Lengths of plasma plume

Admixture of helium and oxygen has been widely used to produce more radials in atmospheric pressure plasma jets31,32,33. The results indicate that, when a small amount of oxygen is introduced, the plasma plume length decreases with increasing the oxygen concentrations. Adding oxygen into the feeding gas tends to diminish the production of radicals due to the electron attachment to oxygen inside the nozzle34. In this work, two types of oxygen injection were used to determine a proper addition method suitable for effective plasma treatment. The measured plume lengths, indicating the distance many reactive species can extend into the ambient atmosphere, are shown as functions of applied voltage and additive oxygen flow rate in Fig. 2. In accordance with previous reports, our results show that the plasma plume length decreases with increasing the oxygen flow rate30. As the applied voltage was increased and oxygen gas flow rate decreased, the plasma plume length increased. Especially, the plume length was reduced gradually in Type 2 (Figure 2B) compared to in Type 1 (Figure 2A) with oxygen flow rate. The Type 1-mixing (direct mixing of helium and oxygen) interferes with each gas flow essential for the discharge stability and requires much higher breakdown voltage due to the presence of oxygen molecules. In Type 2, on the other hand, the helium and oxygen gases are not mixed together before the ignition of helium plasma inside the discharge region. Accordingly, oxygen molecules interact with the helium plasma following the plasma ignition, which leads to relatively easy incorporation of oxygen molecules into the plasma state. Therefore, the use of the capillary tube (Type 2) is found to be favorable to mix the additive oxygen gas efficiently under a stable jet operation.

Figure 2 Comparison of plasma plume lengths from two types of jet. Plume lengths as functions of applied voltage and additive oxygen flow rate (A) in Type 1 and (B) in Type 2. In Type 2, the plasma plume length was reduced gradually with oxygen flow rate compared to Type 1. Full size image

Electrical characteristics

The typical waveforms of the applied voltage and total current for the pulsed discharge operating at a repetition frequency of 50 kHz in Type 2 are shown in Figure 3A. Two distinct discharge current pulses per applied voltage pulse are observed. To find out the actual discharge current, the displacement current I no (without gas flow, plasma off) is subtracted from the total current I tot (plasma on). For the applied voltage amplitude 1.7 kV, the peak value of the primary discharge current is about 0.10 A and the peak value of the secondary discharge is smaller than that of the primary discharge. The secondary discharge ignites because of the voltage induced by the charges which have accumulated on the surface of the quartz tube during the primary discharge30.

Figure 3 Voltage-current characteristics of the discharge. (A) Typical waveforms of voltage (black solid line) and current in Type 2 jet. The operating condition of pulsed-dc plasma jet is the applied voltage 1.7 kV pp , repetition frequency 50 kHz and duty ratio 10%. The inset shows the total current (plasma on; pink solid line) and displacement current (plasma off; gray dashed line). To find out the actual discharge current, the displacement current is subtracted from the total current. (B) Total current as a function of additive oxygen flow rate for two types of oxygen injection. Full size image

Figure 3B represents the total current as a function of oxygen concentration for two types of oxygen injection. This characteristic is similar to the change in the plasma plume length. In the type 1-mixing, when O 2 flow rate reaches 20 SCCM, the voltage and current characteristics were disturbed much in accordance with the change in the plume length shown in Figure 2A. As mentioned above, atmospheric pressure plasma jets based on the direct mixture of helium and oxygen require a higher applied voltage than pure helium plasma due to differing plasma ignition conditions, the type 1-mixing results in an inefficient plasma ignition. The oxygen molecules severely affect the initiation process of the helium plasma in Type 1.

Optical characteristics

Figure 4A shows typical emission spectrum observed in the He plasma jet with 10 SCCM oxygen flow through the capillary electrode (Type 2). The discharge produces a significant UV radiation that belongs to transitions of the NO γ bands at 200–300 nm, the OH band at 308 nm, the atomic oxygen lines at 777 and 844 nm, the N 2 emission bands at 310–440 nm and the N 2 + emission bands at 391–428 nm. Helium plasma jet with a separated oxygen channel was observed to produce more ROS in the plasma. This is attributed to the higher density of oxygen atom introduced in the excitation region. The variations of the intensities of NO, OH, H α and O with additive oxygen flow are shown in Figure 4B. The intensities from these species decreased with additive oxygen flow except a slight increase of the intensity from O 844 nm at 10 SCCM. This is attributable to a decrease in the current with additive oxygen flow shown in Figure 3B. The plasma density can be estimated from the current assuming that the drift velocity is not much changed with additive oxygen flow. From the experimental results of the current and optical intensities, it can be inferred that the plasma density decreases with an increase in the oxygen flow. This is partly attributable to the reduction of electron density due to attachment to oxygen35. It is noted that the addition of oxygen to the He plasma causes a decrease in the light emissions from He and air molecules. This suggests that some electrons are probably consumed to produce O radicals when O 2 is injected. Others then collide with air, water vapor and He to produce N, OH, H α and He radicals34. This phenomenon is consistent with the changes in the plasma plume length and volume mentioned above. When the oxygen flow rate is raised from 0 SCCM to 100 SCCM, the plume color becomes whitened due to the contributions from the light emission at diverse frequencies.

Figure 4 Various reactive species and ozone monitoring in Type 2. (A) Optical emission spectrum of the plasma plume from 250 nm to 850 nm observed in the helium/oxygen plasma at the applied voltage 1.7 kV pp , repetition frequency 50 kHz and duty ratio 10%. (B) Comparisons of the change of the emission intensities from NO, OH, H α , O radicals in different oxygen flow rates increasing from 0 to 100 SCCM. (C) The intensity ratios of the lines from NO, OH, H α , O radicals to the helium line (706 nm). (D) Measurement of ozone concentration for different oxygen flow rates increasing from 0 to 20 SCCM. Full size image

Figure 4C represents the optical emission intensity ratios of the lines from NO, OH, H, O radicals to the helium line (706 nm). Since the amount of He remains unchanged in the plasmas with different oxygen addition, the normalized intensities can represent the excited state densities of the species NO, OH, H and O. The line intensities from NO and OH decreased with additive oxygen flow rate a little further than that of line intensity from He. On the other hand, the line intensities from O and H exhibit the opposite behavior. It is believed that the increased oxygen species in the plasma contributes to the cancer therapy due to its strong oxidative effect on the cells36. Figure 4D shows the ozone production as a function of the additive oxygen flow rate. Ozone is a strong oxidant that generates ROS in cells and even causes DNA damage37. During the plasma treatment, ozone levels increased with the oxygen flow rate. Ozone's damaging effect of the cell can be mediated via an induction of ROS within cells38. Since the reaction O + He + O 2 → He + O 3 is believed as the main way to produce ozone in the discharge region39, it is expected that the ozone density correlates with the densities of atomic oxygen and oxygen molecule. Thus, the ozone density keeps growing with increase in the concentration of O 2 40.

Determination of excitation temperature & oxygen atom density

The excitation temperature was estimated by using a Boltzmann plot method applied to several excited helium emission lines (447, 501, 587, 667, 706 and 728 nm)41. The atomic emission intensity (I pq ) of the transition from level p to level q depends on the transition probability (A pq ) and absolute population of the atomic level (n p ), as shown in the following equation; I pq = n p A pq hv (where h is the Planck constant and v is the photon frequency corresponding to the p → q transition). Assuming a Boltzmann distribution of the population of the atomic level, the emission intensity is expressed as (where E p and g p are the energy and degeneracy of excited level p, respectively, k B is the Boltzmann constant and T exc is the excitation temperature in Kelvin). From the measurement of intensity and wavelength, a Boltzmann plot was obtained for the helium plasma with 40 SCCM additive oxygen gas (Figure 5A). Using this formula, the electron excitation temperature can be estimated. Figure 5B shows that the measured excitation temperature is in the range of 0.22–0.26 eV. The excitation temperature slightly increased with the additive oxygen flow up to 60 SCCM. The presence of oxygen in the plasma, a source of electronegative species, causes an increase of the sustaining voltage and the electric field strength. Therefore, the mean electron kinetic energy is increased and this leads to an increase of the excitation temperature43. The excitation temperature may be used as a rough indicator of the electron temperature. Because it is free electrons which cause the excitation, their energies should be described by a Boltzmann distribution at a given temperature41. The electron temperature increases and the electron density decreases (inferred from the decrease in the optical intensities in Figure 4B) with increasing oxygen concentration. In this range, the contribution of Penning ionization decreases due to the reduction of the available helium metastables in the plasma. This observation is in agreement with the results of the global modeling44.

Figure 5 Excitation temperature and oxygen atom density at different additive oxygen flows in Type 2. A Boltzmann plot method was used to estimate the excitation temperature: (A) Boltzmann plot of atomic helium lines (447, 501, 587, 667, 706 and 728 nm) for the additive oxygen flow rate of 400 SCCM. Variations of (B) the excitation temperature and (C) estimated atomic oxygen density via actinometry as a function of the additive oxygen flow rate. The dotted line indicates the expected estimates when the generation of excited oxygen atom via the collisional reactions involving oxygen metastables and excited oxygen molecules is considered. Full size image

O 2 -containing plasmas are of interest in cancer therapy due to their ROS effect in cells. To examine the change in O-atom density, the fixed feed gas was kept constant at 1 L/min helium with 0.1% trance of argon admixtures (for actinometry) and the oxygen flow rates were varied from 0 to 100 SCCM. Optical emission actinometry is a widely utilized diagnostic tool for in-situ monitoring of spatial and temporal variations of atomic and molecular concentrations. In this method, a known concentration of an impurity is introduced and the intensities of two neighboring spectral lines, one from the known gas and one from the sample, are compared. Since both species are bombarded by the same electron distribution and the concentration of the actinometer is known, the density of the sample can be calculated. To determine the concentration of the ground state oxygen atoms, O (3s 3P − 3p 3S) 844 nm and Ar (3p5 4p (2p 1 ) − 3p5 4s (1s 2 )) 750.4 nm transitions were chosen and the dissociation fraction can be determined by calculating the ratio42,45,46.

Here, n l is the number density of ground species l, is the intensity of emission from excited species l for the transition i → j, is the Einstein coefficient, is the spectral response of the system and is the frequency of the light emission. And is the rate coefficient for electron-impact direct excitation to O* (3s 3P) from the ground-state O, is the rate coefficient for electron-impact direct excitation to Ar (2p 1 ) from the ground-state Ar, is the rate coefficient for electron-impact dissociative excitation to O* (3s 3P) from the ground-state O 2 and k Q is the rate coefficient for collisional quenching. The ratio of rate coefficients and are obtained from the literature42. Neglecting the collisional quenching, the O-atom density is finally evaluated according to

where is the optical decay rate of the upper state i of species l) denotes the effective optical branching ratio of the transition. Figure 5C indicates that the ground state atomic oxygen density slightly increases with the oxygen flow rate. This result is in agreement with the global modeling47. However, it should be noted that the estimation of ground state O-density utilizing Eq. (2) is not precise at the higher oxygen concentrations because non-negligible portion of the excited state atomic oxygen is generated by collisional reactions involving oxygen metastables and excited oxygen molecules40,44,47. Then, the number density of O* (3s 3P), represented by in the denominator of the first term in Eq. (2) (the electron density n e is cancelled with the one in the numerator), increases actually with increase in the oxygen concentration. Therefore, if this contribution is considered, the ground state O-density is likely to behave as indicated by the dot line in Figure 5C, which is in accord with the experimental results of rf-driven atmospheric pressure plasmas39,48.

Plasma-cell interaction: intracellular ROS concentrations

To enhance the intracellular ROS generation in cells, the cells were treated by the helium plasma jet with additive oxygen gas. Figure 6A shows the fluorescence images (upper row) of intracellular ROS concentration in A549 cells (non-treated, He plasma-treated, He/O 2 plasma-treated) immediately after plasma treatment. The figures of lower row represent the bright-field images using an inverted microscopy. It is observed that plasma exposure leads to the increase of the intracellular ROS generation and plasma-induced ROS production can be controlled by additive oxygen gas. The highest intracellular ROS concentration in the plasma-treated cells was seen to occur at an additive oxygen flow rate of 20 SCCM, as shown in Figure 6B. Interestingly, when the oxygen flow rate was increased to 40 SCCM, a slight decrease in intracellular ROS concentration was observed. It means that high oxygen flow rate into the feeding gas influences the plasma discharge diminishing the production of radicals due to the electron attachment to oxygen inside the nozzle. Therefore, plasmas with higher oxygen flow rate than 40 SCCM tend to induce lesser intracellular ROS concentration. The generation of intracellular ROS is not solely determined by the atomic oxygen density but influenced by the concentrations of NO, OH and O 3 .

Figure 6 Intracellular ROS in A549 cells following plasma treatment. A549 cells were treated with DCF-DA and assayed using fluorescence microscopy. Intracellular ROS formation in response to helium plasma-exposed A549 cells for different oxygen flow rate: (A) Fluorescence images (upper row) of intracellular ROS concentration in A549 cells (non-treated, He plasma-treated, He/O 2 plasma-treated) and bright-field images (lower row), (B) the quantification by measuring fluorescence pixel intensity with MetaMorph software (Each point represents the mean ± SD of three replicates. * p < 0.05 and ** p < 0.01). Scale bar = 100 μm. Full size image

Plasma-cell interaction: DNA damage response

To elucidate the effect of cellular damage induced by plasma in cancer cells, the expression level of p53 in cells treated with plasma was investigated. The tumor suppressor protein p53 plays a major role in the cellular response to DNA damage and other genomic aberrations. DNA damage induces phosphorylation of p53 at Ser15 and Ser20. Activation of p53 can lead to cell cycle arrest and apoptosis by activating transcription of many downstream target genes27,28. Reactive oxygen species have been considered as important mediators of the DNA damage after plasma treatment49. At 12 hours after plasma or plasma with oxygen treatment, expression level of p53 was significantly increased compared to those from the gas control (Figure 7A). Since the p53 activation is required for expression of itself we measured the level of phosphorylation of p53 at Ser15 (p-p53, the active form of p53) at 2 hours after the treatment and found that prominent p-p53 expression in plasma-treated cells (Figure 7B). In order to scrutinize the additional oxygen effect on p53 signaling in single cell level, we performed immunocytochemistry using p53 and p-p53 antibodies at 2 hours after the treatment. The results indicate that both the number of p53 and p-p53 positive cells and the fluorescence intensity in each of the oxygen-plasma treated cells were higher compared to those in the plasma only treated cells (Figure 7C & D), implicating more DNA damage accumulation by the additional oxygen in plasma.

Figure 7 Effect of plasma treatment on DNA damage in A549 cells. (A) Protein level of p53 at 12 h after the gas or plasma-treatment was analyzed using an immunoblot assay. GAPDH was probed to indicate the evenness of protein loading, (B) Immunoblottings of phospho-p53 (p-p53), p53 and GAPDH at 2 h after the indicated treatments. Uncropped full scans for A & B are shown in the Supplementary Figure 1. (C) Cells treated with indicated gas or plasma were fixed 2 hour later and immunostained for p53 and p-p53 (green) and DNA were counterstained with Hoechst dye (depicted as dotted lines) and (D) the quantification of (C) by measuring fluorescence pixel intensity (Each point represents the mean ± SD of three replicates. * p < 0.05, * p < 0.01 and *** p < 0.001). Full size image