A comparative study of the major antimicrobial agents against the yeast cells on the tissue model by helium and air surface micro-discharge plasma

Surface micro-discharge (SMD) plasma with a large-area and homogeneous discharge has attracted much attention in the skin disinfection due to its high antimicrobial efficiency and less side effects on tissues. Although SMD plasma sterilization is undisputedly attributed to the reactive oxygen and nitrogen species (RONS), the exact RONS speciation on the tissues and their individual contribution to the plasma inactivation are still not fully understood. Herein, we investigated the generation and distribution of hydroxyl radical (·OH), hydrogen peroxide (H2O2), ozone (O3), nitrite (NO2−), and peroxynitrite/peroxynitrous acid (OONO−/ONOOH) on the agarose tissue model and their contribution to yeast inactivation by helium (He) or air SMD plasma at different irradiation distances. The results show that He and air SMD plasma exhibited different RONS speciation and antimicrobial activity. The He SMD plasma mostly generated ·OH and H2O2 on the tissue model, which were concentrated in every hexagon micro-discharge unit and decreased with the irradiation distance, while the air SMD plasma mainly produced O3, NO2−, and OONO−/ONOOH, which were uniformly distributed on the whole tissue model. More importantly, the ·OH generation on the tissue model by the He SMD plasma was derived from the plasma delivery, while UV photolysis led to the in situ ·OH generation by the air SMD plasma. Additionally, the air SMD plasma has a higher inactivation efficiency than the He SMD plasma and the major antimicrobial agent for He and the air SMD plasma is, respectively, ·OH and O3 in this plasma–tissue interaction system.Surface micro-discharge (SMD) plasma with a large-area and homogeneous discharge has attracted much attention in the skin disinfection due to its high antimicrobial efficiency and less side effects on tissues. Although SMD plasma sterilization is undisputedly attributed to the reactive oxygen and nitrogen species (RONS), the exact RONS speciation on the tissues and their individual contribution to the plasma inactivation are still not fully understood. Herein, we investigated the generation and distribution of hydroxyl radical (·OH), hydrogen peroxide (H2O2), ozone (O3), nitrite (NO2−), and peroxynitrite/peroxynitrous acid (OONO−/ONOOH) on the agarose tissue model and their contribution to yeast inactivation by helium (He) or air SMD plasma at different irradiation distances. The results show that He and air SMD plasma exhibited different RONS speciation and antimicrobial activity. The He SMD plasma mostly generated ·OH and H2O2 on the tissue model, which were concentrated in every hexagon micro-discharge...

Cold atmospheric plasma (CAP) has recently shown great prospects in biomedical applications, such as blood coagulation, 1 skin disinfection, 2 and cancer treatment. [3][4][5] Among these, CAP applied in skin disinfection has attracted much attention due to its fast and successful inactivation of diverse pathogens. 6 Reactive oxygen and nitrogen species (RONS) are considered as the major antimicrobial agents during skin disinfection by CAP. 7 The RONS speciation on the tissue can be derived from the plasma delivery of gas-phase RONS into the tissue (plasma delivery), in situ RONS generation by UV photolysis within the tissue (UV photolysis), and the gas-phase RONS reacting with the water molecules in the tissue (plasma-liquid interaction). 8,9 Moreover, many researchers have found that the plasma-derived RONS can be delivered millimeters into the tissue with a concentration exceeding hundreds of micromoles and the RONS in the tissues are predominately stable secondary RONS [such as hydrogen peroxide (H 2 O 2 ), nitrite (NO 2 − ), and nitrate (NO 3 − )] by using gelatin or agarose gel as the surrogate for real tissues. [10][11][12][13][14] However, the aforementioned research studied the plasmatissue interaction mainly by using plasma jet but rarely using surface micro-discharge (SMD) plasma. The SMD plasma may have a different RONS speciation, distribution pattern, and delivery depth in the tissues compared with plasma jet due to the different transportation modes of RONS. The plasma jet-generated RONS can be delivered over a long distance by the transportation of gas flow, while the SMD plasma-generated RONS are mainly delivered by diffusion with a relatively short transportation distance. 15,16 Meanwhile, for the practical application of CAP to skin disinfection, the plasma jet with a small-area beam may lead to incomplete coverage of the treated tissues, while the SMD plasma with a large-area and homogeneous ARTICLE scitation.org/journal/adv discharge may be more suitable for skin disinfection. [17][18][19] Therefore, it is necessary to investigate the RONS speciation in the SMD plasma-tissue interaction system and their individual contribution to SMD plasma sterilization. Herein, we compared the generation and two-dimensional (2D) distribution of five representative RONS [hydroxyl radical (⋅OH), ozone (O 3 ), H 2 O 2 , NO 2 − , and peroxynitrite/peroxynitrous acid (OONO − /ONOOH)] in helium (He) and air SMD plasma at different irradiation distances by using an agarose tissue model with embedded chemical reporters. Especially, we also evaluated the effects of UV photolysis on the ⋅OH generation in He and air SMD plasma. Additionally, the inactivation efficiency and killing pattern of SMD plasma against yeast cells (as a surrogate for the fungal pathogens) were measured and the Pearson correlation coefficient between the inactivation efficiency and the RONS concentration was analyzed to estimate the contribution of these RONS to yeast inactivation by He or air SMD plasma.  21 The optical emission spectrum (OES) was used to investigate the major excited species generated in He and air SMD plasma. In Fig. 2(a), the spectrum of He SMD plasma was dominated by OH (A → X) band emissions (306-309 nm), N 2 second positive system [N 2 (C → B)] emissions (316 nm, 337 nm, 357 nm, and 380 nm), and He emissions (389 nm, 501 nm, 667 nm, 706 nm, and 728 nm). The OH (A → X) emissions were produced via the water dissociation caused by electrons or metastable He (He * ) impact (reactions 1 and 2) (all the reactions involved in this study are listed in supplementary material S4). 21 The N 2 (C → B) emissions were produced upon collision of the electrons or He * with the tiny residual air molecules in the chamber. For the air SMD plasma, the spectrum was dominated by the N 2 (C → B) emissions at 316 nm, 337 nm, 357 nm, 380 nm, 394 nm, 399 nm, and 405 nm as a result of the direct or stepwise electron impact excitations and the pooling reaction of the metastable N 2 [ Fig. 2(b)]. 22 Compared with the He SMD plasma, the emission intensity of N 2 (C → B) was much higher in the air SMD plasma due to the presence of much more N 2 molecules in the chamber by using air as the working gas, while the emission intensity of the N 2 + first negative system [N 2 + (B → X)] at 391 nm in the air SMD plasma was much lower, which was probably attributed to many other excitation processes to produce N 2 + in the He SMD plasma except electron impact, such as the charge transfer (reaction 3) and He * excitation of the ground state N 2 (reaction 4). Additionally, the emission intensity of ⋅OH in the air SMD plasma was much weaker compared with the He SMD plasma probably due to the following two reasons. One is that O 2 in the air gas can consume the free electrons by reacting with them to form O 2 − , consequently decreasing the density of ⋅OH derived from the water dissociation by electron impact. 23 The other is that the oxygen species such as O and O 3 formed in the air plasma can react with ⋅OH. 21 The OES results indicated that the major excited species in the gasphase He and air SMD plasma were, respectively, ⋅OH and excited N 2 , which can result in the different RONS speciation on the tissue model. Given that skin tissues always contain much water and water evaporation from the skin tissues during plasma treatment could influence the RONS generation, 24-27 the variation of relative humidity in the micro-environment between the tissue model and the mesh electrode during the 10 min He or air plasma discharge process at different irradiation distances was detected. In Figs. 2(c) and 2(d), the relative humidity of the He and air SMD plasma at different irradiation distances was all first increased rapidly and then remained stable, which could be attributed to the water evaporation from the tissue model. Additionally, the average relative humidity was decreased with the irradiation distance for both the He and air SMD plasma due to the reason that the shorter irradiation distance resulted in a higher temperature of the tissue model (see supplementary material S3), which could enhance the water evaporation.
Then, the 2D distribution and relative concentration of ⋅OH, O 3 , H 2 O 2 , NO 2 − , and OONO − /ONOOH on the tissue model after 10 min He or air SMD plasma discharge process at different irradiation distances were measured and the detailed procedures are shown in supplementary material S1. Among the five RONS, ⋅OH is the most powerful oxidizing species (2.8 V), which can drastically react with almost all biomolecules. 19 For the He SMD plasma, ⋅OH was mainly concentrated at the center of every micro-discharge unit at short irradiation distances (1 mm, 2 mm, and 3 mm) and almost disappeared at long irradiation distances (5 mm and 10 mm) [ Fig. 3(a-I)]. The ⋅OH concentration was significantly decreased with the irradiation distance probably due to the following two reasons [ Fig. 3(a-III)]. One is that the ⋅OH has a short diffusion distance due to the short lifetime and high reactivity. The other is that the short irradiation distance has a high relative humidity, which may enhance the ⋅OH generation. We also evaluated the effect of UV photolysis on the ⋅OH generation via reaction 5 9,28 by using

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scitation.org/journal/adv a quartz plate covered on the tissue model, which could block the RONS diffusion and has no effects on the UV transmission. ⋅OH cannot be observed when the tissue model was covered with a quartz plate [ Fig. 3(a-II)], indicating that the ⋅OH on the tissue model was mainly derived from the plasma delivery rather than UV photolysis. Figure 3(a-IV) describes the probable transportation pathway of ⋅OH between the electrode and the tissue model in the He SMD plasma. For the air SMD plasma, ⋅OH was uniformly distributed on the whole tissue model surface and its fluorescence was much weaker compared with the He plasma at short irradiation distances without quartz plate [ Fig. 3(b-I)]. Moreover, ⋅OH could still be observed when the tissue model was covered with a quartz plate [Figs. 3(b-II) and 3(b-III)], indicating that UV photolysis leads to in situ ⋅OH generation on the tissue model in the air SMD plasma. More interestingly, the ⋅OH concentration was slightly increased with the irradiation distance and the tissue model covered with a quartz plate had a higher ⋅OH concentration, which can be explained in Fig. 3(b-IV). It is well known that the water molecules could consume UV, thereby decreasing the UV transmission to the target. Herein, the shorter irradiation distance had a higher relative humidity, which could attenuate the UV intensity. Conversely, the quartz plate could block the water evaporation, consequently enhancing the UV intensity. Unlike ⋅OH, O 3 is a long-lived RONS, which plays important roles in air plasma sterilization. [29][30][31] In Fig. 4, almost no O 3 production can be observed in the gas-phase He plasma and tissue model, while the concentrations of gas-phase and tissue-phase O 3 in the air SMD plasma were both much higher, which was due to the fact that the air gas contains much more oxygen and thus could increase the O 3 production via reaction 6. Moreover, the O 3 was uniformly distributed on the tissue model and the irradiation distance almost had no effects on the O 3 production in the air SMD plasma due to the long diffusion distance of O 3 . It is noteworthy that a slight raise of O 3 in the first 30 s in the He plasma [ Fig. 4(a)] can be attributed to the residual air in the chamber. Meanwhile, the white round shape formed in the He plasma [ Fig. 4(c)] was possibly caused by ⋅OH, which can also bleach the indigo blue color. 32 Besides O 3 , H 2 O 2 is also an important long-lived antimicrobial agent in CAP. It is generally believed that the aqueous H 2 O 2 was derived from the dissolution of gaseous H 2 O 2 and the combination reaction of ⋅OH in water (reaction 7). 33 As shown in Fig. 5(a), H 2 O 2 presents a similar 2D distribution pattern and change trend to that of ⋅OH in the He and air SMD plasma, indicating that ⋅OH combination in the gas-tissue interface contributed to H 2 O 2 generation on the tissue model. H 2 O 2 can react with NO 2 − to form a more bioactive species OONO − /ONOOH (reaction 8). 34 In Fig. 5(b-III), the air SMD plasma had a markedly higher NO 2 − generation on the tissue model compared with the He SMD plasma due to the fact that NO 2 − are mainly derived from the dissolution of gas-phase nitrogen oxides (NOx) formed by the reactions of dissociated N 2 and O 2 . 34 The NO 2 − concentration in the air SMD plasma was significantly decreased with the irradiation distances except 1 mm. The reason may be that although 1 mm is the shortest delivery distance of the gas-phase NOx, the high relative humidity at 1 mm might have adverse effects on the NOx generation. More interestingly, NO 2 − was uniformly distributed on the tissue model in the air SMD plasma, while the magenta-colored azo compounds were not generated in every micro-discharge unit at short irradiation distances in the He SMD plasma [Figs. 5(b-I) and 5(b-II)]. The reason may be that ⋅OH formed in this area can not only oxidize NO 2 to form HNO 3 (reaction 9), 21 but also directly degrade azo dyes. 35 Additionally, the distribution pattern and change trend of OONO − /ONOOH were similar to those of NO 2 − in the air SMD plasma and the OONO − /ONOOH concentration in the air SMD plasma was also higher than that in the He SMD plasma [ Fig. 5(c)].
Next, we investigated the antimicrobial effects of 10 min He or air SMD plasma against yeast cells by measuring the inactivation efficiency and killing pattern (detailed procedures are given in supplementary material S2). Expectedly, the killing pattern of yeast cells corresponded to the distribution of the major RONS on the tissue model. For the He SMD plasma, the colony was not formed in the presence of ⋅OH and H 2 O 2 and the inactivation efficiency was decreased with the irradiation distance [Figs. 6(a) and 6(c)], whose change trend was similar to that of the ⋅OH and H 2 O 2 concentrations, indicating that ⋅OH and H 2 O 2 play important roles in the yeast inactivation by the He SMD plasma. For the air SMD plasma, no yeast colonies appeared on the tissue model due to the uniform distribution of the major RONS on the tissue model [ Fig. 6(b)]. The inactivation efficiency was the same for all irradiation distances, which was consistent with the change trend of ⋅OH, H 2 O 2 , and O 3 concentrations, indicating that they all contribute to the air SMD plasma sterilization.
Furthermore, in order to estimate the individual contribution of these five RONS to yeast inactivation by He or air plasma, we analyzed the Pearson correlation coefficient between the inactivation efficiency and the RONS concentration. According to the results (see supplementary material S5), the most important antimicrobial agent for the He and the air SMD plasma is, respectively, ⋅OH (0.973 * * ) and O 3 (1.000 * * ). Additionally, the air SMD plasma exhibited a markedly stronger antimicrobial activity compared with the He SMD plasma. The air plasma achieved a 4.5 log reduction of yeast cells, while the maximum inactivation efficiency of the He plasma was only 2 log reduction. Despite the excellent inactivation efficiency of the air SMD plasma, it may be not a good choice for skin disinfection due to the reason that the gas-phase O 3 reached a high level (about 16 ppm) during 1 min air plasma treatment, which could seriously threaten the human health. The U.S. Environmental Protection Agency (EPA) reports that exposure to ozone (0.3-2 ppm) can induce inflammatory responses in various animal models. 36 In conclusion, we compared the RONS speciation and antimicrobial effects of the He and the air SMD plasma. The He SMD plasma has a higher ⋅OH and H 2 O 2 generation on the tissue model, which were mainly produced at the center of every hexagon mesh electrode and decreased with the irradiation distance, while the concentrations of O 3 , NO 2 − , and OONO − /ONOOH were higher in the air SMD plasma, which were uniformly distributed on the whole tissue model. Importantly, the ⋅OH generation on the tissue model by the He and the air SMD plasma was, respectively, derived from the plasma delivery and UV photolysis. The air SMD plasma exhibited a stronger antimicrobial activity compared with the He plasma and the key antimicrobial agent for the He and the air SMD plasma is, respectively, ⋅OH and O 3 . This work gives an insight into the major antimicrobial agents in the SMD plasmatissue interaction system by using different working gases, which could promote the development of the SMD plasma applied to skin disinfection.