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Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Plasma-Functionalized Solution: A Potent Antimicrobial Agent for Biomedical Applications from Antibacterial Therapeutics to Biomaterial Surface Engineering Joo Young Park,†,# Sanghoo Park,†,# Wonho Choe,*,†,‡ Hae In Yong,§ Cheorun Jo,§ and Kijung Kim† †

Department of Physics and ‡Department of Nuclear and Quantum Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea § Department of Agricultural Biotechnology, Center for Food and Bioconvergence, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea S Supporting Information *

ABSTRACT: Deadly diseases caused by pathogenic bacteria and viruses have increasingly victimized humans; thus, the importance of disinfection has increased in medical settings as well as in food and agricultural industries. Plasma contains multiple bactericidal agents, including reactive species, charged particles, and photons, which can have synergistic effects. In particular, the chemicals formed in aqueous solution during plasma exposure have the potential for high antibacterial activity against various bacterial infections. Here, we report the antibiotic potency of plasma-treated water (PTW). To illustrate the applicability of PTW for disinfecting biological substances, an Escherichia coli biofilm was used. We sought to identify the chemical species in PTW and investigate their separate effects on biofilm removal. Dielectric barrier discharge in ambient air was used to prepare the PTW and treat the biofilm directly. Hydrogen peroxide, ozone, and nitrites were identified as the long-lived reactive species in the PTW, whereas hydroxyl radicals and superoxide anions were identified as the short-lived reactive species in the PTW; all these species showed an ability to disinfect in biofilm removal. KEYWORDS: dielectric barrier discharge, plasma-treated water, antibiotic agent, biofilm inactivation, reactive oxygen and nitrogen species

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INTRODUCTION Active attempts to apply atmospheric-pressure plasma science and technology to various areas, such as microelectronics,1 purification,2,3 material synthesis,4,5 and more recent biomedical applications,6,7 have produced impressive results and shown the potential for using plasma in industrial fields. Along with these activities, efforts to apply plasma for food decontamination and pasteurization and in agriculture for nitrogen fixation and disinfection (i.e., plasma farming) have been rapidly increasing.8−14 The relationship between electric discharge and agriculture is well-known from practical experience, as reflected in the following Korean proverbial phrase: “It will be a bumper crop year if lightning strikes frequently.” This proverb alludes to lightning fixing atmospheric nitrogen and oxygen, which are essential plant nutrients, into soil and plants via raindrops, thereby resulting in a bumper crop. With similar underlying mechanisms as the interactions between rain and chemical products generated by lightning in nature, contact between atmospheric-pressure plasma and liquid (as well as remote exposure) rapidly dissolves nitrogen and oxygen into the target solution. Recent publications have © XXXX American Chemical Society

demonstrated that atmospheric-pressure plasma-treated water (PTW), which contains a considerable amount of reactive species, has bactericidal activity.15,16 As is well-known, the major factors underlying the effect of atmospheric-pressure plasma are reactive oxygen and nitrogen species (RONS), whereas the influence of photons in the ultraviolet−visible range, a transient electromagnetic field, and energetic charged particles is negligible. Because RONS generated by plasma, such as nitric oxide, hydroxyl radicals, and other intermediate compounds, have a high oxidation potential, atmosphericpressure plasma and PTW likely have an antimicrobial effect against biological substances, such as bacteria and cancer cells. The effect of chemical parameters, such as pH and temperature, on RONS concentrations and the bactericidal activity of PTW have recently been studied.17,18 However, a key relevant issue is identifying the important chemical species and determining the significance of these chemicals in the desired reactions for Received: September 20, 2017 Accepted: November 28, 2017

A

DOI: 10.1021/acsami.7b14276 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces targeted treatments. As a bold attempt to address this issue, we employed a targeted treatment for the inactivation of a biofilm. A biofilm is a cluster of biomaterials and microorganisms, such as viruses, enzymes, fungi, and bacteria, and it selfassembles into a biofilm community and protects itself by secreting a slime and protective film.19 Inside the biofilm, the attached microorganisms generate an exopolysaccharide matrix that attracts suspended solids and nutrients.20,21 A strong attachment between the biofilm and various surfaces of household goods, such as kitchen knives, water pipes, contact lenses, and food containers, causes hygienic problems that result in serious health issues. In this work, we investigated the separate bactericidal effects of long- and short-lived chemical products in PTW on Escherichia coli biofilms, which is one of the biofilms that causes problems related to food safety. Inplane dielectric barrier discharge (DBD) in ambient air was employed, and the concentrations of each chemical product (nitrate, NO3−; nitrite, NO2−; hydrogen peroxide, H2O2; ozone, O3; and hydroxyl radical, •OH) in the PTW were measured. Sixty-three percent of the E. coli biofilm was reduced under plasma exposure at 200 W for 10 min. In the case of long-lived species (NO3−, NO2−, H2O2, and O3), separate bactericidal activity was estimated based on the reduction in the biofilm in chemical solutions that contained the same concentration of each chemical species in the PTW. To estimate the bactericidal effect of short-lived species (•OH and superoxide anion, •O2−), the reduction in the biofilm by plasma treatment in solutions with and without their scavengers were compared. We demonstrated that long-lived species present bactericidal activity against biofilms in the order of H2O2, O3, and NO2−, whereas short-lived species present bactericidal activity against biofilms in the order of •OH and •O2−.

Figure 1. Three different experiments for biofilm treatments. Biofilm samples were treated by (a) direct plasma exposure, (b) immersion in PTW, and (c) immersion in a chemical solution. Three milliliters of solution was poured into a 40 mm-diameter fused silica dish in each case.

nitrogen oxides react immediately and irreversibly with water molecules and form HNO2 and HNO2 via the following reactions

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RESULTS AND DISCUSSION As illustrated in Figure 1, the E. coli biofilm samples were treated in three different ways: (1) by exposure to the plasma (see Figure 1a), (2) by immersion in PTW (Figure 1b), and (3) by immersion in solutions containing specific chemical species at the same concentrations as in the PTW (Figure 1c). On the basis of the results of methods (2) and (3), we identified the chemical species that are important for biofilm reduction. Among these different treatments, the plasma treatment was most effective for biofilm reduction. Constituents of PTW. Treating distilled water with plasma produces various types of chemicals.15,22 In the case of air discharge, nitrogen oxides, such as NO, NO2, and N2O3, are mainly produced at the discharge layer near the electrode through the reactions given below O(3P) + N*2 (Σ+g , v) → NO(2Π) + N( 4S)

(1)

NO + O2 (O3) → NO2 + O(O2 )

(2)

2NO + O2 → 2NO2

(3)

2NO2 ⇌ N2O4

(4)

NO + NO2 ⇌ N2O3

(5)

2NO2 + H 2O → HNO2 + HNO3

(6)

N2O3 + H 2O → 2HNO2

(7)

N2O4 + H 2O → HNO2 + HNO3

(8)

HNO2 ⇌ H+ + NO2−

(9)

HNO3 ⇌ H+ + NO3−

(10)

Because HNO3 (the negative logarithm of the acid dissociation constant, pKa ≅ −1.4) and HNO2 (pKa ≅ 2.8− 3.2), which release H+ ions via deprotonation reactions at a higher pH than pKa, are produced at nonnegligible concentrations, the pH of the PTW gradually decreases during plasma treatment, and acidification of the PTW promises the bactericidal activity of NO2− against a number of organisms.23,24 In our experiment, the pH of 3 mL of distilled water decreased from 7.0 to 3.5 after 10 min of plasma treatment at 200 W. Long-lived species other than nitrogen-related species in PTW are also created by air discharge as follows •OH + •OH + M → H 2O2 + M

(M = N2 or O2 ) (11)

O2 + O + M → O3 + M

(M = N2 or O2 )

(12)

Reaction 11 shows the generation of H2O2 in a gaseous phase, which then permeates into water. The previous works25,26 discussed a detailed mechanism of the direct synthesis of H2O2 from plasma−water interactions. The main process of gaseous O3 formation is the three-body reaction shown in reaction 12; aqueous O3 is mainly formed via the fast dissolution of gaseous O3 into aqueous solution. H2O2 and O3

These gaseous species rapidly diffuse and permeate into the target solution (distilled water in this work) and initiate many chemical reactions at the surface of the solution. Nitric oxide (NO) is rarely soluble in water, whereas most dissolved B

DOI: 10.1021/acsami.7b14276 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 2. Removal ability of DBD exposure. The fraction of the removed biofilm after the plasma treatment with respect to (a) treatment time (input power, 200 W) and (b) input power (treatment time, 10 min). The remaining biofilm values after treatment were normalized to 100% for an untreated biofilm sample. In addition, the removed biofilm values were calculated by subtracting the remaining values from 100%. The input power was fixed at 200 W, whereas the treatment time varied to assess the treatment time effect; additionally, the treatment time was fixed at 10 min, whereas the input power was varied to assess the input power effect.

are usually considered key chemicals and are already used as strong oxidizers in various industries. Short-lived species in PTW may also play a key role in biofilm inactivation. However, these species are not considered in this study, except for •OH and •O2−, because their contribution to biofilm removal activity is small because of their low concentrations;15 in addition, their final or intermediate products are related to •OH and •O2− (e.g., ONOOH → •OH + NO2). Although gaseous •OH does not easily permeate into water because of its short lifetime, •OH is generated in liquid via reproduction processes, such as the following

Table 1. Concentration of RONS Produced in 3 mL of Distilled Water after 10 min of Plasma Treatment RONS −

NO2 NO3− H2O2 O3 OH a

(13)

hν

HNO2 → •NO + •OH

(14)

O3 + HO2 → 2O2 + •OH

(15)

SEMa

5 mM 1 mM 1 mM 10 μM 0.1 μM

0.2 0.06 0.04 0.03 0.015

Standard error of the mean (n = 5).

plasma treatment using benzene as a chemical probe. The concentration of •OH reported here is the total quantity produced during the plasma operation (10 min) and not the steady-state concentration. Separate Effects of Long-Lived RONS in PTW on Biofilm Inactivation. On the basis of the examined constituents of the PTW, the experiments presented in Figure 1b,c were conducted to identify the plasma-assisted chemicals in the PTW that can reduce biofilm. As explained in detail in the previous subsection, the effective long-lived species in PTW are NO2−, NO3−, H2O2, and O3. To identify the separate bactericidal activities of these species, we separately prepared chemical solutions with the same concentrations as those in the PTW (plasma treatment at 200 W for 10 min): 1 mM H2O2, 1 mM NO3−, 5 mM NO2−, and 10 μM O3. Then, the biofilm was selectively immersed in a 40 mm-diameter fused silica dish in 3 mL of each solution for 10 min. Because the bactericidal activity of PTW depends on the pH and temperature,17,18 the pH and temperature of the samples were adjusted to those of the PTW. The pH of all solutions was adjusted to 3.5 by adding hydrogen chloride (HCl), and the temperature of the solutions was maintained at 25 °C throughout the experiment. The degree of biofilm reduction for each species is depicted in Figure 3a. It is worth noting that the concentrations of long-lived species in the case of plasma treatment are lower than those of long-lived species in PTW and chemical solutions. In other words, longlived species are continuously produced by plasma during the 10 min treatment, and the final concentrations are the same as those in the PTW and chemical solutions. Therefore, the biofilm removal of PTW and each chemical solution cannot be directly compared with that of the plasma treatment. Nitrate. NO3− has a weak effect on biofilm reduction by less than 1% (not shown here). At the gas−liquid interface, HNO3 is produced via reactions between nitrogen oxides and water, and it is immediately deprotonated (reaction 10) at pH 3.5 because of a low pKa. The concentration of NO3− remains constant and does not vary with storage time or treatment time.

hν

H 2O2 → •OH + •OH

Concentration

Ultraviolet photolysis of the dissolved H2O2 and HNO2 leads to the formation of •OH (reactions 13 and 14), and reactions of HO2 with O3 also contribute to the release of •OH in PTW (reaction 15). Removal Efficacy of Air Discharge under Different Durations and Input Power. As discussed in the previous section, the generation of antibacterial chemical agents in PTW enables biofilm reduction. First, we sought to establish the operating conditions of the plasma treatment because the concentrations of the aforementioned chemicals strongly depend on the plasma conditions. Figure 2 shows biofilm reduction by method (1) as a function of treatment time and input power. The biofilm reductions were quantified using a crystal-violet (CV) staining method, which will be described in the Experimental Section in detail. The fraction of removed biofilm was apparently increased with increasing treatment time and input power. Considering the practical aspect of treatment time and input power, a treatment time greater than 10 min and an input power greater than 200 W were avoided in this work. Thus, plasma treatment was performed at 200 W for 10 min to produce the representative PTW for comparison with the results obtained by methods (2) and (3). Prior to conducting the detailed study, the constituents of PTW were identified. Five chemicals were identified in 3 mL of distilled water treated at 200 W for 10 min, and their concentrations were measured, as listed in Table 1. We classified the chemical agents that induce biofilm reduction as long- and short-lived species. The long-lived species, H2O2, O3, NO2−, and NO3−, were measured after the plasma treatment, and the short-lived species, •OH, was measured during the C

DOI: 10.1021/acsami.7b14276 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 3. (a) Comparison of the individual H2O2, NO2−, and O3 solutions, the mixed H2O2, NO2−, and O3 solution, and PTW. (b) Comparison of the Fenton reactions, scavenger treatments and plasma treatment. (c) Quantification of •OH radicals produced in a 1 mM H2O2 solution via the Fenton reaction. (d) Biofilm inactivation caused by the Fenton reactions according to the •OH concentration.

Indeed, NO3− is chemically stable alone, which means that it does not produce other chemicals and react with biological substances. However, the concentration of NO3− in PTW continuously increases with storage time because NO3− is a final product of intermediates released from acidified NO2− and other chemicals in acidic solution. Although NO3− is not a key species in biofilm inactivation, the acidification of PTW by HNO3 is important for the chemical reaction of other products in PTW and is a key requirement for the bactericidal activity of PTW, which will be discussed later. Nitrite. The 5 mM NO2− solution reduced the biofilm by approximately 9%. NO2 − is known to have a strong antimicrobial effect under acidic conditions. Acidified NO2− is protonated and decomposes moderately fast into NO, NO2, and H 2 O, and the two formal intermediates of NO 2 − decomposition are strongly toxic to cells. In addition, NO can be released via the photolysis of NO2− as a reaction 14,27 although this reaction pathway is not considered in this work. Ozone. O3 has the highest oxidation potential among the long-lived species in PTW, which explains why O3 is the second major component (a biofilm reduction of approximately 14%) for biofilm reduction, despite having a much lower concentration than the other long-lived species in the PTW. Hydrogen Peroxide. H2O2 induced by plasma and water has attracted considerable attention, and its antibiotic contribution in PTW is well-established.28,29 As expected, H2O2 is the most effective agent among the long-lived reactive species and reduced the biofilm by approximately 25%. PTW. To investigate the synergistic effect of the combination of the identified species related to the intermediates and reaction products, biofilms were immersed in a mixed solution and PTW. The mixed solution contained the four long-lived species described above. The PTW used in this experiment was obtained by exposing 3 mL of distilled water to 10 min of plasma at 200 W in the absence of a biofilm. One minute after the plasma treatment, the biofilm was immersed in the solution, as shown in Figure 1b. Because one minute of delay is much longer than the decay time of the short-lived species, the contribution of these short-lived species is negligible, whereas

the intermediate products can be released via postdischarge reactions among long-lived species. As shown in Figure 3a, there was a little difference in biofilm inactivation among the results obtained from the mixed solution, PTW, and the combined results of the separate experiments for NO2−, O3, and H2O2. This result indicates that the synergistic effects induced by long-lived species in PTW were negligible in our experiment, although papers have reported synergistic effects of H2O2 and NO2− on antibacterial activity.30 To the best of our knowledge, different experimental conditions and concentrations of chemical products in PTW produce different amounts of chemicals and cause the PTW to have varying antibiotic ability. For instance, the intermediates rapidly react with other species rather than with the target biological substances. Moreover, trace amounts of other aqueous chemical products, which inhibit the synergistic effects of H2O2 and NO2−, can exist in PTW. It is worth noting that the CV staining method is normally used for total biomass quantification, and thus, the antimicrobial activity, which is demonstrated using the CV method, can be underestimated. To assess the viability of biofilm after treatments, the remaining viable bacteria were also determined as colony-forming units (cfu). As shown in Figure S1 in the Supporting Information, 1.9 and 3.9 log cfu cm−2 of biofilm were reduced by PTW treatment and direct plasma treatment at 200 W for 10 min, respectively. Separate Effects of Short-Lived RONS Produced during Plasma Treatment on Biofilm Inactivation. As discussed in the previous subsection, despite the lower concentrations of long-lived species produced by plasma during biofilm treatment than those in the PTW, more biofilms were inactivated by plasma treatment than by the PTW. From this result, we can expect the generation of short-lived species whose antimicrobial effect on biofilm inactivation is noticeable during plasma treatment. In the case of the plasma treatment applied to the biofilm immersed in distilled water, short-lived species, such as •OH and •O2−, as well as long-lived species contribute to biofilm removal. Biofilm reductions related to •OH and •O2− produced during the plasma treatment were D

DOI: 10.1021/acsami.7b14276 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 4. Relative removal potential of plasma treatment and short-lived species (i.e., •OH, •O2−). The lowest red bar, which represents the difference between two cases, indicates the existence of other effective species on biofilm inactivation in PTW or the importance of other short-lived species and intermediates produced by long-lived species in disinfection.

Figure 4 presents the normalized biofilm removal efficacy of •OH, •O2−, and other species. As depicted by the red-colored bar in the figure, there was a large discrepancy between the plasma treatment and the two short-lived species, indicating the complexity of PTW as well as atmospheric-pressure plasmas. This discrepancy can be attributed to long-lived species, as demonstrated here (although it cannot be compared with the results of the plasma treatment, as discussed above), and shortlived species other than •OH and •O2−; NO and NO2, which are released at concentrations comparable to that of •OH, are likely two of these species in PTW. As shown in reaction 14, the photolysis of HNO2 leads to the production of NO and •OH. One well-accepted origin is peroxynitrous acid (HOONO), which decomposes into NO2 and •OH.

estimated via method (1). Two representative scavengers were used in this experiment: 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which is usually used as a scavenger of •OH and •O 2 − , and superoxide dismutase (SOD), which is a representative scavenger of •O2−. During the plasma treatment with DMPO (denoted by “iii” in Figure 3b), •OH and •O2− are eliminated in the solution; during the plasma treatment with SOD (denoted by “ii” in Figure 3b), only •O2− is eliminated. Thus, the removal activity of each species can be easily estimated because (i)−(ii) is a •O2− and (ii)−(iii) is a •OH. Additional experiments were dedicated to confirming the separate influence of •OH on biocidal activity, as follows. We prepared a nitrogen-free •OH solution using the Fenton reaction (Fe2+ + H2O2 → Fe3+ + •OH + OH−), and the biofilm was immersed into this solution via method (3). Iron(II) sulfate heptahydrate (Sigma-Aldrich 215422) and H2O2 (Junsei 23150-0350) were used in this experiment. Plasma Treatment for Biofilm Reduction Using the Scavengers DMPO and SOD: Effect of Hydroxyl Radicals and Superoxide Anions on Biofilm Removal. Figure 3b compares the Fenton reactions, the addition of scavengers to the plasma treatment, the PTW treatment, and the direct plasma treatment. Biofilm reductions of 60 and 50% were observed in the presence of SOD and DMPO, respectively. Consequently, compared with direct treatment (63%), 3% of the biofilm reduction was achieved by •O2− and 10% by •OH. Effect of Hydroxyl Radicals Produced via the Fenton Reaction. First, the production yield of •OH within 10 min of storage time was obtained as a function of the FeSO4 concentration in 1 mM H2O2. As shown in Figure 3c, 0.1 μM •OH was produced by 10 μM FeSO4. We are sure that less than 200 μM FeSO4 does not affect biofilm reduction alone. A Fenton solution containing 1 mM H2O2 and 10 μM FeSO4 was prepared, and the biofilm was treated in the same manner as illustrated in Figure 1c. The experimental conditions were the same as in the previous experiments. Figure 3b shows the results of the Fenton solutions, in which biofilm reduction was achieved by •OH and H2O2 simultaneously in the aqueous phase. A 25% reduction of the biofilm was achieved by 10 μM H2O2 without the iron ions, whereas an additional 10% of biofilm reduction was achieved by the Fenton solutions, which can be considered a separate effect of •OH in PTW. Although the concentration of •OH (0.1 μM) is much lower than that of the long-lived species in PTW, •OH showed a notable performance similar to that of NO2−, as discussed above. This result indicates that •OH is the most important and influential species among the RONS in PTW for the inactivation of biological substances. As given in Figure 3d, the fraction of reduced biofilm increased linearly with increases in the •OH concentration.

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CONCLUSIONS Because devastating diseases caused by lethal pathogens represent major health concerns, disinfecting treatments are of great importance in food and agricultural industries, as well as in the biomedical field. PTW, in particular, is considered an attractive material for disinfection; thus, the important bactericidal agents in PTW must be identified, and their relative efficacy for disinfection must be determined. We clearly demonstrated the bactericidal activity of reactive chemical species in PTW and determined the relative significance of reactive species in biofilm inactivation. Over 63% of an E. coli (DH5a) biofilm was reduced by plasma treatment at 200 W of input power. The major species of PTW that led to the biofilm reduction were •OH, H2O2, HNO2, and O3. H2O2 was the most influential species and reduced the biofilm by 25%, while O3 and HNO2 reduced the biofilm by 14 and 9%, respectively. The results showed that the mixed solution containing H2O2, HNO2, and O3 had the same removal ability against the E. coli biofilm as the PTW. •OH and •O2−, which are representative short-lived species, resulted in 10 and 3% biofilm reduction, respectively. According to our experiments (not shown here), the chemical species discussed in this paper have the same bactericidal activity against Listeria monocytogenes and Salmonella typhimurium as they do against E. coli, and those results will be separately reported later. These results should provide informative and valuable references for related future studies and industries.

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EXPERIMENTAL SECTION

Specification of the Plasma Treatment System. Figure 5a shows the plasma treatment system, which consisted of a DBD source and a gas-tight chamber. The plasma source consisted of two electrodes, covering each side of a 1 mm-thick and 10 × 10 cm2 fused silica plate, as detailed in Figure 5b. One electrode was connected to the power supply that provided a bipolar square-wave at a frequency of 15 kHz. The ground electrode in direct contact with the discharge was E

DOI: 10.1021/acsami.7b14276 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

cuvette (Sigma-Aldrich Z276650), a halogen−deuterium lamp (Ocean Optics ISS-UV/VIS), and a spectrometer (Ocean Optics Maya2000 Pro), and a standard curve for H2O2 was obtained. To determine the H2O2 concentration in the PTW, 1.5 mL of the PTW sample was added instead of the H2O2 standard solution to the metavanadate solution, and the absorption spectrum of the mixture was obtained. The concentration of H2O2 was calculated from the standard curve. Hydroxyl Radicals. The •OH inside the PTW was scavenged by benzene, and phenol was formed as a product. A 75 ± 15% yield of phenol produced from the reaction between benzene and •OH was previously reported.33 Therefore, the concentration of •OH was determined by [phenol]/0.75. Phenol quantification was conducted via high-performance liquid chromatography (Agilent HP 1260) using a 250 × 4.6 mm2 Superco Sil C-18 reverse-phase column and a 220 nm ultraviolet light source.33 A 50% acetonitrile (CH3CN) solution was used as a solvent for benzene (Sigma-Aldrich 270709) and phenol (Sigma-Aldrich P1037). Ozone. The concentration of O3 in the PTW was measured with a colorimeter (Oaktan C-108). Preparation of the Biofilm Sample. The Luria−Bertani (LB) broth medium was prepared by dissolving 10 g of Bacto-tryptone, 5 g of Bacto-yeast, and 5 g of NaCl in 1000 mL of distilled water, and it was adjusted to pH 7.5 with 10 N NaOH. E. coli (DH5a) was cultured in the LB medium and grown to a stationary phase on an orbital shaker (120 rpm, Sejongplus SJP-500SI) at 37 °C for 48 h.34 The activated cell cultures were centrifuged (at 2795g for 15 min) at 4 °C in a refrigerated centrifuge (Hanil Continent-512R), and the pellets obtained were washed twice with sterile saline solution (0.85%). The pellets were finally suspended in sterile saline solution to a final cell concentration of approximately 108 to 109 cfu mL−1. Stainless steel pieces (1 × 1 cm2) washed with 70% ethanol and sterile saline were dried at room temperature. Prepared pieces were immersed in test-culture suspension (25 mL) and incubated at 37 °C for 24 h. Then, the samples were air-dried in a clean bench for 30 min. Preparation of the Chemical Solution for Long-Lived Species. NO2− and NO3− solutions were prepared using NaNO2 (Sigma-Aldrich 563218) and NaNO3 (Sigma-Aldrich 229938), respectively. The H2O2 solution was prepared by diluting 30% of the H2O2 solution (Junsei 23150-0350). The O3 solution was produced using an ozone generator (Ozonetech OT-012), whose outlet was soaked in distilled water. The pH of all chemical solutions was adjusted to 3.5, which was the pH of the PTW, by the addition of HCl (Sigma-Aldrich 295426). Quantification of the Remaining Biofilm Assay Using Crystal Violet Staining. To quantify the remaining static biofilm after treatment, we followed the procedure described in the previous work.34 The treated samples were washed three times with distilled water, and then, the biofilm was stained with a crystal violet (SigmaAldrich C0775) solution (0.1%, 300 μL). After 20 min at room temperature, ethanol (95%, 300 μL) was added to the sample to dissolve the stained biofilm. The remaining biofilm was quantified from the optical density of the stained biofilm measured at 570 nm using a halogen−deuterium lamp (Ocean Optics ISS-UV/VIS) and a spectrometer (Ocean Optics Maya2000 Pro). All optical densities were normalized to those of the untreated biofilm. Quantification of the Remaining Biofilm Assay as log cfu cm−2. After PTW and plasma treatment, stainless steel pieces were immersed in 10 mL of sterile saline solution. Then, the samples were sonicated (Branson CPX1800H-E) in saline solution for 5 min and vortexed for 1 min to detach the bacterial cells. Then, appropriate dilutions were prepared in sterile saline and plated onto the LB medium. The plates were incubated at 37 °C for 48 h, and the microbial counts were expressed as log cfu cm−2.

Figure 5. Experimental setup for the PTW. (a) Photographs of the airtight plasma chamber and a DBD device with a square-patterned electrode for producing PTW. (b) Schematic illustration of the plasma system and arrangements of the biofilm samples. composed of a nickel−chromium alloy to prevent oxidation of the electrode because of the presence of highly oxidative species. The second and third photographs shown in Figure 5a are real images of the 7 × 7 mm2 square-patterned ground electrode with and without plasma, respectively. Air surface discharge was generated at the open surface of the patterned ground electrode. Plasma was produced in ambient air without any specific gas supply. Experimental Arrangement for Plasma Treatment. A schematic illustration of the experimental setup is shown in Figure 5b. A 40 mm-diameter fused silica dish was placed beneath the plasma source, and 3 mL of distilled water was poured into the dish to produce the PTW. A biofilm cultured on a 1 × 1 cm2 stainless steel plate was placed inside the dish. The distance between the biofilm samples (or the water surface) and the electrode surface was kept constant at 10 mm throughout the experiment. The duration of the plasma exposure to produce the PTW was 10 min, and the duration for the plasma treatment, PTW treatment, and chemical solution treatment for the biofilm was also 10 min. For the plasma treatment of biofilm, 3 mL of distilled water was spread over the biofilm surface to avoid desiccation, and it was then treated with a 100−200 W plasma source for 10 min. According to the experimentally obtained charge− voltage Lissajous diagram, the power dissipated by the plasma was 61 W at 200 W of input power. Quantification of RONS in PTW. Nitrite and Nitrate. Concentrations of aqueous NO2− and NO3− were measured using UV−visible absorption spectroscopy. The standard solutions of NO2− and NO3− were prepared by dissolving NaNO2 (Sigma-Aldrich 563218) and NaNO3 (Sigma-Aldrich 229938) in distilled water, respectively, and using these solutions with HCl (Sigma-Aldrich 295426), standard curves were obtained at different pH levels ranging from 1 to 7. A total of 3 mL of solution was prepared in a 45 × 12.5 × 12.5 mm3 quartz cuvette (Sigma-Aldrich Z276650), and the absorption spectrum in the wavelength range from 200 to 450 nm was obtained using a halogen−deuterium lamp (Ocean Optics ISSUV/VIS) and a spectrometer (Ocean Optics Maya2000 Pro). Hydrogen Peroxide. The H2O2 concentration in the PTW was determined using the metavanadate method based on the reaction of H2O2 with ammonium metavanadate (NH4VO3). In acidified metavanadate solution,31,32 peroxovanadium cations (VO23+), which absorb 450 nm light, were produced as follows

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VO3− + 4H+ + H 2O2 → VO2 3 + + 3H 2O In this work, a combination of 1.5 mL of 20 mM NH4VO3 (SigmaAldrich 398128), 0.5 mL of 1 M H2SO4 (Junsei 83010-0030), and 1.5 mL H2O2 (Junsei 23150-0350) standard solutions was employed. The absorption spectrum of this mixed solution was measured using a

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b14276. F

DOI: 10.1021/acsami.7b14276 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Quantification of the remaining biofilm with crystal violet and cfu counts; NO2−, NO3−, and H2O2 measurements in the aqueous phase (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Joo Young Park: 0000-0002-4884-3301 Cheorun Jo: 0000-0003-2109-3798 Author Contributions #

J.Y.P. and S.P. contributed equally to this work. J.Y.P., S.P., and W.C. conceived the experiments; J.Y.P. conducted all experiments; K.K. and H.I.Y. cultivated the biofilms; C.J. cultivated and quantified the biofilms; J.Y.P., S.P., and W.C. analyzed the results; J.Y.P. and S.P. prepared the manuscript and figures; J.Y.P., S.P., and W.C. contributed to the compilation and review of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS All authors in this work were supported by the R&D Programs of “Plasma Advanced Technology for Agriculture and Food (Plasma Farming)” through the National Fusion Research Institute of Korea (NFRI) funded by government funds. This work was also partially supported by the R&BD programs of “Development of high-speed plasma sterilizer for medical devices (N0002038)” through the Ministry of Trade, Industry and Energy (MOTIE) of the Republic of Korea.

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DOI: 10.1021/acsami.7b14276 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsami.7b14276 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX