The biological effect of the physical energy of plasma

Since the publication of the initial paper on atmospheric pressure plasma sterilization by Dr Laroussi in 1996, researchers have contributed to the field with an extensive number of papers on plasma medicine. However, these studies have primarily concentrated on the biological impacts of the chemical reactive components generated by plasma, specifically focusing on the effects of reactive oxygen and nitrogen species. Conversely, when plasma directly interacts with biological organisms, there are additional physical energies involved, such as electric fields, ultraviolet (UV)/vacuum ultraviolet (VUV) radiation, heat, etc., which may also play crucial roles in their interaction. This paper delves into this aspect by using the simplest bactericidal effect as a model for biological effects. Three dielectrics—Al2O3, quartz, and MgF2 glass—are employed to isolate the chemical active components, enabling the examination of the bactericidal effects of the electric field, UV, and VUV, respectively. The findings indicate that the plasma-induced electric field can induce irreversible electroporation, effectively eliminating bacteria at 27 kV cm−1. Notably, at a plasma-induced electric field of 40 kV cm−1, sterilization efficiency experiences a significant enhancement. The bactericidal effects of UV and VUV are closely linked to the choice of the plasma’s working gas. Specifically, when Ar is the working gas, the bactericidal effect of UV surpasses that of using only the plasma-induced electric field by two orders of magnitude, while using He results in only a one-order increase. Despite VUV radiation being considerably weaker than UV, its bactericidal effect remains substantial. In instances where He plasma is utilized, the addition of VUV doubles the bactericidal effect. In short, this paper pioneers the exploration of the biological effects of plasma’s physical energy, providing essential insights for the advancement of plasma medicine.

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In the context of most cold plasma applications in plasma medicine, the gas temperature is either at or close to room temperature [15,16], rendering the consideration of 'Thermal radiation' unnecessary.
Plasma propagates because of applied and space charge electric field.When plasma contacting with a human, it delivers intense electric field.This electric field may induce the rearrangement of the phospholipid bilayer structure, leading to lipid membrane peroxidation.Subsequent to the application of the electric field, nanometer-sized pores, referred to as plasmaporation, may form in the cell membrane structure.This process could ultimately result in the leakage of cytoplasm if the electric field is high enough [17].However, to date, there is only few simulation studies reported on such effect [18].
UV/VUV, with its ability to penetrate and weaken cell walls by breaking unsaturated fatty acids, inflicts damage on the internal cellular structure.Bactericidal effects from UV/VUV generated by plasma primarily result from the induction of single and double-strand DNA breaks, effectively inhibiting bacterial replication [23][24][25].Earlier studies suggest that a radiation dosage of 9 mJ cm −2 can eradicate 1 log10 of E. coli, while 7.8 mJ cm −2 can produce same effect for S. aureus [26].
VUV, possessing higher energy than UV due to its shorter wavelength, has not been fully explored in atmospheric air due to its easy absorption by oxygen below 195 nm and nitrogen below 145 nm.Few studies have investigated the bactericidal effects of UV (>220 nm) in plasma [24,26], but to date, there is no reported study on the bactericidal effects of VUV of plasma.
In this paper, the experiment was designed to investigate the biological effects of physical energy, encompassing both the plasmaporation effect and the VUV bactericidal effect in plasma.
In this research, an atmospheric pressure plasma jet (APPJ) was utilized, as depicted in figure 1.The high-voltage (HV) electrode consisted of a tungsten needle positioned along the central axis of a quartz tube (outer diameter 12 mm, inner diameter 10 mm).The quartz tube featured a nozzle with an outer diameter of 3 mm and an inner diameter of 1 mm.The tip of the tungsten needle was positioned 8 mm away from the tube's nozzle.Helium (He) was introduced into the quartz tube at a flow rate of 2 standard liters per minute (slm).
A HV microsecond pulse power supply was employed to drive the He APPJ.The experimental arrangement is depicted in figure 1.A circular substrate composed of rounded MgF 2 /quartz glass/alumina ceramic with a diameter of 14 mm was coated with 5 µl of E. coli bacterial solution at a concentration of 5 × 10 8 ml −1 .To ensure the bacterial solution formed a film on the dielectric surface without flowing, it was allowed to dry for 20 min.The dielectric was positioned on a platform featuring a circular hole (diameter 14 mm) at its center, with the side coated with the bacterial solution facing downward.This experimental design was implemented to mitigate the effects of RONS and focus solely on the effect of the physical energy of the plasma.The distance between the nozzle of the quartz tube and the bottom of the rounded dielectric was maintained at 6 mm.
MgF 2 /quartz glass/alumina ceramic exhibit distinct transmission ranges, as outlined in table 1.The alumina ceramic only allows the electric field to interact with bacteria.Quartz glass facilitates the action of both the electric field and UV on bacteria.MgF 2 enables the impact of the electric field, UV, and VUV at wavelengths beyond 110 nm on bacteria.It is important to note that while light with a wavelength exceeding 380 nm can pass through quartz, it has minimal germicidal effects, and thus, this aspect will not be taken into consideration.Because MgF 2 /quartz glass/alumina ceramic have different dielectric constants, the magnitude of the electric field at the bottom of the dielectric may vary.To ensure that bacteria on the different dielectrics are subjected to the same magnitude of electric field, three dielectrics of different thicknesses have been selected.The electric field was measured using the electro-optical effect.
As depicted in figure 2, a diode-pumped solid-state laser (MGL-III-532, Continuous-wave) was employed to generate linearly polarized light in the Y-axis direction (wavelength: 532 ± 1 nm, beam diameter at the aperture 1/e2: 1.2 mm).The laser beam, oriented along the z-axis, traversed through a polarized beam splitter (PBS) (Thorlabs, America) and entered the BGO crystal.The distance between the quartz tube nozzle and the bottom of the BGO crystal was approximately 7.5 mm, and the distance between the nozzle and the bottom of the dielectric was 6 mm.
The laser beam passed through the BGO crystal and underwent reflection due to a reflective layer covering one side of the BGO crystal.Only the X-axis component of the reflected light could be reflected by the PBS.Subsequently, it traversed through a polarizer and was collected by the photomultiplier tube.The intensity of the light signal could be measured as the electric field changes.
To measure the voltage and current waveforms of the plasma jet, a high voltage probe (Tektronix 6015A) and a current probe (Tektronix 312A) were employed.The signals were recorded using a digital oscilloscope (Tektronix MSO 3054).The current waveforms in figures 3 and 7 are plasma discharge currents, which are obtained by subtracting the displacement currents from the total currents.
The electric field under 1 mm thickness of MgF 2 , 0.8 mm thickness of quartz glass, and 1.4 mm thickness of alumina ceramic were measured, and the results are presented in figure 3. The electric field under the three dielectrics is essentially the same.In figure 3, the current rapidly reaches its peak after the rising edge of the voltage, then gradually decreases to near-zero values.During this period, the electric field swiftly increases to its maximum value.This electric field is sustained at this maximum value until the onset of the voltage's falling edge, at which point the current exhibits a negative pulse, and the electric field rapidly drops to zero in tandem with this current.This observation indicates that the primary cause of this electric field is the accumulation of charges on the dielectric during the discharge process.Laplacian electric field is transferred from the electrode region where voltage is applied to the target through the plasma column connecting the high voltage electrode with the tip of the plasma jet [27,28].Except the Laplacian electric field and charge deposition induced electric field measured in this report, there still exist the transient electric field, space charge electric field, associated with the ionization wave propagation.Some reports reveal that plasma propagates in region where an intense longitudinal electric field component exists a few cm ahead the ionization front [29][30][31].This is the limitation of the present work for the reason that biological effects may be dependent on the electric field strength and temporal evolution.Figure 4 displays the electric field at the BGO-dielectric interface as the applied voltage varies from 3 kV to 8 kV, with the corresponding electric field varying from 24 kV cm −1 to 40 kV cm −1 .
In the subsequent sections, E. coli (ATCC 25922) is chosen as the reference organism to investigate the physical effects in plasma.
In the experimental group, 5 µl of E. coli was applied to each of the three dielectrics.The bacteria were allowed to dry until forming a solidified film on the dielectric surface, preventing flow.Subsequently, the bacteria were exposed to plasma treatment for 10 min.Following the plasma treatment, the dielectric was immersed in 3 ml of saline for 15 min to wash off the adhered bacteria.The resulting mixture of bacteria and saline was then diluted to various concentrations and applied to tryptose soya agar media.After cultivation in a bacterial incubator for 24 h, the bacterial count was determined.
In the control group, plasma treatment was substituted with a 10 min period of non-treatment.The initial bacterial count in the control group was approximately 2 × 10 6 .
The applied voltage is a key factor influencing the discharge current, thereby affecting plasma characteristics, including the induced electric field and UV/VUV emission intensity.The association between the number of bacteria on the dielectric and the applied voltage is depicted in figure 5, with six replicate experiments conducted for each data point.When using alumina ceramic, only the electric field is effective in the sterilization process.Therefore, the BRF of electric field (BRF EF ) can be obtained.When using quartz glass, both the electric field and UV play a role.By subtracting the contribution of the electric field, the sterilization factor of UV (BRF UV ) can be obtained.Similarly, the sterilization factor for VUV (BRF VUV ) can be obtained in a comparable manner.
When Al 2 O 3 is used, as shown in figure 5, even with an applied voltage of 3 kV, the number of bacteria on the Al 2 O 3 is significantly lower compared to the control group (2 × 10 6 ).This suggests that electroporation induced by the electric field plays a role in this process at such a low voltage level.
From the figure 5, it can be observed that as the voltage increases to 6-7 kV, the sterilization rate using MgF 2 glass is approximately an order of magnitude higher than that with quartz glass, while the sterilization efficiency with quartz glass is slightly higher than that with alumina ceramic.This indicates that the VUV generated by the plasma at this point has a strong sterilizing effect, and UV also plays a certain role in sterilization.However, when the voltage increases to 8 kV, the differences in sterilization among these three dielectric materials become smaller.The sterilization efficacy significantly improves when using alumina ceramic and quartz glass, while the increase in sterilization efficiency with MgF 2 is only marginal.This may be attributed to the much stronger electric field-induced electroporation effect at this voltage level, where the corresponding electric field is 40 kV cm −1 as shown in  In order to better differentiate the three physical effects, the treatment time of plasma has been prolonged.Based on the findings from figure 5, an applied voltage of 6 kV has been chosen to investigate the correlation between plasma treatment time and bactericidal effects, given the relatively larger difference in bactericidal effects among the three cases.Five groups with varying treatment times (0, 10, 20, 30, and 60 min) were established to assess the bactericidal efficacy of the plasma after passing through quartz glass/MgF 2 /alumina ceramic.Three replicate experiments were conducted for each data point, and the results are illustrated in figure 6.
Figure 6(a) shows that, for the treatment time of 60 min, BRF EF = 1.65,BRF UV = 1.33,BRF VUV = 1.12.It is evident that all three of these physical factors contribute to the disinfection process.As shown in figure 6(b), the bactericidal effect of the electric field approaches saturation after 20-30 min, while the bactericidal effect of UV/VUV is linearly correlated with treatment time.The saturation situation may due to the charge accumulation during long time exposure which has impact on the plasma jet delivery.Electrostatic repulsion may indeed occur during this process [32,33].
Furthermore, as we know, the UV/VUV emission is dependent on the working gas.The emission of UV/VUV could vary significantly for different working gases.Therefore, in the next phase, the bactericidal effects of using He and Ar working gases will be investigated.
Given that the discharge current is much higher when He is used compared to Ar for the same applied voltage, and the electric field induced by the plasma is related to the total charges deposited on the dielectric, in the subsequent experiments, different voltages will be selected for different working gases to ensure an equal amount of charges obtained from the discharge current.
The measured results are presented in figure 7. When He is used as the working gas, the pulse width of the plasma current is notably wider compared to Ar.The integral of the plasma current at an applied voltage of 5 kV and with He as the working gas is comparable to the discharge with Ar at a voltage of 8.5 kV.Photographs of the discharges for the two gases are displayed in figure 8.
The plasma treatment duration was set to 30 min.The bactericidal outcomes under the two working gases are illustrated in figure 9.When He is used as the working gas, the distinguishable bactericidal effect produced after passing through the three dielectrics can be observed.Through calculation, BRF EF = 0.79, BRF UV = 1.10, and BRF VUV = 0.34.It can be inferred that the electric field, UV, and VUV all have a certain effect in He plasma.
When Ar is the working gas, BRF EF = 0.76, BRF UV = 2.12, BRF VUV = 0.12.It is evident that UV plays a vital role in the bactericidal process, while VUV has much weaker effect on bacteria.
Moreover, the bactericidal effects of the electric field for both He and Ar plasma are similar, indicating that our choice of the applied voltages to achieve the same integrated charges is reasonable.
To further investigate the mechanism for the differing bactericidal effects of the two gases in figure 9, the emission spectra of the plasma for both gases were measured.The wavelength range from 110 nm to 200 nm was measured using a VUV spectrometer H30-UVL (Horiba Jobin Yvon, America), with a slit width set at 300 µm.The results are displayed in figure 10.The following integral data for spectra has considered the spectral sensitivity response of the spectrometer.
The waveform of figure 10(a) is integrated to obtain a total area of 13.25, while the waveform of figure 10(b) is integrated to obtain a total area of 8.75.This indicates that the VUV intensity of He plasma is slightly higher than that of Ar plasma under the conditions set by this experiment.
The optical emission spectrum from 200 nm to 500 nm was acquired using a half-meter spectrometer (Acton Research Corporation SpectraPro2500i), with a slit width set at 100 µm.The results are presented in figure 11.
In the spectrum of Ar, the ArO * excimers appeared at 308 nm due to the ambient air [34].
The waveform from 200 nm to 380 nm (the UV range) of figure 11(a) is integrated to obtain a total area of 9299.The waveform from 200 nm to 380 nm of figure 11(b) is integrated to obtain a total area of 56 695.This indicates that the UV intensity of the Ar plasma is higher than that of the He plasma under the conditions set by this experiment.
In summary, an experiment was conducted to investigate the biological effects of plasma-induced physical energy, specifically focusing on the impacts of electric fields, UV radiation (220-380 nm), and VUV radiation (110-220 nm).The results revealed that, under certain experimental conditions, all three processes contribute to the biological effect.
Regarding the electroporation effect potentially caused by electric fields, it is crucial to note that the voltage frequency employed in traditional electroporation research is typically lower, usually ranging from a few Hz to tens of Hz.In contrast, when plasma is utilized, the voltage frequency aligns with the frequency of the plasma driving power supply, typically ranging from a few kHz to tens of kHz.Many studies have found that the pulse repetition play a key role in electroporation [35,36].Consequently, the electric field strength needed to achieve irreversible electroporation for bacteria elimination during plasma treatment may be considerably lower than that required for traditional electroporation.According to [37], for a 3microsecond pulse, the minimum electric field strength needed for irreversible electroporation using traditional methods is approximately 40 kV cm −1 .Based on the experimental results in this paper, even with an applied voltage of 4 kV, the corresponding electric field is around 27 kV cm −1 , at which point the bactericidal effect due to irreversible electroporation can be observed.When the applied voltage is increased to 8 kV, the corresponding electric field strength reaches 40 kV cm −1 , resulting in a more pronounced bactericidal effect.At this juncture, BRF EF = 1.59,BRF UV = 0.38, BRF VUV = 0.28, indicating that the electric field predominantly influences the bactericidal process under the experimental conditions.
Furthermore, it should be noted that in reality, the plasma could come into direct contact with the biological sample rather than being located on the back of the dielectric, as discussed in this paper.When the plasma is in direct contact with the biological sample, the induced electric field in the  biological sample could be even higher, potentially resulting in a more significant plasma-induced electroporation effect.
The role of UV/VUV in bacterial eradication is significantly tied to the working gas.When Ar gas is employed, the UV light intensity is approximately six times that of He gas discharge, leading to a one-order-of-magnitude higher bactericidal effect within 30 min.Considering the contribution of VUV, the VUV radiation intensity of He plasma is about 1.5 times that of Ar.Consequently, the bactericidal effect of He plasma is further doubled, while the bactericidal effect of Ar plasma increases by about 20%.It is important to note that research on VUV killing is limited due to the rapid absorption of VUV in air.Conversely, as VUV is scarce in nature, bacteria are less resistant and are more easily inactivated by such radiation [34].

Figure 2 .
Figure 2. Measurement system for electric field.

Figure 5 .
Figure 5. Variation curve of the number of bacteria on the dielectric with treatment voltages.Repetition rate: 8 kHz; pulse width: 3 µs; gas flow rate: He at 2 slm; plasma treatment time: 10 min.

Figure 6 .
Figure 6.(a) Variation curve of the number of bacteria on the dielectric with treatment times.(b) Variation curve of the BRF of the three physical factors with treatment times.Applied voltage: 6 kV; repetition rate: 8 kHz; pulse width: 3 µs; gas flow rate: He at 2 slm.

Figure 8 .
Figure 8. Photographs of discharges of different working gases.(a) He discharge at a driving voltage of 5 kV, 8 kHz, 3 µs.(b) Ar discharge at a driving voltage of 8.5 kV, 8 kHz, 3 µs.

Figure 9 .
Figure 9.The bactericidal effects of plasma under different working gases.Plasma treatment time: 30 min; gas flow rate: 2 slm; Blue bar: He with a voltage of 5 kV, 8 kHz, 3 µs.Red bar: Ar with a voltage of 8.5 kV, 8 kHz, 3 µs.

Figure 10 .
Figure 10.The spectrum from 110 nm to 200 nm of different working gases with the acquisition time of 2 s per point.Slit width: 300 µm.Gas flow rate: 2 slm.(a) He with a voltage of 5 kV, 8 kHz, 3 µs.(b) Ar with a voltage of 8.5 kV, 8 kHz, 3 µs.

Table 1 .
Properties of materials.