On the design and characterization of a new cold atmospheric pressure plasma jet and its applications on cancer cells treatment Morteza Akhlaghi, Hajar Rajayi, Amir Shahriar Mashayekh, Mohammadreza Khani, Zuhair Mohammad Hassan, and Babak Shokri Citation: Biointerphases 10, 029510 (2015); doi: 10.1116/1.4918806 View online: http://dx.doi.org/10.1116/1.4918806 View Table of Contents: http://scitation.aip.org/content/avs/journal/bip/10/2?ver=pdfcov Published by the AVS: Science & Technology of Materials, Interfaces, and Processing Articles you may be interested in Comparison of the characteristics of atmospheric pressure plasma jets using different working gases and applications to plasma-cancer cell interactions AIP Advances 3, 092128 (2013); 10.1063/1.4823484 DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets Appl. Phys. Lett. 102, 233703 (2013); 10.1063/1.4809830 Reactive oxygen species-related plasma effects on the apoptosis of human bladder cancer cells in atmospheric pressure pulsed plasma jets Appl. Phys. Lett. 101, 053703 (2012); 10.1063/1.4742742 Deactivation of A549 cancer cells in vitro by a dielectric barrier discharge plasma needle J. Appl. Phys. 109, 053305 (2011); 10.1063/1.3553873 Atmospheric-pressure plasma-jet from micronozzle array and its biological effects on living cells for cancer therapy Appl. Phys. Lett. 98, 073701 (2011); 10.1063/1.3555434
On the design and characterization of a new cold atmospheric pressure plasma jet and its applications on cancer cells treatment Morteza Akhlaghi Department of Physics, Shahid Beheshti University, Tehran 19839, Iran
Hajar Rajayi Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115, Iran
Amir Shahriar Mashayekh Department of Physics, Shahid Beheshti University, Tehran 19839, Iran
Mohammadreza Khani Laser and Plasma Research Institute, Shahid Beheshti University 19839, Tehran, Iran
Zuhair Mohammad Hassan Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115, Iran
Babak Shokria) Department of Physics, Shahid Beheshti University, 19839 Tehran, Iran and Laser-Plasma Research Institute, Shahid Beheshti University, 19839 Tehran, Iran
(Received 31 January 2015; accepted 10 April 2015; published 23 April 2015) In this paper, a new configuration of a cold atmospheric pressure plasma jet has been designed and constructed. Poly-methyl-methacrylate was used as a new dielectric in this configuration which in comparison to other dielectrics is inexpensive, more resistant against break, and also more shapeable. Then, the plasma jet parameters such as plume temperature, rotational and vibrational temperatures, power, electrical behavior (voltage and current profile), electron density, and the produced reactive species were characterized. In order to determine the jet temperature and the amount of reactive species, effects of applied voltage, gas flow rate, and distance from the nozzle were studied. The power of the jet was specified using Lissajous curve approach. The plume temperature of the plasma jet was about the room temperature. Optical emission spectroscopy determined the type of reactive species, and also electron density and its corresponding plasma frequency (6.4 1013 cm3 and 4.52 1011 Hz). Because of producing different reactive species, the device can be used in different applications, especially in plasma medicine. Thus, 4T1 cancer cells were treated using this plasma jet. The results showed that this plasma jet has a great potential C 2015 American Vacuum to kill one of the most aggressive and resistant cancerous cell lines. V Society. [http://dx.doi.org/10.1116/1.4918806] I. INTRODUCTION Cold atmospheric pressure plasma (CAP) as a kind of nonequilibrium plasma can be produced in different configurations employed for the particular application.1 Cold plasmas are usually produced by a dielectric barrier discharge (DBD).2 The presence of a dielectric between electrodes prevents the transition to an arc discharge and makes plasma cold. Nowadays, this kind of plasma has been used in many fields, especially in biomedical applications.3 Because of producing reactive oxygen and nitrogen species, especially atomic oxygen, hydroxyl radicals, and nitrogen oxides, these systems can affect live cells and organisms, microbes, etc. However, it should be mentioned that some applications of plasma in medicine relied mainly on its thermal effects.4 It was suggested that some species have “killing” or “healing” effects.5 One can use desirable configuration for different purposes, e.g., wound healing,6 decontamination of bacteria,7 cancer treatment,8 allergen inactivation,9 root canal disinfection,10 and vegetables teatment.11 The use of plasmas a)
Electronic mail: [email protected]
029510-1 Biointerphases 10(2), June 2015
for biomedical purposes can be tracked back to the 1990s where Laroussi used atmospheric pressure plasma to inactivate microorganisms.12 At the early ages of 2000s, Stoffels et al. introduced the plasma needle for fine surface treatment of biomaterials.13 Since then, various devices and configurations were presented by different groups.1,14 In nonequilibrium plasmas, the electrons drive the plasma chemistry while the heavy particles remain at low temperature. So, there is no need for elevated gas temperatures.15 Determining the electron density by probe methods is prevalent. However, probe diagnostics are not only hampered by the small dimensions of some atmospheric pressure plasmas but also by the difficulty of interpretation of the measurements due to the collisionality of plasma.16 There is another indirect way using optical emission spectroscopy (OES).17 In this method, Stark broadening is used to determine the electron density and plasma frequency. OES provides a means to determine some reactive species produced in plasma and also shows the role of important parameters such as applied voltage, gas flow, and distance from the nozzle in producing reactive species.18
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C 2015 American Vacuum Society V
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029510-2 Akhlaghi et al.: On the design and characterization of a new cold atmospheric pressure plasma jet
Temperature is one of the major parameters especially for biomedical applications where a temperature less than body temperature is required. There are several ways and tools to measure it.19,20 A thermometer as well as OES was used to measure the temperature. Also voltage, gas flow rate, and distance from the nozzle as three parameters affecting the temperature were considered. As mentioned above, these parameters have effects on producing reactive species as well. Rotational and vibrational temperatures were determined by OES. CAP systems usually consume low power, a maximum of order of several watts. For power measurement, there are different ways. The most common way is the measurement of voltage and current by probes and calculation of the power using these parameters. However, there is Lissajous curve approach, which is more precise and has been customary recently.21 In this method, a capacitor is series with the DBD reactor. Measuring its voltage and a brief calculation would give the power.22 Voltage and current profiles were obtained as usual by voltage and current probes. Many scientists use a metal rod or pipe inside a dielectric tube as inner electrode and a metal ring as outer electrode to produce CAP.23,24 With this configuration, the electric field along the plasma plume is enhanced. Walsh and Kong showed that a high electric field along the plasma plume is favorable for generating long plasma plumes and more active plasma chemistry.25 In biological and biomedical applications, it is important that plasma be without electric shocks. Therefore, the grounded ring electrode was placed at the end of the torch, around the plasma jet exit aperture to neutralize the jet and reduce the risk of electric shocks. Quartz, glass, and ceramic are used frequently as dielectrics to produce CAP, but there are some difficulties. First, these materials are very brittle. In addition, it is difficult to form them into desired shape, and above all they are expensive (especially quartz). Thus, in this work, poly-methylmethacrylate (PMMA) polymer was used as a new dielectric which in comparison to other dielectrics is inexpensive, more resistant against break, and also more shapeable. Another advantage of PMMA respect to other materials and dielectrics is its transparency characteristic, which let us see what happens inside the discharge zone. In order to show the great potential of the plasma jet, murine breast cancer cell line (4T1) was treated with a series of doses. Scientists have studied different breast cancer cell lines earlier. Wang et al. showed that CAP can selectively ablate metastatic BrCa cells in vitro without damaging healthy human mesenchymal stem cells.26 Mirpour et al. studied MCF7 cell line and showed that longer treatment time had serious damage to cancer cells while it had no significant effect on normal cells.27 Another study on MCF7 was performed by Kim et al., which demonstrated that a significant portion of human breast cancer cells exhibits apoptotic fragmentation by applying a pulsed atmospheric pressure plasma jet.28 This paper introduced a new configuration for generating a cold atmospheric pressure plasma jet with a new dielectric, Biointerphases, Vol. 10, No. 2, June 2015
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temperature of order of room temperature, low power consumption, stable performance, and without electrical shocks. In addition, properties such as jet temperature, electron density, rotational and vibrational temperatures, electrical behavior, power consumption, and produced reactive species have been considered by the newest methods. It will be shown that against other configurations, in this configuration, current pulses would be eliminated. As a new application, the plasma jet then treated 4T1 cancer cells, one of the most aggressive and resistant cell lines. The Methylthiazol Tetrazolium (MTT) assay results demonstrated its great potential and proficiency. II. EXPERIMENTAL SETUP AND METHODOLOGY A. Configuration of the plasma jet reactor
To generate CAP, a copper pipe and a steel ring were used as electrodes. As mentioned earlier, the grounded ring electrode was placed at the end of the torch, around the plasma jet exit aperture to neutralize the plasma jet and reduce the risk of electric shocks. As shown in Fig. 1, in this situation, the grounded electrode was located between the high voltage electrode and sample, and works as a beam neutralizer. The PMMA tube was used as the dielectric between electrodes. Its dielectric properties were characterized by Basavaraja Sannakki and Anita.29 In comparison to other dielectrics, PMMA is inexpensive, more resistant against break, and also more shapeable. Because of low breakdown voltage and capability of producing uniform plasma, helium was selected as the carrier gas. A mass flow controller (APEX, AX-MC-5SLPM-D) was used to control the gas flow. A high-voltage high-frequency power supply (Information Unlimited Corporation, DIDRIV 10) with sinusoidal waveform ran the plasma jet. Its voltage and frequency were variable, but all experiments were done at 25 kHz. The plasma reactor could give stable
FIG. 1. (a) Side view, (b) three-dimension shape, and (c) generated plasma of the reactor.
029510-3 Akhlaghi et al.: On the design and characterization of a new cold atmospheric pressure plasma jet
plasma with almost 35 mm plume length at 4 standard liter per minute (SLPM) gas flow.
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capacitor and capacitor start storing electrical charges. The following equation would give us the power: q ¼ Cc Vc ;
B. Characterization methods
The first important parameter of plasma in biomedical and biological applications is its temperature. The plasma jet temperature was measured by a digital thermometer (JUMO, dtron 304) with a ceramic sensor (PT-100). Measurements were done in different conditions. In all experiments, the plasma jet temperature was measured in 1, 2, 3, 4, and 5 min after running the reactor. Other parameters affecting the plasma jet temperature are voltage, gas flow, and distance from the tip of the nozzle. In order to consider these parameters, the gas flow was fixed at 4 SLPM, and then, the temperature was measured at different voltages and distances. In order to determine the effect of gas flow, the voltage was fixed at 4 kV, and the temperature was measured at 15 mm distance at different gas flows. Another important parameter is electrical behavior, which is determined by time variation of voltage and current curves studied by a high voltage probe (Tekteronix, P6015A) and a current probe (Tektronix, TCP202). Displacement and discharge currents can be specified separately by using these probes. Displacement current is created when a voltage is applied to the separated electrodes. The discharge current refers to the motion of the charged particles. Therefore, when plasma is on, we have both displacement and discharge currents. Turning off plasma eliminates the discharge current. So, to determine displacement and discharge currents, the current should be measured in both plasma on and plasma off conditions Plasma On :
Ion ¼ Idispl þ Idisch ;
(1)
Plasma Off :
Ioff ¼ Idispl ;
(2)
Idisch ¼ Ion Ioff :
(3)
The power of the reactor was measured by Lissajous curve approach. In this approach, a capacitor is series with the plasma jet reactor, as shown in Fig. 2. Therefore, whole transmitted current from the reactor would pass through the
Cr ¼
q Vreactor
(4) ;
(5)
þ þ dq dt ¼ f Cr V ðtÞdV; Power ¼ f E ¼ f V ðtÞ dt
(6)
T
where q is amount of charge stored in capacitor, Cc is the capacitance of the capacitor, Vc is voltage of the capacitor, Cr is capacitance of the reactor, V(t) is the applied voltage, and f is the applied voltage frequency.22 The last and also the most important property is capability of producing different and suitable reactive species. This feature and also electron density usually was considered by optical emission spectroscopy. Here, a spectrometer (AVANTES, AvaSpec 3648) was used for recording the emitted spectrum and determining the produced reactive species. There are several parameters that may affect reactive species production. Therefore, in the present work, the effects of voltage, gas flow, and distance from nozzle were considered. One could characterize the rotational and vibrational temperatures through comparing experimental and simulated spectra. Here, wavelengths 370–381 nm (nitrogen second positive system transition) and specair software were used for simulation and comparison of spectra. The software shows the rotational and vibrational temperatures when the experimental and simulated spectra are fitted. The electron and ion bombardments may destroy the bacteria cell membrane and then the activated oxygen and nitrogen species penetrating into the cell kill the bacteria.30 Therefore, electrons play an important role in bioplasma. OES can be used to determine the electron density and the plasma frequency likewise. The electron density corresponds to Stark broadening as follows:31 Dkstark ¼ 2 1011 ðn2=3 e Þ; where ne is in cm3, and plasma frequency is32 pffiffiffiffiffi xp ¼ 5:65 104 ne :
(7)
(8)
Therefore, knowing Stark broadening leads to compute the electron density. Stark broadening was considered at wavelength 434 nm for H-gamma. Stark broadening is related to Lorentz and van der Waals broadenings as follows: DkLorentz ¼ DkStark þ Dkvan der Waals :
(9)
van der Waals broadening corresponds to gas temperature31 Dkvan der Waals ¼ 3:6
FIG. 2. Power measurement circuit (Lissajous curve approach). Biointerphases, Vol. 10, No. 2, June 2015
P ; T 0:7
(10)
where p is the pressure in atmosphere and T is the gas temperature in Kelvin.
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To know Lorentz broadening, one could fit the experimental spectrum with Voigt profile which is superposition of Gauss and Lorentz broadenings. Fitting was performed using Origin software and Lorentzian full width at half maximum was determined. Thus, knowing Lorentz and van der Waals broadenings, Stark broadening is determined, and as a result, the electron density and plasma frequency can be found. C. Cells preparation and assay
In order to investigate anticancer effects of the plasma jet, murine breast cancer cell line (4T1) was obtained from National Cell Bank of Pasteur Institute of Iran. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM L-glutamine, supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 100 U/ml penicillin, 100 lg/ml streptomycin solution (Gibco, USA) in 5% CO2 and 95% O2 at 37 C. The cytotoxicity of plasma and produced reactive species was studied by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. 4T1 cells at density of 16 000 per well in a volume of 200 ll DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin solution (Gibco, USA) were seeded on three 96-well cell culture plates (Grinner, Germany). They were treated with a series of doses (30, 60, 90, 120, 150, and 180 s plasma treatment). Also, an MgF2 filter, which only transmits UV wavelength, was used to consider UV production by the plasma jet and its effects on cancer cells. Then, MTT at the final concentration of 0.5 mg/ml was added to each well at different times (0, 24, and 48 h) after treatment. After incubation of 4 h and removing the medium of each well, 100 ll dimethyl sulfoxide was added to dissolve the blue formazan crystals. The absorbance was further measured at 540 nm using enzyme-linked immunosorbent assay reader (Multiscan, Finland). The viability of cells was presented as the percentage of control as follows: Viability ¼
OD ðsamplesÞ ½plasma treated cell lines 100 : OD ðcontrolsÞ ½untreated cell lines (11)
FIG. 3. Temperature variation vs distance from nozzle at different voltages.
rotational excitations of molecules are strongly related to electron energy. Therefore, the coefficient rate of these reactions, the number of reactive species, and the temperature of the plasma jet are increased. Hence, heating of the gas is not only the result of the larger number of collisions between electrons and neutrals as the discharges become more intense but also the additional heating might be the result of more energetic collisions. For example, the plasma temperature at 10 mm distance in 5 kV is 5 higher than its value in 3 kV. In addition, the diagram shows that by increasing the distance from the nozzle, the temperature far from the electrodes is reduced. In this case, electrons lose their energy and recombination of ions and electrons increases. In fact, by increasing the distance from the nozzle, electron energy is not high enough to excite the vibrational and rotational states, and consequently recombination of electrons increases.32 It is noticeable that the laboratory temperature during experiments was 24 C. Gas flow rate is another important parameter. Figure 4 shows that higher gas flow is equivalent with lower temperature, because in higher gas flows, there are more particles in the discharge zone and then the electrical energy should be divided between more particles. In other words, particle
Statistical analyses were performed using GraphPad Prism version 6.01 (Prism, USA), to compare different groups and p values less than 0.05 were considered as statistically significant. III. RESULTS AND DISCUSSION A. Plasma jet characterization
It was observed that the temperature was constant during the time. The plasma jet temperature was measured in 1, 2, 3, 4, and 5 min after running the reactor, and no difference was seen, which indicates stable performance of the reactor. The effect of the applied voltage and distance from the nozzle is plotted in Fig. 3. According to Fig. 3, increasing the applied voltage increases the number of electrons. Also, vibrational and Biointerphases, Vol. 10, No. 2, June 2015
FIG. 4. Temperature variation vs gas flow.
029510-5 Akhlaghi et al.: On the design and characterization of a new cold atmospheric pressure plasma jet
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FIG. 5. Variation of voltage and current during time.
FIG. 7. Typical spectra emitted from the plasma jet.
energy contribution (the energy per particle) decreases. This results in particles energy reduction and therefore decreases the temperature. The next investigated parameters were voltage and current profiles. It can be seen in Fig. 5 that the variation of both voltage and current is periodic and sinusoidal, which again indicates stable performance. It is remarkable that against other configurations in which there are pulse discharges in microsecond intervals, here the current profile is sinusoidal and current value is very low (less than 20 and 120 mA for discharge and displacement currents, respectively). The discharge power obtained using Lissajous curve approach was less than 1 W (0.45 W). This low power is a reason for low temperature of the plasma jet. The calculation was performed in 5 kV applied voltage (Fig. 6). It is notable that changing applied voltage made no significant change in the power; in other words, the power was almost constant against applied voltage. OES indicated that the designed plasma reactor can generate different reactive species. Obtained spectrum reveals that the nitrogen and oxygen radicals have maximum abundance, shown in Fig. 7. Hydroxyl radical (OH), a little amount of ozone, and excited helium atoms are the other
reactive species produced. Production of reactive oxygen and nitrogen species has great importance in medical and biological applications because they can run different types of biological processes such as preoxidation, peroxidation, and cell death. Similar to temperature, three effective parameters on reactive species production were considered. The wavelengths 308, 309, 390, 705, and 777 nm were used to investigate production of O3, OH, N2, He, and O, respectively.33 Figure 8 shows that by increasing the distance from the nozzle the concentration of reactive species decreases because at this distance the strength of the electric field at electrodes is low and both the electron density and energy are not high enough to participate in ionization and excitation. Therefore, the intensity of excited species in this region is lower. At the tip of the nozzle (0 mm), the density of nitrogen and oxygen is not high due to flux of helium. This is why an increment in N2 intensity in 5 mm has been observed compared to 0 mm. Therefore, between 5 and 15 mm, the decreasing of intensity is clear. The major role of voltage was demonstrated in Fig. 9 in the way that increasing voltage enhances reactive species
FIG. 6. Obtained Lissajous curve in 5 kV.
FIG. 8. Reactive species concentration against distance from nozzle.
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FIG. 11. Comparing experimental and simulated spectra.
density of ne ¼ 6.4 1013 cm3 and the plasma frequency of xp ¼ 4.52 1011 Hz. B. Cytotoxicity effects of plasma FIG. 9. Reactive species concentration against voltage.
production because the more the voltage is, more ionization happens and therefore more reactive species will be produced. Figure 10 reveals that although increasing gas flow is accompanied by an increment in reactive species, because at higher gas flow rates there are more particles and consequently more collisions happen but its effect is not remarkable. As in the previous case, at higher gas flow rates the electrical energy should be divided between more particles; hence, particle energy contribution (the energy per particle) will decrease. Determination of the rotational and vibrational temperatures was performed by comparing experimental and simulated spectra using SpecAir (edition 2.2) software as shown in Fig. 11. Best fitting was at rotational temperature of 300 K and the vibrational temperature of 3900 K. As mentioned before, Lorentz broadening was obtained by fitting the experimental spectrum to Voigt profile (DkLorentz ¼ 0:098 nm), as shown in Fig. 12. Using 1 atm and 300 K, van der Waals broadening was determined as Dkvan der Waals ¼ 0:066 nm. Therefore, Stark broadening would be 0.032 nm, which corresponds to the electron
FIG. 10. Reactive species concentration against gas flow rate. Biointerphases, Vol. 10, No. 2, June 2015
The cytotoxicity effect of the designed plasma jet on 4T1 cell line was demonstrated in a dose dependent manner. As shown in Fig. 13, by increasing treatment time, more cells were killed because of more reactive species. In addition, more cell death occurred at the interval time (between treatment and analysis) of 24 h in comparison to 0 h, which reveals that plasma has delayed effects. Although no significant difference was observed in viability percentage between the interval time of 24 and 48 h, which indicates that the plasma jet probably affects the proliferation process, it more likely blocks the cell cycle. The last important result was about safety of produced plasma in the sense that it is UVfree. As exhibited in Fig. 13(d), there was no significant difference between the control group and the UV-groups which received only UV radiation through the MgF2 filter. The results showed that if there was UV produced in plasma, it is apparently not responsible for the observed decrease in viability. IV. SUMMARY AND CONCLUSIONS A new configuration using a new dielectric for generating a cold atmospheric pressure homogenous plasma jet was designed and constructed. Its characteristics such as plume temperature, rotational and vibrational temperatures, power,
FIG. 12. Comparison of experimental data and Voigt profile.
029510-7 Akhlaghi et al.: On the design and characterization of a new cold atmospheric pressure plasma jet
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FIG. 13. Cancer cells viability against treatment time and interval time between treatment and analysis; MTT assay performed (a) after treatment, (b) 24 h after treatment, (c) 48 h after treatment. UV effectless can be observed in (d).
electrical behavior (voltage and current profile), electron density, plasma frequency, and produced reactive species were determined. The properties were almost time independent and were summarized in Table I. Furthermore, the effect of voltage, gas flow rate, and distance from the nozzle on the plasma jet temperature and reactive species production were considered. The role of voltage and distance was more effective than that of gas flow rate in both cases. To demonstrate the device potential in cancerous cells treatment, 4T1 cell line, one of the most aggressive cell lines, was selected. It
was observed that increasing treatment time and interval time between treatment and assay causes more cell death. Using an MgF2 filter demonstrated safety of plasma in the sense that it is UV-free. It will be valuable to carry out cancer cell treatment in more details and advanced analysis. In future works, we will focus on cancer cells treatment. The effects of three parameters on cancerous cells and the plasma jet on health cells will be considered. Also, more biological analysis will be performed to see what actually happens. 1
TABLE I. Summary of plasma parameter. Parameter Plasma jet temperature Rotational temperature Vibrational temperature Power Voltage and current profile Displacement current Discharge current Electron density Plasma frequency
Biointerphases, Vol. 10, No. 2, June 2015
Value Room temperature 300 K 3900 K 0.45 W Sinusoidal and periodic 120 mA (peak to peak)
On the design and characterization of a new cold atmospheric pressure plasma jet and its applications on cancer cells treatment Morteza Akhlaghi Department of Physics, Shahid Beheshti University, Tehran 19839, Iran
Hajar Rajayi Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115, Iran
Amir Shahriar Mashayekh Department of Physics, Shahid Beheshti University, Tehran 19839, Iran
Mohammadreza Khani Laser and Plasma Research Institute, Shahid Beheshti University 19839, Tehran, Iran
Zuhair Mohammad Hassan Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115, Iran
Babak Shokria) Department of Physics, Shahid Beheshti University, 19839 Tehran, Iran and Laser-Plasma Research Institute, Shahid Beheshti University, 19839 Tehran, Iran
(Received 31 January 2015; accepted 10 April 2015; published 23 April 2015) In this paper, a new configuration of a cold atmospheric pressure plasma jet has been designed and constructed. Poly-methyl-methacrylate was used as a new dielectric in this configuration which in comparison to other dielectrics is inexpensive, more resistant against break, and also more shapeable. Then, the plasma jet parameters such as plume temperature, rotational and vibrational temperatures, power, electrical behavior (voltage and current profile), electron density, and the produced reactive species were characterized. In order to determine the jet temperature and the amount of reactive species, effects of applied voltage, gas flow rate, and distance from the nozzle were studied. The power of the jet was specified using Lissajous curve approach. The plume temperature of the plasma jet was about the room temperature. Optical emission spectroscopy determined the type of reactive species, and also electron density and its corresponding plasma frequency (6.4 1013 cm3 and 4.52 1011 Hz). Because of producing different reactive species, the device can be used in different applications, especially in plasma medicine. Thus, 4T1 cancer cells were treated using this plasma jet. The results showed that this plasma jet has a great potential C 2015 American Vacuum to kill one of the most aggressive and resistant cancerous cell lines. V Society. [http://dx.doi.org/10.1116/1.4918806] I. INTRODUCTION Cold atmospheric pressure plasma (CAP) as a kind of nonequilibrium plasma can be produced in different configurations employed for the particular application.1 Cold plasmas are usually produced by a dielectric barrier discharge (DBD).2 The presence of a dielectric between electrodes prevents the transition to an arc discharge and makes plasma cold. Nowadays, this kind of plasma has been used in many fields, especially in biomedical applications.3 Because of producing reactive oxygen and nitrogen species, especially atomic oxygen, hydroxyl radicals, and nitrogen oxides, these systems can affect live cells and organisms, microbes, etc. However, it should be mentioned that some applications of plasma in medicine relied mainly on its thermal effects.4 It was suggested that some species have “killing” or “healing” effects.5 One can use desirable configuration for different purposes, e.g., wound healing,6 decontamination of bacteria,7 cancer treatment,8 allergen inactivation,9 root canal disinfection,10 and vegetables teatment.11 The use of plasmas a)
Electronic mail: [email protected]
029510-1 Biointerphases 10(2), June 2015
for biomedical purposes can be tracked back to the 1990s where Laroussi used atmospheric pressure plasma to inactivate microorganisms.12 At the early ages of 2000s, Stoffels et al. introduced the plasma needle for fine surface treatment of biomaterials.13 Since then, various devices and configurations were presented by different groups.1,14 In nonequilibrium plasmas, the electrons drive the plasma chemistry while the heavy particles remain at low temperature. So, there is no need for elevated gas temperatures.15 Determining the electron density by probe methods is prevalent. However, probe diagnostics are not only hampered by the small dimensions of some atmospheric pressure plasmas but also by the difficulty of interpretation of the measurements due to the collisionality of plasma.16 There is another indirect way using optical emission spectroscopy (OES).17 In this method, Stark broadening is used to determine the electron density and plasma frequency. OES provides a means to determine some reactive species produced in plasma and also shows the role of important parameters such as applied voltage, gas flow, and distance from the nozzle in producing reactive species.18
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C 2015 American Vacuum Society V
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029510-2 Akhlaghi et al.: On the design and characterization of a new cold atmospheric pressure plasma jet
Temperature is one of the major parameters especially for biomedical applications where a temperature less than body temperature is required. There are several ways and tools to measure it.19,20 A thermometer as well as OES was used to measure the temperature. Also voltage, gas flow rate, and distance from the nozzle as three parameters affecting the temperature were considered. As mentioned above, these parameters have effects on producing reactive species as well. Rotational and vibrational temperatures were determined by OES. CAP systems usually consume low power, a maximum of order of several watts. For power measurement, there are different ways. The most common way is the measurement of voltage and current by probes and calculation of the power using these parameters. However, there is Lissajous curve approach, which is more precise and has been customary recently.21 In this method, a capacitor is series with the DBD reactor. Measuring its voltage and a brief calculation would give the power.22 Voltage and current profiles were obtained as usual by voltage and current probes. Many scientists use a metal rod or pipe inside a dielectric tube as inner electrode and a metal ring as outer electrode to produce CAP.23,24 With this configuration, the electric field along the plasma plume is enhanced. Walsh and Kong showed that a high electric field along the plasma plume is favorable for generating long plasma plumes and more active plasma chemistry.25 In biological and biomedical applications, it is important that plasma be without electric shocks. Therefore, the grounded ring electrode was placed at the end of the torch, around the plasma jet exit aperture to neutralize the jet and reduce the risk of electric shocks. Quartz, glass, and ceramic are used frequently as dielectrics to produce CAP, but there are some difficulties. First, these materials are very brittle. In addition, it is difficult to form them into desired shape, and above all they are expensive (especially quartz). Thus, in this work, poly-methylmethacrylate (PMMA) polymer was used as a new dielectric which in comparison to other dielectrics is inexpensive, more resistant against break, and also more shapeable. Another advantage of PMMA respect to other materials and dielectrics is its transparency characteristic, which let us see what happens inside the discharge zone. In order to show the great potential of the plasma jet, murine breast cancer cell line (4T1) was treated with a series of doses. Scientists have studied different breast cancer cell lines earlier. Wang et al. showed that CAP can selectively ablate metastatic BrCa cells in vitro without damaging healthy human mesenchymal stem cells.26 Mirpour et al. studied MCF7 cell line and showed that longer treatment time had serious damage to cancer cells while it had no significant effect on normal cells.27 Another study on MCF7 was performed by Kim et al., which demonstrated that a significant portion of human breast cancer cells exhibits apoptotic fragmentation by applying a pulsed atmospheric pressure plasma jet.28 This paper introduced a new configuration for generating a cold atmospheric pressure plasma jet with a new dielectric, Biointerphases, Vol. 10, No. 2, June 2015
029510-2
temperature of order of room temperature, low power consumption, stable performance, and without electrical shocks. In addition, properties such as jet temperature, electron density, rotational and vibrational temperatures, electrical behavior, power consumption, and produced reactive species have been considered by the newest methods. It will be shown that against other configurations, in this configuration, current pulses would be eliminated. As a new application, the plasma jet then treated 4T1 cancer cells, one of the most aggressive and resistant cell lines. The Methylthiazol Tetrazolium (MTT) assay results demonstrated its great potential and proficiency. II. EXPERIMENTAL SETUP AND METHODOLOGY A. Configuration of the plasma jet reactor
To generate CAP, a copper pipe and a steel ring were used as electrodes. As mentioned earlier, the grounded ring electrode was placed at the end of the torch, around the plasma jet exit aperture to neutralize the plasma jet and reduce the risk of electric shocks. As shown in Fig. 1, in this situation, the grounded electrode was located between the high voltage electrode and sample, and works as a beam neutralizer. The PMMA tube was used as the dielectric between electrodes. Its dielectric properties were characterized by Basavaraja Sannakki and Anita.29 In comparison to other dielectrics, PMMA is inexpensive, more resistant against break, and also more shapeable. Because of low breakdown voltage and capability of producing uniform plasma, helium was selected as the carrier gas. A mass flow controller (APEX, AX-MC-5SLPM-D) was used to control the gas flow. A high-voltage high-frequency power supply (Information Unlimited Corporation, DIDRIV 10) with sinusoidal waveform ran the plasma jet. Its voltage and frequency were variable, but all experiments were done at 25 kHz. The plasma reactor could give stable
FIG. 1. (a) Side view, (b) three-dimension shape, and (c) generated plasma of the reactor.
029510-3 Akhlaghi et al.: On the design and characterization of a new cold atmospheric pressure plasma jet
plasma with almost 35 mm plume length at 4 standard liter per minute (SLPM) gas flow.
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capacitor and capacitor start storing electrical charges. The following equation would give us the power: q ¼ Cc Vc ;
B. Characterization methods
The first important parameter of plasma in biomedical and biological applications is its temperature. The plasma jet temperature was measured by a digital thermometer (JUMO, dtron 304) with a ceramic sensor (PT-100). Measurements were done in different conditions. In all experiments, the plasma jet temperature was measured in 1, 2, 3, 4, and 5 min after running the reactor. Other parameters affecting the plasma jet temperature are voltage, gas flow, and distance from the tip of the nozzle. In order to consider these parameters, the gas flow was fixed at 4 SLPM, and then, the temperature was measured at different voltages and distances. In order to determine the effect of gas flow, the voltage was fixed at 4 kV, and the temperature was measured at 15 mm distance at different gas flows. Another important parameter is electrical behavior, which is determined by time variation of voltage and current curves studied by a high voltage probe (Tekteronix, P6015A) and a current probe (Tektronix, TCP202). Displacement and discharge currents can be specified separately by using these probes. Displacement current is created when a voltage is applied to the separated electrodes. The discharge current refers to the motion of the charged particles. Therefore, when plasma is on, we have both displacement and discharge currents. Turning off plasma eliminates the discharge current. So, to determine displacement and discharge currents, the current should be measured in both plasma on and plasma off conditions Plasma On :
Ion ¼ Idispl þ Idisch ;
(1)
Plasma Off :
Ioff ¼ Idispl ;
(2)
Idisch ¼ Ion Ioff :
(3)
The power of the reactor was measured by Lissajous curve approach. In this approach, a capacitor is series with the plasma jet reactor, as shown in Fig. 2. Therefore, whole transmitted current from the reactor would pass through the
Cr ¼
q Vreactor
(4) ;
(5)
þ þ dq dt ¼ f Cr V ðtÞdV; Power ¼ f E ¼ f V ðtÞ dt
(6)
T
where q is amount of charge stored in capacitor, Cc is the capacitance of the capacitor, Vc is voltage of the capacitor, Cr is capacitance of the reactor, V(t) is the applied voltage, and f is the applied voltage frequency.22 The last and also the most important property is capability of producing different and suitable reactive species. This feature and also electron density usually was considered by optical emission spectroscopy. Here, a spectrometer (AVANTES, AvaSpec 3648) was used for recording the emitted spectrum and determining the produced reactive species. There are several parameters that may affect reactive species production. Therefore, in the present work, the effects of voltage, gas flow, and distance from nozzle were considered. One could characterize the rotational and vibrational temperatures through comparing experimental and simulated spectra. Here, wavelengths 370–381 nm (nitrogen second positive system transition) and specair software were used for simulation and comparison of spectra. The software shows the rotational and vibrational temperatures when the experimental and simulated spectra are fitted. The electron and ion bombardments may destroy the bacteria cell membrane and then the activated oxygen and nitrogen species penetrating into the cell kill the bacteria.30 Therefore, electrons play an important role in bioplasma. OES can be used to determine the electron density and the plasma frequency likewise. The electron density corresponds to Stark broadening as follows:31 Dkstark ¼ 2 1011 ðn2=3 e Þ; where ne is in cm3, and plasma frequency is32 pffiffiffiffiffi xp ¼ 5:65 104 ne :
(7)
(8)
Therefore, knowing Stark broadening leads to compute the electron density. Stark broadening was considered at wavelength 434 nm for H-gamma. Stark broadening is related to Lorentz and van der Waals broadenings as follows: DkLorentz ¼ DkStark þ Dkvan der Waals :
(9)
van der Waals broadening corresponds to gas temperature31 Dkvan der Waals ¼ 3:6
FIG. 2. Power measurement circuit (Lissajous curve approach). Biointerphases, Vol. 10, No. 2, June 2015
P ; T 0:7
(10)
where p is the pressure in atmosphere and T is the gas temperature in Kelvin.
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To know Lorentz broadening, one could fit the experimental spectrum with Voigt profile which is superposition of Gauss and Lorentz broadenings. Fitting was performed using Origin software and Lorentzian full width at half maximum was determined. Thus, knowing Lorentz and van der Waals broadenings, Stark broadening is determined, and as a result, the electron density and plasma frequency can be found. C. Cells preparation and assay
In order to investigate anticancer effects of the plasma jet, murine breast cancer cell line (4T1) was obtained from National Cell Bank of Pasteur Institute of Iran. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM L-glutamine, supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 100 U/ml penicillin, 100 lg/ml streptomycin solution (Gibco, USA) in 5% CO2 and 95% O2 at 37 C. The cytotoxicity of plasma and produced reactive species was studied by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. 4T1 cells at density of 16 000 per well in a volume of 200 ll DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin solution (Gibco, USA) were seeded on three 96-well cell culture plates (Grinner, Germany). They were treated with a series of doses (30, 60, 90, 120, 150, and 180 s plasma treatment). Also, an MgF2 filter, which only transmits UV wavelength, was used to consider UV production by the plasma jet and its effects on cancer cells. Then, MTT at the final concentration of 0.5 mg/ml was added to each well at different times (0, 24, and 48 h) after treatment. After incubation of 4 h and removing the medium of each well, 100 ll dimethyl sulfoxide was added to dissolve the blue formazan crystals. The absorbance was further measured at 540 nm using enzyme-linked immunosorbent assay reader (Multiscan, Finland). The viability of cells was presented as the percentage of control as follows: Viability ¼
OD ðsamplesÞ ½plasma treated cell lines 100 : OD ðcontrolsÞ ½untreated cell lines (11)
FIG. 3. Temperature variation vs distance from nozzle at different voltages.
rotational excitations of molecules are strongly related to electron energy. Therefore, the coefficient rate of these reactions, the number of reactive species, and the temperature of the plasma jet are increased. Hence, heating of the gas is not only the result of the larger number of collisions between electrons and neutrals as the discharges become more intense but also the additional heating might be the result of more energetic collisions. For example, the plasma temperature at 10 mm distance in 5 kV is 5 higher than its value in 3 kV. In addition, the diagram shows that by increasing the distance from the nozzle, the temperature far from the electrodes is reduced. In this case, electrons lose their energy and recombination of ions and electrons increases. In fact, by increasing the distance from the nozzle, electron energy is not high enough to excite the vibrational and rotational states, and consequently recombination of electrons increases.32 It is noticeable that the laboratory temperature during experiments was 24 C. Gas flow rate is another important parameter. Figure 4 shows that higher gas flow is equivalent with lower temperature, because in higher gas flows, there are more particles in the discharge zone and then the electrical energy should be divided between more particles. In other words, particle
Statistical analyses were performed using GraphPad Prism version 6.01 (Prism, USA), to compare different groups and p values less than 0.05 were considered as statistically significant. III. RESULTS AND DISCUSSION A. Plasma jet characterization
It was observed that the temperature was constant during the time. The plasma jet temperature was measured in 1, 2, 3, 4, and 5 min after running the reactor, and no difference was seen, which indicates stable performance of the reactor. The effect of the applied voltage and distance from the nozzle is plotted in Fig. 3. According to Fig. 3, increasing the applied voltage increases the number of electrons. Also, vibrational and Biointerphases, Vol. 10, No. 2, June 2015
FIG. 4. Temperature variation vs gas flow.
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FIG. 5. Variation of voltage and current during time.
FIG. 7. Typical spectra emitted from the plasma jet.
energy contribution (the energy per particle) decreases. This results in particles energy reduction and therefore decreases the temperature. The next investigated parameters were voltage and current profiles. It can be seen in Fig. 5 that the variation of both voltage and current is periodic and sinusoidal, which again indicates stable performance. It is remarkable that against other configurations in which there are pulse discharges in microsecond intervals, here the current profile is sinusoidal and current value is very low (less than 20 and 120 mA for discharge and displacement currents, respectively). The discharge power obtained using Lissajous curve approach was less than 1 W (0.45 W). This low power is a reason for low temperature of the plasma jet. The calculation was performed in 5 kV applied voltage (Fig. 6). It is notable that changing applied voltage made no significant change in the power; in other words, the power was almost constant against applied voltage. OES indicated that the designed plasma reactor can generate different reactive species. Obtained spectrum reveals that the nitrogen and oxygen radicals have maximum abundance, shown in Fig. 7. Hydroxyl radical (OH), a little amount of ozone, and excited helium atoms are the other
reactive species produced. Production of reactive oxygen and nitrogen species has great importance in medical and biological applications because they can run different types of biological processes such as preoxidation, peroxidation, and cell death. Similar to temperature, three effective parameters on reactive species production were considered. The wavelengths 308, 309, 390, 705, and 777 nm were used to investigate production of O3, OH, N2, He, and O, respectively.33 Figure 8 shows that by increasing the distance from the nozzle the concentration of reactive species decreases because at this distance the strength of the electric field at electrodes is low and both the electron density and energy are not high enough to participate in ionization and excitation. Therefore, the intensity of excited species in this region is lower. At the tip of the nozzle (0 mm), the density of nitrogen and oxygen is not high due to flux of helium. This is why an increment in N2 intensity in 5 mm has been observed compared to 0 mm. Therefore, between 5 and 15 mm, the decreasing of intensity is clear. The major role of voltage was demonstrated in Fig. 9 in the way that increasing voltage enhances reactive species
FIG. 6. Obtained Lissajous curve in 5 kV.
FIG. 8. Reactive species concentration against distance from nozzle.
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FIG. 11. Comparing experimental and simulated spectra.
density of ne ¼ 6.4 1013 cm3 and the plasma frequency of xp ¼ 4.52 1011 Hz. B. Cytotoxicity effects of plasma FIG. 9. Reactive species concentration against voltage.
production because the more the voltage is, more ionization happens and therefore more reactive species will be produced. Figure 10 reveals that although increasing gas flow is accompanied by an increment in reactive species, because at higher gas flow rates there are more particles and consequently more collisions happen but its effect is not remarkable. As in the previous case, at higher gas flow rates the electrical energy should be divided between more particles; hence, particle energy contribution (the energy per particle) will decrease. Determination of the rotational and vibrational temperatures was performed by comparing experimental and simulated spectra using SpecAir (edition 2.2) software as shown in Fig. 11. Best fitting was at rotational temperature of 300 K and the vibrational temperature of 3900 K. As mentioned before, Lorentz broadening was obtained by fitting the experimental spectrum to Voigt profile (DkLorentz ¼ 0:098 nm), as shown in Fig. 12. Using 1 atm and 300 K, van der Waals broadening was determined as Dkvan der Waals ¼ 0:066 nm. Therefore, Stark broadening would be 0.032 nm, which corresponds to the electron
FIG. 10. Reactive species concentration against gas flow rate. Biointerphases, Vol. 10, No. 2, June 2015
The cytotoxicity effect of the designed plasma jet on 4T1 cell line was demonstrated in a dose dependent manner. As shown in Fig. 13, by increasing treatment time, more cells were killed because of more reactive species. In addition, more cell death occurred at the interval time (between treatment and analysis) of 24 h in comparison to 0 h, which reveals that plasma has delayed effects. Although no significant difference was observed in viability percentage between the interval time of 24 and 48 h, which indicates that the plasma jet probably affects the proliferation process, it more likely blocks the cell cycle. The last important result was about safety of produced plasma in the sense that it is UVfree. As exhibited in Fig. 13(d), there was no significant difference between the control group and the UV-groups which received only UV radiation through the MgF2 filter. The results showed that if there was UV produced in plasma, it is apparently not responsible for the observed decrease in viability. IV. SUMMARY AND CONCLUSIONS A new configuration using a new dielectric for generating a cold atmospheric pressure homogenous plasma jet was designed and constructed. Its characteristics such as plume temperature, rotational and vibrational temperatures, power,
FIG. 12. Comparison of experimental data and Voigt profile.
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FIG. 13. Cancer cells viability against treatment time and interval time between treatment and analysis; MTT assay performed (a) after treatment, (b) 24 h after treatment, (c) 48 h after treatment. UV effectless can be observed in (d).
electrical behavior (voltage and current profile), electron density, plasma frequency, and produced reactive species were determined. The properties were almost time independent and were summarized in Table I. Furthermore, the effect of voltage, gas flow rate, and distance from the nozzle on the plasma jet temperature and reactive species production were considered. The role of voltage and distance was more effective than that of gas flow rate in both cases. To demonstrate the device potential in cancerous cells treatment, 4T1 cell line, one of the most aggressive cell lines, was selected. It
was observed that increasing treatment time and interval time between treatment and assay causes more cell death. Using an MgF2 filter demonstrated safety of plasma in the sense that it is UV-free. It will be valuable to carry out cancer cell treatment in more details and advanced analysis. In future works, we will focus on cancer cells treatment. The effects of three parameters on cancerous cells and the plasma jet on health cells will be considered. Also, more biological analysis will be performed to see what actually happens. 1
TABLE I. Summary of plasma parameter. Parameter Plasma jet temperature Rotational temperature Vibrational temperature Power Voltage and current profile Displacement current Discharge current Electron density Plasma frequency
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Value Room temperature 300 K 3900 K 0.45 W Sinusoidal and periodic 120 mA (peak to peak)
