
17
W. V. Boxem, J. Van der Paal, Y. Gorbanev, S. Vanuytsel, E. Smits, S.
Dewilde, and A. Bogaerts, “Anti-cancer capacity of plasma treated PBS:
Effect of chemical composition on cancer cell cytotoxicity,” Sci. Rep. 7,
16478 (2017).
18
J. Ma, H. Zhang, C. Cheng, J. Shen, L. Bao, and W. Han, “Contribution of
hydrogen peroxide to non-thermal atmospheric pressure plasma induced
A549 lung cancer cell damage,” Plasma Process Polym. 14, e1600162
(2017).
19
R. Furuta, N. Kurake, K. Ishikawa, K. Takeda, H. Hashizume, H. Tanaka,
H. Kondo, M. Sekine, and M. Hori, “Intracellular responses to reactive
oxygen and nitrogen species, and lipid peroxidation in apoptotic cells cul-
tivated in plasma-activated medium,” Plasma Process Polym. 14,
e1700123 (2017).
20
P. Lu, D. Boehm, P. Bourke, and P. J. Cullen, “Achieving reactive species
specificity within plasma-activated water through selective generation
using air spark and glow discharges,” Plasma Process Polym. 14,
e1600207 (2017).
21
J. Duan, X. Lu, and G. He, “The selective effect of plasma activated
medium in an in vitro co-culture of liver cancer and normal cells,” J. Appl.
Phys. 121, 013302 (2017).
22
S. J. Kim, T. H. Chung, H. M. Joh, J. H. Cha, I. S. Eom, and H. J. Lee,
“Characteristics of multiple plasma plumes and formation of bullets in an
atmospheric-pressure plasma jet array,” IEEE Trans. Plasma Sci. 43, 753
(2015).
23
P. Lukes, E. Dolezalova, I. Sisrova, and M. Clupek, “Aqueous-phase
chemistry and bactericidal effects from an air discharge plasma in contact
with water: Evidence for the formation of peroxynitrite through a pseudo-
second-order post-discharge reaction of H2O2 and HNO2,” Plasma Sources
Sci. Technol. 23, 015019 (2014).
24
K. Ninomiya, T. Ishijima, M. Imamura, T. Yamahara, H. Enomoto, K.
Takahashi, Y. Tanaka, Y. Uesugi, and N. Shimizu, “Evaluation of extra-
and intracellular OH radical generation, cancer cell injury, and apoptosis
induced by a non-thermal atmospheric-pressure plasma jet,” J. Phys. D:
Appl. Phys. 46, 425401 (2013).
25
A. R. Gibson, H. O. McCarthy, A. A. Ali, D. O’Connell, and W. G.
Graham, “Interactions of a non-thermal atmospheric pressure plasma efflu-
ent with PC-3 prostate cancer cells,” Plasma Process. Polym. 11, 1142
(2014).
Phys. Plasmas 25, 073505 (2018)
26
A. Sobota, O. Guaitella, G. B. Sretenović, I. B. Krstić, V. V. Kovačević,
A. Obrusnık, Y. N. Nguyen, L. Zajıčkova, B. M. Obradović, and M. M.
Kuraica, “Electric field measurements in a kHz-driven He jet—The influ-
ence of the gas flow speed,” Plasma Sources Sci. Technol. 25, 065026
(2016).
27
X. Lu, G. V. Naidis, M. Laroussi, and K. Ostrikov, “Guided ionization
waves: Theory and experiments,” Phys. Rep. 540, 123 (2014).
28
G. Uchida, K. Takenaka, and Y. Setsuhara, “Influence of voltage pulse
width on the discharge characteristics in an atmospheric dielectric-barrier-
discharge plasma jet,” Jpn. J. Appl. Phys., Part 1 55, 01AH03 (2016).
29
E. J. Baek, H. M. Joh, S. J. Kim, and T. H. Chung, “Effects of the electri-
cal parameters and gas flow rate on the generation of reactive species in
liquids exposed to atmospheric pressure plasma jets,” Phys. Plasmas 23,
073515 (2016).
30
Y. Yue, X. Pei, and X. Lu, “OH density optimization in atmospheric-
pressure plasma jet by using multiple ring electrodes,” J. Appl. Phys. 119,
033301 (2016).
31
H. M. Joh, J. Y. Choi, S. J. Kim, T. H. Kang, and T. H. Chung, “Effects of
the pulse width on the reactive species production and DNA damage in
cancer cells exposed to atmospheric pressure microsecond-pulsed helium
plasma jets,” AIP Adv. 7, 085106 (2017).
32
K. Yambe, S. Muraoka, T. Nihei, and S. Abe, “Estimation of excitation
temperature by duty ratio of observed period in nonequilibrium plasma,”
Phys. Plasmas 24, 063512 (2017).
33
J. Jarrige, M. Laroussi, and E. Karakas, “Formation and dynamics of
plasma bullets in a non-thermal plasma jet: Influence of the high-voltage
parameters on the plume characteristics,” Plasma Sources Sci. Technol.
19, 065005 (2010).
34
K. Yambe and S. Satou, “Investigation of helium plasma temperature in
atmospheric-pressure plasma plume using line pair method,” Phys.
Plasmas 23, 023509 (2016).
35
A. Greb, K. Niemi, D. O’Connell, and T. Gans, “Energy resolved actinom-
etry for simultaneous measurement of atomic oxygen densities and local
mean electron energies in radio-frequency driven plasmas,” Appl. Phys.
Lett. 105, 234105 (2014).
36
J.-S. Boisvert, L. Stafford, N. Naude, J. Margot, and F. Massines,
“Electron density and temperature in an atmospheric-pressure helium dif-
fuse dielectric barrier discharge from kHz to MHz,” Plasma Sources Sci.
Technol. 27, 035005 (2018).
37
E. Desjardins, M. Laurent, A. Durocher-Jean, G. Laroche, N. Gherardi, N.
Naude, and L. Stafford, “Time-resolved study of the electron temperature
and number density of argon metastable atoms in argon-based dielectric
barrier discharges,” Plasma Sources Sci. Technol. 27, 015015 (2018).
38
E. J. Szili, J.-S. Oh, S.,-H. Hong, A. Hatta, and R. D. Short, “Probing the
transport of plasma-generated RONS in an Agarose target as surrogate for
real tissue: Dependency on time, distance and material composition,”
J. Phys. D: Appl. Phys. 48, 202001 (2015).
39
K. Wende, S. Bekeschus, A. Schmidt, L. Jatsch, S. Hasse, K. D.
Weltmann, K. Masura, and T. von Woedtke, “Risk assessment of a cold
argon plasma jet in respect to its mutagenicity,” Mutat. Res. 798–799, 48
(2016).
40
M. Horiba, T. Kamiya, H. Hara, and T. Adachi, “Cytoprotective effects of
mild plasma-activated medium against oxidative stress in human skin
fibroblasts,” Sci. Rep. 7, 42208 (2017).
41
T. Adachi, S. Nonomura, M. Horiba, T. Hirayama, T. Kamiya, H.
Nagasawa, and H. Hara, “Iron stimulates plasma-activated medium-
induced A549 cell injury,” Sci. Rep. 6, 20928 (2016).