Int. J. Plasma Environ. Sci. Technol. 17 (2023) e01003 (9pp)

Regular Paper

DOI: 10.34343/ijpest.2023.17.e01003

Effect of supplied gas on ROS transport throughliquid targets by plasma jet irradiation
Heping Shi, Kecheng Shen, Toshiyuki Kawasaki*
Department of Electrical Engineering, Nishinippon Institute of Technology, Japan* Corresponding author: tkawasak@nishitech.ac.jp (Toshiyuki Kawasaki)
Received: 21 November 2022
Revised: 16 February 2023
Accepted: 20 February 2023
Published online: 21 February 2023

Abstract
Recently, plasma jet technology has been developed for biomedical applications, so we must control the supply of reactive
oxygen species (ROSs) by plasma to use it safely and effectively. In previous studies, several experiments were conductedusing a He plasma jet. However, we have not investigated whether there is any difference when supplying different typesof gas. In this study, we studied the effects of supplied gases (He and Ar) on ROS distribution in liquid and tissue models
induced by plasma jet irradiation. In this experiment, we visualized the ROS distribution pattern after passing through awater layer or tissue model and obtained the relative ROS concentration profiles using absorbance measurement. The
results show that the transport characteristics of ROSs produced by He or Ar plasma jets have nothing in common. We
also analyzed the components of water after plasma jet irradiation.
Keywords: ROS transport, water analysis, 2-dimentsional distribution.

1. Introduction
Atmospheric pressure plasma has received extensive attention in plasma-induced reactive oxygen species(ROS) transportation, which is critical in recent plasma science and technology [1–3]. An atmospheric pressureplasma jet can be directly irradiated to target liquids, including human skin and biological tissues inatmospheric air, with no disadvantages or damages. Clarifying the interaction between plasma and liquids isimportant for advancing plasma science in areas where liquids are intensely involved such as plasma medicine,
plasma agriculture, and water treatment. In these cases, the transportation of ROS to target cells through liquid
or biomedical tissue is an important application of plasma technology. Therefore, ROS transportation throughliquid targets must be investigated for the safe and effective use of this technology.
Several reports have emphasized the supply and transport of reactive species in various plasma-liquid
systems [4]. In previous studies, the transport characteristics of reactive oxygen species and its penetration
depth through water layers or tissue models had different effects when He gas was supplied for a coldatmospheric plasma jet generation under different conditions [5–8]. In this experiment, we found that a twodimensional (2D) distribution of ROS is completely different when Ar or He gas is used. We conducteddifferent water experiments and analyzed to better understand its mechanism. Based on the water analysis
difference between the Ar and He irradiated water, we discovered the reason for the different situations of
ROS transportation.
2. Experimental
2.1 Plasma-jet
Fig. 1 shows schematics of the plasma generator used in this experiment. It is set up with a glass-coated rod
high voltage electrode installed in the center of a glass tube with an inner diameter of 4 mm, and a ground1Int. J. Plasma Environ. Sci. Technol. 17 (2023) e01003

H. Shi et al.

electrode was wrapped around the outside of the glass tube 10 mm away from the tip of the high voltage
electrode. A plasma-generating gas He or Ar was supplied to the gap between the glass tube and the rod
electrode at a flow rate of 3 L min–1 using a mass-flow controller (Kofloc 8500 MC). We used a function
generator (NF WF1948) and an amplifier (Trek 10/40A) to generate high voltage. The voltages were set to 14
kVp-p and 15 kVp-p for He and Ar gases, respectively. The frequency was set to 10 kHz in both cases. The
corresponding discharge power was approximately 3 W, as measured using an oscilloscope (Iwatsu DS-8104).
The irradiation distance between the open end of the tube and the target surface was set at 10 mm.
Fig. 2 shows the typical voltage/current waveforms and discharge photographs of (a) Ar and (b) He gases.

Fig. 1. Schematics of the experiment setup.

Fig. 2. Typical voltage/current waveforms and discharge photographs of (a) Ar and (b) He.

2.2 Visualization of Two-dimensional distribution
Fig. 3 (a) shows the KI-starch gel ROS detection reagent for determining the 2D distribution of ROS. This
reagent was divided into 20-g portions in Petri dishes with a diameter of 90 mm. The KI-starch gel ROS
detection reagent undergoes an oxidation reaction with the active species generated by plasma irradiation,
resulting in a purple color change. An example of the color reaction is shown in Fig. 3 (b).
To obtain the ROS distribution through several layers, we first irradiated the KI-starch gel reagent for 5 s
directly, then measured the reagent absorbance [9-12]. We also prepared distilled water at a thickness of 2 mm
using the KI-starch gel reagent [13]. The surfaces of the water layer are irradiated with a plasma jet underopen-air conditions. The ROS are transported through the water layer to the KI-starch gel reagent in a 15-min
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plasma irradiation. Then, the water was removed from the KI-starch gel reagent after irradiation, and wevisualize the ROS distribution patterns after plasma irradiation. In addition, we used agarose (0.4%) as thetissue model (thickness: 1 mm) irradiated by plasma jet for five min and stand by plasma-off for five min, thenremoved the tissue model, and finally, the color reaction of the KI-starch reagent was observed.

(a) Before irradiation(b) After irradiation(c) Absorbance measurement to obtain relative ROS concentration

Fig. 3. Two-dimensional measurement system for gel reagent (a) before and (b) after plasma
irradiation, (c) absorbance measurement to obtain relative ROS concentration.

Fig. 3 (c) shows the 2D measurement system used in our laboratory to measure the absorbance of the color
reaction. After plasma irradiation, the gel-like ROS detection reagent is irradiated by a light source simulating
sunlight underneath, and the relative ROS concentration was measured using the Beer-Lambert law from thetransmitted light and the light intensity of the light source. A common and practical expression of the BeerLambert law relates the optical attenuation of a physical material containing a single attenuating species of
uniform concentration to the optical path length through the sample and absorptivity of the species. This
expression is as follows:
𝑨 𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆

log

𝜀𝐿𝑐

(1)

where A is the absorbance, 𝜀 is the molar attenuation coefficient or absorptivity of the attenuating species, L isthe optical path length in cm, 𝑐 is the concentration of the attenuating species, 𝐼 is the radiant flux transmitted
by the material sample, 𝐼 is the radiant flux received by the material sample.
In this experiment, ε and L are constant values, so the relative ROS concentration can be obtained from theabsorbance.
2.3 Water analysis
In this experiment, we prepared 40 ml of distilled water in the same Petri dishes. Then, we irradiated the surfaceof the liquid. The irradiation time was varied from 5 to 30 min to determine what happened in the liquid after
plasma irradiation. We used densitometers (Kyoritsu chemical-check lab) and absorption photometry with achromogenic reagent to measure the concentration of hydrogen peroxide (densitometers: DPM2-H2O2,
reagent: WAK-H2O2) and nitrous acid (densitometers: DPM2-NO2, reagent: WAK-NO2). We also measured
the pH and liquid conductivity of the water after plasma irradiation.
3. Results and discussion
In this study, some ROS generated by plasma irradiation can reach the target through the liquid. Therefore,
focused on the distribution of transported ROS through liquids by plasma irradiation.
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Fig. 4 shows the result of a 2D distribution experiment in air without any water or water layers. A
visualization of the oxidation reaction on the target surface in the air is shown in Figs. 4 (a) and 4 (b). The
color reaction of the KI-starch reagent using an Ar plasma jet is shown in Fig. 4 (a), and the color reaction by
the He plasma jet is shown in Fig. 4 (b). The color reaction of the Ar plasma generator is different from thatof the He plasma generator. Fig. 4 (a) shows a small color reaction at the center of the KI-starch reagent, andtwo bigger circular color reactions around the center. While Fig. 4 (b) shows only a small circle at the centerof the KI-starch reagent. Based on the color reaction results, we obtained the relative ROS concentration
distributions by measuring the absorbance along lines A to B (Figs. 4 (a) and 4 (b)). The relative ROS
concentrations when the surface of the KI-starch reagent is irradiated with an Ar or He plasma generator underopen-air conditions are shown in Fig. 4 (c).

Fig. 4. Results of two-dimensional distribution experiment without a water layer or tissue modelfor (a) Ar gas, (b) He gas, and (c) corresponding relative ROS concentration obtained byabsorbance measurement along the AB lines.

Fig. 5 shows the result of a 2D distribution experiment through the water. The visualization of the oxidation
reaction on the target surface through water is shown in Figs. 5 (a) and 5 (b). The color reaction of the KIstarch reagent using an Ar plasma jet is shown in Fig. 5 (a), while the color reaction by the He plasma jet isshown in Fig. 5 (b). The color reaction takes the shape of donuts when using the Ar plasma generator (Figs.
5(a) and 5 (b)). However, the color reaction cannot be observed when using the He plasma generator in thisexperiment.

Fig. 5. Results of two-dimensional distribution experiment through the water layer for (a) Ar gas,(b) He gas and (c) corresponding relative ROS concentrations obtained by absorbancemeasurement along the AB lines.

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Based on the color reaction results, we obtained the relative ROS concentration distributions by measuring
the absorbance along lines A to B (Figs. 5 (a) and 5 (b)). The relative ROS concentration transported throughthe water layer to reach the KI-starch gel reagent beneath when the surface of the water layer is irradiated withan Ar or He plasma generator under open-air conditions is shown in Fig. 5 (c).
Fig. 6 shows the result of the 2D distribution experiment through a tissue model. A visualization of theoxidation reaction on the target surface is shown in Figs. 6 (a) and 6 (b). The color reaction of the KI-starch
reagent using an Ar plasma jet is shown in Fig. 6 (a), while the color reaction by the He plasma jet is shownin Fig. 6 (b). The color reaction by the Ar plasma generator is different from that by the He plasma generator.
A dark and circular color reaction could be observed at the center of the KI-starch reagent (Fig. 6 (a)), whereas
only a thick and circular color reaction could be observed at the center of the KI-starch reagent (Fig. 6 (b)).
Based on the color reaction results, we obtained the relative ROS concentration distributions by measuring theabsorbance along lines A to B (Figs. 6 (a) and 6 (b)). The relative ROS concentrations when the surface of thetissue model is irradiated with a plasma generator under open-air conditions are shown in Fig. 6 (c).
Comparing the 2D distribution results of Ar plasma irradiation with the results of He plasma irradiation,
we found some differences between Ar and He plasma. For the color reaction results of water layers shown in
Fig. 5, it has been reported that this donut-shaped distribution is related to the plasma-induced flow [13, 14].
The transport of ROS through water layer is affected by the plasma-induced flows. And in this experiment, theplasma-induced flows may cause the diffusion of ROS in water easily because of the low viscosity of water.
However, for the color reaction results of tissue models shown in Fig. 6, the transport of ROS through tissue
model in this experiment is hard to spread because of the high viscosity of tissue model. Based on the resultsof two-dimensional distribution, we focused on plasma differences through the water layer. We investigatedthe ROS supplied or produced by Ar plasma irradiation transport to the bottom of the liquid (Fig. 5 (a)).
However, we cannot observe anything on the surface of the KI-starch reagent (Fig. 5 (b)). To explain these
phenomena, we analyzed the liquid component after Ar or He plasma jet irradiation.

Fig. 6. Results of two-dimensional distribution experiment through the tissue model for (a) Ar gas,(b) He gas and (c) corresponding relative ROS concentrations obtained by absorbancemeasurement along the AB lines.

In previous studies, Ar and He plasma, upon contact with water, initiate the chemical reaction that generates
ROS such as OH radical to produce hydrogen peroxide. It also generates RNS variations due to the interaction
with the surrounding atmosphere to produce NO2− and NO3−. The collision of the electrons can produce thefollowing reactions [15]:Ar + e → Ar* + e

(2)

[16]

He + e → He*+ e

(3)

[17]

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H. Shi et al.

O2 + e → 2O + e

(4)

[18]

N2 + e → 2N + e

(5)

[19]

H2O + e → H˙ + HO• + e

(6)

[20]

Aqueous and gaseous hydrogen peroxide can result from the recombination of two hydroxyl radicals in aliquid.
Ar* + H2O → Ar + H˙ + HO˙

(7)

[16]

He* + H2O → He + H˙ + HO˙

(8)

[21]

2N + 2H2O → N2 + 2H˙ + 2HO˙

(9)

[22]

O + H2O → 2HO˙

(10)

[23]

HO˙ + HO˙ → H2O2

(11)

[24]

The untypical trend of nitrite concentration in water during plasma activation is the result of a competition
between nitrites and nitrates produced in a hermetic enclosure [23]. The formation of nitrogen oxide in the gas
phase is described by the Zeldovich reactions. In the atmospheric plasma, NO is rapidly turned into nitrogendioxide by three reactions with oxygen. In the second phase, nitrogen dioxide is dissolved in water, resulting
in the formation of nitrites, nitrates, and hydronium ions [25].
N + O → NO + N

(12)

[26]

N + O → NO + O

(13)

[26]

NO + O˙ + M → NO2 + M

(14)

[26]

2NO2 + H2O → NO2− + NO3 + 2H+

(15)

[27]

NO + NO2 + H2O → 2NO2− + 2H+

(16)

[28]

Consequently, the rate of nitrite production decreases with activation time while two reactions transform
nitrites into nitrates using O3, as follows:
NO2− + O3 → NO3− + O2

(17)[27,29]The chemical reactions mentioned here are well known as representative chemical reactions in theinteraction between the plasma irradiation and the liquid phase, as in this experiment. Therefore, we focusedon the concentration of hydrogen peroxide and nitrous acid in water after plasma irradiation in this experiment.
Figs. 7 (a) and 7 (b) show the concentration of nitrous acid and hydrogen peroxide after plasma irradiationfor 5 to 30 min, respectively. The results of the color reaction of the chromogenic reagent are shown in Figs.
7(a) and 7 (b). The concentration of nitrous acid and hydrogen peroxide after Ar plasma irradiation is higher
than the results after He plasma irradiation (Figs. 7 (a) and 7 (b)). In addition, the liquid conductivity and pHof the liquid after plasma irradiation are shown in Figs. 7 (c) and 7 (d), respectively. The liquid conductivity
after Ar plasma irradiation is higher than the results after He plasma irradiation, while the pH after Ar plasma
irradiation is lower than the results after He plasma irradiation.
Several reports have shown that the number of OH radicals generated using Ar plasma is greater than the
results generated using He plasma [1]. Ar plasma jet provides higher efficiency in generating hydrogen
peroxide [30], as well as more ozone and nitrogen oxides than He plasma jets [31]. Therefore, the results of
the water analysis in this experiment can be confirmed using these reports.

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Fig. 7. Results of water analysis for (a) NO2⁻ concentration, (b) H2O2 concentration, (c) liquid
conductivity and (d) pH of water by plasma irradiation from 5 to 30 mins.

Based on the results of the water analysis, there was a correlation between ROS transport and the resultsof the water analysis. We can understand that there are more ROS transported to the bottom through waterlayer or tissue model when supply Ar gas. Therefore, the ROS does not have enough power to be transported
deeply when using a He plasma jet.
However, the results of irradiation in air without a water layer or tissue model as shown in Fig. 4, it does
not mean the He plasma jet provides less concentration of ROS than the plasma generated by Ar gas. Since
these relationships cannot be explained at present, this will be a subject for our future work.
4. Conclusion
In this study, we investigated the effect of the supplied gas on ROS transport in the liquid after plasma jet
irradiation. Under the present experimental conditions, the amount of ROS that reached the bottom of the waterlayer or tissue model transported by Ar plasma jet is larger than the results by He plasma jet. In addition, water
analysis experiments were performed, and there was a correlation between the ROS transport and the amountof hydrogen peroxide or nitrous acid in the liquid produced by plasma irradiation. In conclusion of thecomparison, hydrogen peroxide and nitrous acid generated or supplied by plasma irradiation promote ROS
transport toward the depth direction in liquids.
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