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Deactivation of A549 cancer cells in vitro by a dielectric barrier discharge
plasma needle
Jun Huang,
1,a)
Wei Chen,
1
Hui Li,
1
Xing-Quan Wang,
1
Guo-Hua Lv,
1
M. Latif Khosa,
3
Ming Guo,
4
Ke-Cheng Feng,
4
Peng-Ye Wang,
1
and Si-Ze Yang
1,2
1
The Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China
2
Fujian Key Laboratory for Plasma and Magnetic Resonance, Department of Aeronautics, School of Physics
and Mechanical & Electrical Engineering, Xiamen University, Xiamen 361005, China
3
Pakistan Institute of Technology for Mineral and Advanced Engineering Material, Ferozepur Road,
Lahore-54600, Pakistan
4
College of Science, Changchun University of Science and Technology, Changchun 130022, China
(Received 16 October 2010; accepted 7 January 2011; published online 14 March 2011)
An inactivation mechanism study on A549 cancer cells by means of a dielectric barrier discharge
plasma needle is presented. The neutral red uptake assay provides a quantitative estimation of cell
viability after plasma treatment. Experimental results show that the efficiency of argon plasma for the
inactivation process is very dependent on power and treatment time. A 27 W power and 120 s
treatment time along with 900 standard cubic centimeter per minute Ar flow and a nozzle-to-sample
separation of 3 mm are the best parameters of the process. According to the argon emission spectra
of the plasma jet and the optical microscope images of the A549 cells after plasma treatment, it is
concluded that the reactive species (for example, OH and O) in the argon plasma play a major role in
the cell deactivation. V
C2011 American Institute of Physics. [doi:10.1063/1.3553873]
I. INTRODUCTION
Over the past 20 years, there has been a growing interest
in developing nonthermal atmospheric-pressure plasmas,
particularly for biomedical applications
1–23
such as bacterial
inactivation,
1,5,10,13,16–18,22
wound healing,
7
dental bleach-
ing,
8
and treatment of cancer cells.
6,9,23
Other conventional treatments for cancers mainly include
intravascular interventional therapeutic
24
and percutaneous
physical ablation.
25
The former has relatively severe side
effect,
26
and the latter takes longer time than the former.
27
An alternative method for cancer treatment is adopting
nonthermal plasma, which has been proven to have less side
effects and high efficiency. Plasma treatments offer the possi-
bility of disposing pathological cells (cancer) and unwanted tis-
sues without inflammation and excessive damage to the body.
Such finess and precision are difficult to realize in conventional
surgical methods. A plasma needle that was first developed in
the Eindhoven University of Technology
28
in 2002 for biomed-
ical applications is a refined surgical tool that can generate a
nonthermal plasma at atmospheric pressure.
15,17,18
The dielectric barrier discharge (DBD) is a type of silent
discharge, and its electron emission is mainly caused by the
ion-induced secondary emission from the dielectric barrier.
Its unique advantage is that low excited atomic and molecu-
lar species, free radicals, and excimers with several electron
volts of energy can be produced. Under most operating con-
ditions, a DBD consists of a (large) number of discharge fila-
ments, which have a nanosecond duration and are randomly
distributed over the dielectric surface. These filaments, also
known as microdischarges, are the active regions of a DBD
in which active chemical species and UV/VUV radiation can
be produced. These microdischarges act as individual dis-
charges that work independently of one another.
In this paper, a sinusoidal alternating voltage was
applied to the electrodes to generate the filamentary dis-
charge. The funnel-shaped nozzle of plasma needle guaran-
tees a low gas temperature. In addition, the local treatment
with high precision can be realized. Here we study the fol-
lowing three aspects: electrical properties, inactivation effec-
tiveness, and optical emission spectra. The rest of the paper
is organized as follows. The experimental setup is described
in Sec. II. Section III presents the experimental results and
discussion related to the preceding three aspects. Finally,
conclusions are given in Sec. IV.
II. EXPERIMENT
A. Plasma needle
Figure 1(a) shows the schematic diagram of the experi-
mental setup. A similar plasma needle device has been
reported in our previous work.
22
It comprises five parts: a
quartz tube, two electrodes, a rubber plug, an inner Teflon
fittings, and a funnel-shaped nozzle. A quartz tube (6 mm
i.d. 8 mm o.d. 100 mm L) serves as the dielectric barrier
layer, which acts as a current limiter and prevents the transi-
tion to an arc. At the center of the quartz tube, a stainless
steel tube (0.9 mm i.d. 1/16 in. o.d., fixed by a perforated
Teflon fitting) is used as the inner electrode. Meanwhile, the
steel tube is also fixed by a rubber plug that provides an air-
tight seal for the device. A copper braid is used as the outer
electrode partially covering the outer surface of the quartz
tube. A funnel-shaped quartz nozzle (inner diameter tapering
from 6 to 1 mm) is attached to the end of the quartz tube.
This specially designed nozzle provides a focusing effect so
a)
Author to whom correspondence should be addressed. Electronic mail:
hjflower@aphy.iphy.ac.cn.
0021-8979/2011/109(5)/053305/6/$30.00 V
C2011 American Institute of Physics109, 053305-1
JOURNAL OF APPLIED PHYSICS 109, 053305 (2011)
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that more plasma activated species can be directed toward the
sample. The separation between the end of the inner steel
electrode and the end of the quartz tube is 2 mm. Both elec-
trodes are connected to an ac power with a peak-peak voltage
up to 30 kV with the frequency ranging from 8 to 40 kHz.
Throughout the experimental procedure, the working gas
used was argon with a flow rate of 900 standard cubic centi-
meter per minute (SCCM) and a frequency of 11.55 kHz. The
separation between the nozzle and the surface of Dulbecco’s
modified Eagle medium (DMEM) is 1 mm and the thickness
of DMEM is 2 mm.
Figure 1(b) shows the image of the plasma jet with 900
SCCM Ar (P
j
¼18 W). The plasma plume filled with pure
argon has a length of 27 mm.
B. Sample preparation
Human lung cancer A549 cells (Cell Resource Center,
IBMS, CAMS/PUMS) were maintained in Dulbecco’s modified
Eagle medium (DMEM, GIBCO) with 1 0% fetal bovine serum
(FBS, GIBCO) and 1% penicillin-streptomycin (GIBCO) at
37 Cin5%CO
2
incubator. The cells were seeded in 96-well
culture plate at the density of 5 104cells ml
1
, 1 day before
the experiment.
III. RESULTS AND DISCUSSION
A. Characterization of the plume
Figure 2shows the effect of power on plume length.
Before the power value reaches a critical point (27 W), the
plume length becomes longer with increasing power. How-
ever, the plume length begins to decrease and form a reverse
Y-shape at the reactor outlet after a power point (27 W). As
the power continuously increases, the plasma plume
becomes shorter, and its color becomes darker and deeper. In
the absence of plasma, the mean Ar velocity in the reactor
outlet is about 19.1 ms
1
, with a corresponding Reynolds
number (Re) of 24.3, which is far lower than the Re (205)
detected by Le´veille´ and Clulombe.
29
Therefore the Ar
shows a laminar flow condition, and it is helps to form a uni-
form plasma plume. We may conclude that the microfila-
ment discharge has no obvious effect on the plasma plume,
and the ionized gas flow is still in a laminar state in the initial
stage. With the power increasing, the discharge becomes
stronger, and the microfilaments changes to micro-arcs and
cruises more frequently at the end of the inner electrode.
These factors lead to more vortices being produced, which
transform the laminar flow to a turbulent one at the outlet of
the reactor. This may be the main reason why the plasma
plume becomes short, asymmetric, and unstable.
Figure 3represents the waveforms of applied voltage
and discharge current. We applied a sinusoidal resonant
power supply to the two electrodes to ignite the discharges in
Ar. In this work, the operating parameters of the sine wave
ac power supply were set at 14.2 kV for the peak to peak
voltage and 11.55 kHz for the frequency. The high voltage
applied to the discharging electrode of the DBD plasma nee-
dle was measured by a 1000:1 high voltage probe (Tektronix
P6015A, maximum input voltage: dc 20 kV, bandwidth:
FIG. 1. (Color online) (a) Schematic of the experimental setup. (b) Image of
the plasma jet with 900 SCCM Ar (P
j
¼18 W).
FIG. 2. (Color online) The effect of power on plume length.
053305-2 Huang et al. J. Appl. Phys. 109, 053305 (2011)
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75 MHz), and the discharge current was measured by a cur-
rent probe (Tektronix P6021, maximum discharge current:
15A, bandwidth: 60 MHz). The signals collected by the
probes were recorded by a digital Tektronix TDS 210 oscillo-
scope. It can be seen that the breakdown of working gas in
DBD, resulting in a large number of current filaments, the so-
called microdischarges, which are randomly distributed both
in time and space. The number of microdischarges is propor-
tional to the voltage applied on the electrodes. In this filamen-
tary mode, the discharge starts with local gas breakdown at
many points in the discharge volume. This mode is character-
ized by a periodic current constituted by many discharge
pulses in each half cycle. An inverse current peak is also
observed when the polarity of the applied voltage changes.
B. Neutral red uptake assay for the estimation of cell
viability
Theneutralreduptakeassaywasusedtoobtainaquanti-
tative estimation of the number of viable cells after plasma
treatment. It is based on the ability of viable cells to incorpo-
rate and bind the supravital dye neutral red in the lysosomes.
When the cell dies, the dye cannot be retained. It is thus possi-
ble to distinguish among viable, damaged, or dead cells
according to their specific lysosomal capacity for taking up the
dye. According to the published protocol,
29
there is a linear
relationship between the neutral red extractable from a culture
and the number of viable cells in that culture. Consequently,
the optical density (OD) value of neutral red extract is propor-
tional to the number of viable cells.
In our experiment, 4000 human lung cancer A549 cells
were seeded into each well bottom of 96-well plate and cul-
tured 2 mm deep in DMEM. The thin, long nozzle was put in
one of the wells of 96-well plat. The separation between the
plasma nozzle and the medium surface was 1 mm and the gas
flow rate was 900 SCCM. The applied power and the expo-
sure time were adjusted. It should be noted that the control ex-
perimental cells were treated by the working gas flowing at
the same flow rate with plasma off. After plasma treatment,
the plate was continuously incubated for 24 h at appropriate
condition. Then we aspirated off medium from cells, washed
the cells with phosphate buffered saline (PBS) per well, and
removed the washing solution by gentle tapping. The neutral
red medium was added to each well of the plate, the plate
were incubated for 2 h at the appropriate culture conditions.
Later we inspected the plate with an inverted microscope
(Olympus IX70), removed the neutral red medium, and
washed the cells with PBS per well. The neutral red destain
solution was added to each well. Subsequently, the plate was
rapidly shook on a microtiter plate shaker until the neutral red
has been extracted from the cells and has formed a homogene-
ous solution. At last the OD of neutral red extract was meas-
ured at 540 nm in a spectrophotometer (U-3010, Hitachi),
using blanks that contain no cells as a reference.
Figure 4is a typical picture of dead and viable A549
cells after plasma treatment. They usually differ markedly in
morphology. Apparently, dead cells have the following char-
acteristics: the cytoskeleton is broken down, the cells shrink
and display blebs, and finally they fall apart and become
shapeless cell fragments. However, the viable cells show
regular cell growth and can incorporate the supravital dye
neutral red. A boundary is clearly seen between the dead and
viable cells. A 1 mm diameter gas outlet nozzle ensures the
accuracy of the treated areas. Thus all cells within the treated
spot were destroyed, while adjacent cells outside the bound-
ary were left unaffected.
Figure 5shows the survival curves of A549 cells in ar-
gon plasma with different applied powers and exposure time.
The vertical axis is a ratio of OD value to the OD
0
value of
the untreated cells, where the OD and OD
0
are the average
of triplet wells. The reference value of OD/OD
0
represents
survival ratio of the treated cells compared to the control
group. It can be seen that the efficiency was somewhat
FIG. 3. (Color online) Typical oscillograms of applied voltage and dis-
charge current of a filamentary discharge (FD) in Ar at 11.55 kHZ excitation
frequency.
FIG. 4. (Color online) Morphological differences between the dead cells
and the viable cells stained with neutral red under inverted microscope
(Olympus IX70).
053305-3 Huang et al. J. Appl. Phys. 109, 053305 (2011)
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proportional to the applied power and the exposure time.
When the power was increased to 27 W, the value of OD/
OD
0
became 6% at 120 s. Beyond 27 W, the plasma became
unstable. The experimental data show that the optimal effi-
ciency of cancer cells deactivation by Ar plasma can be
obtained when 27 W applied power and 120 s exposure time
are used.
It is agreed that the active species of the plasma (radicals
and ions) undergo interactions with the living cells and
induce specific responses at the cellular level.
31–33
Because
these species are unstable and easily consumed in various
reactions, the effects of the plasma treatment are strictly
localized. However, the penetration depth can be extended
by prolonging the treatment time;
19
this agrees well with our
experiments results.
There was a typical saturation effect in plasma-cell
interactions. Results were obtained above a certain threshold,
and further increase of plasma dosage was of no avail.
C. Optical-emission spectra
Optical emission spectroscopy (OES) was applied to
identify the various reactive species generated by the plasma
plume. The wavelength and band lines of the spectra were
characteristic of the excited species. They were quite useful
for diagnostic purposes.
Figure 6is a typical UV-visible emission spectrum (Stel-
larnet, EPP-2000C) in the 200–850 nm regions from sine wave
ac driven (11.55 kHz) DBD plasma at atmospheric pressure.
The optical emission spectra shows three main regions. In the
200–300 nm region, the emission is dominated by the vibra-
tional bands of the NO-r system (A–X),
34
whereas its emission
intensity is quite weak as compared with that of the 300–400
and 690–850 nm regions. The exact reaction mechanism in the
plasma could not be unveiled yet. However, it is expected
that there are two possibilities of the nitric oxide production.
One possibility is that NO is formed by electron-impact disso-
ciation of the ambient N
2
and O
2
gases, which can penetrate
into the active discharge region, and subsequently three-body
recombination N þOþX!NO þXoccurs.
35
Another possi-
bility is believed to be the breaking of oxygen molecules in
plasma to react with nitrogen as given in the reaction
NþO
2
!NO þO.
35
This is in fact a likely explanation for
weak intensity of NO because of the limited presence of air
mixture; on the other hand, it is further oxidized to NO
2
by O
radicals in the plasma or O
2
from the surroundings.
In contrast, the total emitted optical power is the highest
in the 300–400 nm region. We cannot find any atomic argon
emission lines in this region. The strongest lines originate
from the OH radical and molecular nitrogen N
2
(C–B). We
assign the feature at 309 nm to the OH (A
2
R
þ
X
2
Q) tran-
sition
36,37
the formation of which is attributed to the reaction
of excited O with water vapor (either from ambient air or as
a feed gas impurity) (H
2
OþO*!2OH)
29
and the electron
impact dissociation (H
2
Oþe
!HþOH þe
).
29
The pres-
ence of a N
2
line clearly reveals the air entrainment in the re-
actor. Prominent molecular nitrogen lines in this spectral
region include bands from the second positive systems [N
2
(C
3
Q
u
!B
3
Q
g
)] at 337, 358, and 380 nm. N
2
(C
3
Q
u
)
is produced by direct excitation, whereas production of
N
2
(B
3
Q
g
) is caused by direct excitation and transition from
N
2
(C
3
Q
u
).
38
Strong bands of the first negative system of
N
2þ
(B
2
Rþ
u–X
2
Rþ
g) near 391.4 nm are not detected in this
region because argon metastables (in the argon plasmas)
have insufficient energy to create N
2þ
levels.
34
Excitation of
species depends mainly on their collisions with electrons.
For the (0,0) vibrational bands, the energy levels for OH
(A
2
R
þ
), N
2
(C
3
Q
u
) and N
2þ
(B
2
Rþ
u) are approximately 3.7,
11, and 18.76 eV, respectively (relative to their neutral
ground states). Therefore the presence of spectral line OH
(A
2
R
þ
X
2
Q) and N
2
(C
3
Q
u
!B
3
Q
g
) demonstrates that
part of the electron’s energy is higher than 11 eV in the
plasma. The absence of the N
2þ
(B
2
Rþ
u) line indicates that
the electron’s energy is rarely higher than 18.76 eV in the
plasma. In principle, one can directly detect the N
2
(A
3
Rþ
u)
state the excitation potential of which is lower than that of
N
2
(C
3
Q
u
) when emitted from the Vegard–Kaplan system
[N
2
(A
3
Rþ
uX
1
Rþ
g)]. However, in atmospheric pressure
FIG. 5. (Color online) Survival curves of A549 cells in 900 SCCM Ar with
different applied powers and exposure time at a nozzle-to-sample separation
of 3 mm.
FIG. 6. (Color online) Emission spectrum of plasma with 900 SCCM Ar
taken at 3 mm bottom the plume and P
j
¼18 W.
053305-4 Huang et al. J. Appl. Phys. 109, 053305 (2011)
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plasmas, the Vegard–Kaplan system is usually dominated by
the second positive system, N
2
(C
3
Q
u
!B
3
Q
g
),
39
which is
the case in our plasmas. Furthermore, argon metastables can
selectively populate higher excited states of nitrogen, N
2
(C
3
Q
u
) and N
2
(B
3
Q
g
) by two-body collisions that cascade
radiatively down to N
2
(A) level.
40,41
In the 690–850 nm region, the emitted plasma light con-
sists mainly of the excited argon atoms Ar* (709, 729, 797,
811, and 826 nm), the primary argon ion Ar
þ
(803 nm), and
the secondary argon ion Ar
2þ
(774 nm) lines. This shows the
argon atom is easily excited and even ionized in argon dis-
charge. Due to the lower ionization energy of the argon
atom, it is favorable to form abundant active particles in ar-
gon plasmas. These particles can be obtained through reac-
tions as follows:
eþAr !Ar* þe(1)
htþAr !Ar* (2)
eþAr !Arþþ2e (3)
htþAr !Arþþe (4)
eþArþ!Ar2þþ2e (5)
htþArþ!Ar2þþe (6)
Additionally, the optical emission spectroscopy also reveals
the presence of atomic nitrogen (698 nm, 740 nm), primary
nitrogen ion N
þ
(765 nm), and OI (844 nm) lines. Although
the short-lived atomic nitrogen can more easily recombine to
form molecular nitrogen N
2
,
42
the atomic nitrogen lines can
be still detected in the spectral lines because an optical fiber
head is nearby the plasma region. As mentioned before, the
absence of the N
2þ
(B
2
Rþ
u) line indicates that the electron’s
energy is rarely higher than 18.76 eV in the plasma, but the
presence of N
þ
shows that part of the electron’s energy is
higher than 18.07 eV. This is because N
þ
is primarily pro-
duced via the following reactions where the energies are
higher than 18.07 eV:
43
eþN2¼NþþNþ2e DH¼24:29 eV (7)
eþN2ðAÞ¼NþþNþ2e DH¼18:07 eV (8)
Atomic oxygen is generally generated by a dissociative colli-
sion between an oxygen molecule and an electron
(e
þO
2
!OþOþe
).
44
O may also be generated through
Penning ionization (N
2
*þO
2
!N
2
þOþO).
45,46
Figure 7shows the emission spectrum of the plasma
with 900 SCCM Ar flow taken at 2 mm deep in DMEM
along axis.
In the atmosphere and in DMEM, the spectra are obvi-
ously different. In the 200–300 nm region, no molecular NO
emission lines are found. The intensity of the UV radiation
in this region is undetectable. In addition, the inactivation
effect on a cell by ultraviolet radiation is mainly related to
the DNA/RNA damage in UV-C (200–280 nm).
47
This indi-
cates that UV emission does not play a significant role in the
inactivation process. In the 300–400 nm region, the intensity
of all the N
2
line at 337,358,380 nm reduced rapidly, while
the OH line (309 nm) has no remarkable change. This dem-
onstrates that the DMEM solution absorbs the N
2
line but
cannot absorb OH radicals. Because A549 cells were seeded
at the bottom of wells of 96-well plate, the N
2
species cannot
approach the bottom of the solution and cannot play any role
in killing the cancer cells. The OH radicals can enter DMEM
and diffuse around, so they can act on cells and cause cell
death. In the 690–850 nm region, the intensities of all lines
have a little rise as compared to those detected in atmos-
phere. It is considered that the high electron density allows
the argon plasma jet to produce more radical or excited oxy-
gen species and allows the heavy argon gas to create a path-
way more convenient for active species (for example, O or
OH) to reach the cell.
48
This mechanism plays an important
role in cell death.
49,50
It indicated that O and OH that can
pass through the cell membrane and are critical to cell death
because of a stronger oxidation mechanism. We suppose that
the charged species also play a role in plasma-cell interac-
tions. The action of ions may be similar to that of the radicals
because ions can produce radicals by dissociating water mol-
ecules in the vicinity of the cell. If an ion reaches the
exposed A549 cell membrane, affects the membrane poten-
tial, and disturbs the Na-K pump,
19
the result might be fatal
for the cell.
To increase the concentration of OH and O radicals, we
can increase the power. However, the length of plasma
plume shortened rapidly when the power is above 27 W. As
a result, the intensity of needle plasma becomes weaker.
This leads to less ultraviolet rays and radicals in solution cor-
respondingly. Besides, with increasing treatment time, the
chemically active species may cause DNA and gene damage,
which can induce cell apoptosis process.
51
In our experi-
ment, the results showed that the power of 27 W and a treat-
ment time of 120 s were the best parameters of operation.
IV. CONCLUSION
In conclusion, inactivating human lung cancer A549
cells by a DBD plasma needle with a funnel-shaped nozzle is
presented. The typical oscillograms of applied voltage and
FIG. 7. (Color online) Emission spectrum of the plasma with 900 SCCM Ar
taken at 2 mm deep in DMEM and P
j
¼18 W.
053305-5 Huang et al. J. Appl. Phys. 109, 053305 (2011)
Downloaded 10 Jun 2011 to 159.226.35.233. Redistribution subject to AIP license or copyright; see http://jap.aip.org/about/rights_and_permissions
discharge current reveal the characteristics of plasma plume.
After plasma treatments, obvious morphological changes in
A549 cancer cells can be observed under inverted micro-
scope, and neutral red uptake assay provides a quantitative
estimation of cell viability. The experimental results show
that increasing applied power and prolonging exposure time
can improve the efficiency of ablating the cultured A549
cancer cells in vitro. In addition, optical emission spectros-
copy clearly indicates that excited OH, O, N
2
,N
þ
, Ar, Ar
þ
,
and Ar
2þ
exist in the plasma plume. These radicals are
mainly responsible for cell deactivation.
ACKNOWLEDGMENTS
This work were partially supported by the National Nat-
ural Science Foundation of China under Grant No. 10735090
and the Young Scientists Fund of the National Natural Sci-
ence Foundation of China under Grant No. 11005151.
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