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Authors Accepted Manuscript

Research on plasma medicine-relevant plasma-


liquid interaction: What happened in the past five
years?

Helena Jablonowski, Thomas von Woedtke

www.elsevier.com/locate/cpme

PII: S2212-8166(15)00054-2
DOI: http://dx.doi.org/10.1016/j.cpme.2015.11.003
Reference: CPME40
To appear in: Clinical Plasma Medicine
Cite this article as: Helena Jablonowski and Thomas von Woedtke, Research on
plasma medicine-relevant plasma-liquid interaction: What happened in the past
five years?, Clinical Plasma Medicine,
http://dx.doi.org/10.1016/j.cpme.2015.11.003
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Research on plasma medicine-relevant plasma-liquid interaction:

What happened in the past five years?

Helena Jablonowski1 and Thomas von Woedtke1,2

1
Leibniz Institute for Plasma Science and Technology (INP Greifswald e.V.), Greifswald, Germany

2
Greifswald University Medicine, Greifswald, Germany

Abstract

During the last five years mechanisms of plasma-induced change of liquid chemistry have drawn

huge attention in basic research in plasma medicine because liquid phase processes have been

identified to be the main key to understand detailed mechanisms of atmospheric pressure plasma

effects on living systems. Moreover, plasma-activated liquids are considered to be useful for several

applications also in the medical field. This review gives a compilation of the work done in the past five

years mainly from an analytical point of view to reveal both the actual knowledge and the still missing

parts for a more complete understanding of the plasma-liquid-tissue interaction.

In general, independent both on the different atmospheric pressure plasma sources (dielectric barrier

discharges, plasma jets; different working gases) and the different liquid systems (water, non-buffered

and buffered saline solutions, cell culture media) investigated, hydrogen peroxide (H2O2) as well as

nitrite (NO2-) and nitrate (NO3-) were detected as stable reactive oxygen and nitrogen species (ROS,

RNS/RONS). In non-buffered systems, pH decrease was found. It is hypothesized that the basic

pathways of generation of reactive species in liquids after treatment with atmospheric pressure

plasmas can be generalized. These stable and easy to detect ROS and RNS/RONS are considered to be

representative for more or less complex reactions chains with the participation of other more reactive

and short-lived reactive oxygen and nitrogen precursor species which are induced in liquids by

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plasma treatment and may play dominant roles in biological plasma effects. As most important

precursors of hydrogen peroxide as well as nitrite and nitrate, hydroxyl radicals (OH), superoxide

anion radicals (O2-), singlet oxygen (1O2), and nitric oxide (NO) were identified and partially detected

in different plasma treated liquids. By combination of experimental data and theoretical considerations

including modelling approaches first steps to identify reaction pathways were realized yet. Above all,

peroxynitrite (ONOO-) was identified to play a crucial role for biological effects of plasma-treated

liquids. In future, innovative and sophisticated analytics possibly at least partially beyond the classical

chemical and pharmaceutical techniques have to found to improve the fundamental understanding of

liquid phase-transmitted mechanisms of plasma effects on living cells and tissue and its consequences

for complex physiological as well as pathophysiological processes in the organism. This is essential

both for the consolidation of plasma medicine on a sound scientific basis and to open up innovative

fields of plasma application in medicine.

Keywords: plasma-liquid interactions, reactive oxygen species reactive nitrogen species liquid

analytics

1. Introduction

A fundamental understanding of mechanisms of plasma effects on living cells and tissue and its

consequences for complex physiological as well as pathophysiological processes in the organism is

essential for a clinical plasma medicine on a sound scientific basis. Under in vivo conditions, living

cells and tissue are surrounded by a liquid environment. Consequently, it was an earlier idea in

concepts of basic research in plasma medicine to investigate the role of liquid phases for the

transmission of biological effects of plasma to cells and tissue [1]. Two fundamental insights about

mechanisms of plasma-cell and plasma-tissue interactions are the result of recent research [2]:

1. Biological plasma effects are significantly caused by plasma induced changes of the liquid

environment of cells.

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2. Redox-active species (reactive oxygen and nitrogen species - ROS, RNS/RONS) generated in

or transferred into liquid phases play a dominating role in biological plasma effects.

The impact of these reactive oxygen and nitrogen species to mammalian systems is well known in

biology and medicine [3-6].

In connection with this basic research on biological plasma effects and the role of the vital liquid

environment of cells and tissue it could be demonstrated that plasma treatment of liquids may result in

an at least transient biological activation of these liquids [7]. This opens up another innovative field of

medical plasma application that was called plasma pharmacy, i.e. the plasma-based generation,

improvement and/or stabilization of liquids containing biologically active components [8].

However, one of the main challenges of actual research is to explain in detail what reactive species are

active in plasma treated liquids. A lot of work has been done in recent years to come closer to an

answer to this fundamental question.

The aim of this report is to summarize the state of the art in research of plasma medicine-related

plasma-liquid interaction and to report how these results can be intertwined despite the fact that most

groups work with different atmospheric pressure plasma sources using different working gases (argon,

helium air, gas mixtures containing oxygen and nitrogen, etc.) and a variety of liquid media. Based on

last five years publications in this field the following questions should be answered:

1. What species were detected in which kind of liquid media?

2. Are formation pathways already verified for these species or what are the existing assumptions

of its origin?

Concluding, open tasks should be pointed out, with expecting being neither exclusive nor complete.

With this article, the reader should get an insight into the complexity and the challenges of analytics

of plasma-treated liquids.

The article is organized in three parts to answer the highlighted questions above and to summarize

the knowledge gained from the evaluated studies.

2. Detected reactive species in different liquids

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As this article should give an overview about what happened in the past years in the field of plasma

medical research on plasma-liquid interaction, the first question is: which species were generated by

plasma treatment and are they relevant for plasma biomedicine?

The chapter is structured according to the complexity of the investigated liquids. It starts with water

and moves on to non-buffered and afterwards buffered saline solution. Last but not least cell culture

media are discussed.

2.1. Water

To state that water is not a complex system is definitely caused by the perspective. In comparison

with the other liquid media considered here it is at least the less complex system as in general the only

ingredients are water molecules (H2O) and possible dissolved air species such as molecular oxygen

(O2) or nitrogen (N2). If the water is just tab water, it will contain minerals additionally. Deionized (DI

water), distilled water (dH2O) or ultrapure water (also called milliQ water) are different in the state of

purification.

The plasma treatment can take place above or in close vicinity to the liquid surface, or sometimes

also inside the liquid. Treatment inside the liquid does not mean discharges in liquids as known for

instance form waste water treatments [9-12]. In this article treatment inside the liquid means that e.g.

plasma jets were dipped into the liquid forming a kind of gas surrounding around the plasma plume

due to the gas flux, so that the plasma is contacting the liquid but not ignited inside the liquid

phase [13, 14].

In most cases the different plasma treatments lead to an acidification of the water [13, 15-19].

Beside the pH decrease also reactive oxygen species were measured in many studies by different

methods. The reactive oxygen species which is mainly investigated due to its stability and hence its

comparatively easy detectability is hydrogen peroxide (H2O2). H2O2 is often determined by color

forming reactions and spectrophotometric measurements. One common chemical analytic method is

the reaction of hydrogen peroxide with titanium(IV) oxysulfate (TiOSO4). This assay is also known as

titanylsulfate assay. TiOSO4 reacts with H2O2 to a yellow-orange complex and the absorption can be

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observed at 407 nm with a spectrophotometer either in cuvettes or in well plates. This colorimetric

assay was used e.g. in the studies of Burlica et al. [20] and von Woedtke et al. [21]. Another common

method is the iodometric titration [13, 14, 17, 19, 22], which is based on the reaction of hydrogen

peroxide with potassium iodide to iodine in the presence of acidic pH and ammonium molybdate as

catalyst. With sodium thiosulfate as titrant, color change from blue to faint yellow or more or less

colorless results from this reaction. Also commercial test strips for the detection of H2O2 were used,

above all to get a first indication for the presence of reactive oxygen species (ROS). It was shown by

Winter et al. [23] that with some effort a small-step calibration and enhanced read-out and careful

evaluation test strips can be also used when the sample volume is too less for the other methods [24].

Hydrogen peroxide is a stable end-product of more or less complex reactions with the participation

of other reactive oxygen species. Therefore, to get a more detailed insight into the ongoing oxygen

chemistry in plasma treated liquids, other species need to be identified. A few groups started to

investigate the precursors of hydrogen peroxide: hydroxyl (OH) and superoxide anion (O2-) radicals.

Due to their radical character they are short-lived and hence difficult to detect. One approach is the

colorimetric detection. For a qualitative measurement the used detection reagents need to be selective,

which is not fulfilled for several dyes. For instance methylene blue (MB) is a potential redox indicator

used in biomedicine [25]. MB is known to be decolorized in presence of hydroxyl radicals, but

unfortunately this reaction is not specific to OH. If ozone (O3) is present it will yield decoloration of

MB more efficient then hydroxyl radicals [26]. Kanazawa et al. used chemical dosimetry based on

terephthalic acid (TA) [27]. They reported that TA is a well-known hydroxyl radical scavenger, which

should not react neither with other oxygen free radicals such as superoxide anion or hydroperoxyl

(HO2) radicals nor with hydrogen peroxide. The product of OH with TA, hydroxyterephthalic acid

(HTA) is emitting light (=425 nm) if it is irradiated by ultraviolet light (=310 nm). Under the same

condition TA will not emit radiation. The HTA emitted light can be measured and also quantified with

sufficient calibration. One drawback of this method is that alkaline pH is necessary for dissolving TA.

Lukes et al. [28] used phenol as a chemical probe to detect hydroxyl radicals. Phenol reacts with

hydroxyl radicals but also with ozone (O3) and nitric monoxide (NO, also called nitric oxide) and

nitrogen dioxide radicals (NO2) giving very specific oxidized or nitrated phenol products which can

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be detected by high performance liquid chromatography (HPLC). In this study these species were used

in combination with a post-plasma treatment kinetic study as an indirect verification for the presence

of peroxynitrite (ONOO-), which decays partially into OH and NO2.

For direct measurement of free radicals electron paramagnetic resonance (EPR) spectroscopy can be

used. This technique is determining the paramagnetic property of a species leading to a characteristic

spectrum, which is similar to a finger print of this radical. Theoretically the oxygen free radicals can

be detected directly without any further chemicals. Unfortunately, the reality is a bit more

complicated. The life time of such a radical is really short in the range of micro to nanoseconds,

depending on the liquid surrounding [4]. Additionally, the concentrations are quite low, so that they

are often below the lower detection limit of EPR (approximately 10-6 M [29]). Short spin relaxation

time of a radical can also hinder radical detection by EPR although its concentration is sufficient [29].

The radicals of interest in case of plasma-liquid interactions for plasma medicine, OH and O2-,

unfortunately are among these hard to detect ones. Therefore, the measurement of these radicals has to

be facilitated by the use of so-called spin traps. These chemicals react with the radical and form a

specific and still paramagnetic adduct [30]. A common spin trap used for oxygen free radicals is

DMPO (5,5-dimethyl-1-pyrroline-N-oxide). A disadvantage of DMPO in a system where both

superoxide anion as well as hydroxyl radicals are present is, first of all, the different reaction rate for

these two species. The reaction rate of DMPO with OH is kOH > 109 M-1s-1 [4, 31], whereas for

superoxide it is kO2- < 102 M-1s-1, only [4]. Furthermore, the spin trap adduct of DMPO with

superoxide anion is decaying fast into the same adduct hydroxyl radicals are forming with DMPO.

Consequently, the use of DMPO gives only information about the sum of hydroxyl and superoxide

radicals in a liquid. Sun et al. [14] tried to overcome the problem by the addition of scavengers such as

mannitol for hydroxyl radicals and superoxide dismutase for superoxide radicals. An alternative to the

use of scavengers is the utilization of other spin traps such as BMPO (5-tert-butoxycarbonyl-5-methyl-

1-pyrroline-N-oxide) [32], DEPMPO (5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide) [33], or

CYPMPO (5-(2,2-dimethyl-1,3-propoxy-5-methyl-1-pyrroline N-oxide) [34, 35]. All these spin traps

form adducts with superoxide anion radicals which do not decay into a corresponding OH adduct.

They differ in the half-life of the formed adduct, in the complexity of the spectra, in their liquid

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solubility as well as in their ability to pass membranes. For every single analytical case the proper spin

trap needs to be determined with caution, since all of them have advantages and drawbacks.

By the use of one of the mentioned spin traps, hydroxyl and superoxide anion radicals were

successfully detected in plasma-treated water [14, 17, 19, 22, 24, 34, 35]. Okazaki et al. [36] used a

different spin trap, 3,3,5,5-tetramethylpyrroline-N-oxide (M4PO), for hydroxyl radical detection. It

can theoretically also detect both hydroxyl as well as superoxide anion radicals, but the half-life of the

O2--adduct is already lower than that for the DMPO-O2- [37].

Sun et al. [14, 17, 22] also detected singlet oxygen (1O2) by the use of spin trap enhanced EPR

spectroscopy. Singlet oxygen itself is not paramagnetic but by its reaction with a spin trap, TEMP

(2,2,6,6-tetramethylpiperidine), an EPR-active molecule is produced which gives a 1O2-specific

spectrum.

Pua et al. [33] also measured hydrogen radicals (H) in plasma treated water by the use of

DEPMPO as spin trap.

Albeit water itself consists of hydrogen and oxygen, only, also nitrogen is relevant in plasma

treatment of liquids. On the one hand it will be present in the liquid due to solvation from air nitrogen

regarding Henrys law. On the other hand, also with plasma sources using argon (Ar) or helium (He)

as working gases, because of its operation under atmospheric conditions, admixture of atmospheric air

into the plasma phase must be taken into consideration which may result in generation and transfer of

both reactive oxygen and nitrogen species into the plasma-treated liquid.

Zhang et al. [22] and Pua et al. [33] detected nitric oxide (NO) by EPR with spin trapping. The

spin trap they used was diethyldithiocarbamate (DETC) in combination with Fe2+ giving DETC-Fe2+.

This compound reacts with NO and yield an EPR spectrum, but is has a quite low solubility in

water [38].

A common way to get a basic idea of the presence of reactive nitrogen species in plasma-treated

liquids is similar to the way that is common for reactive oxygen species: the detection of the more

stable end products of complex reactive nitrogen species reactions. In the case of nitric monoxide

these easier to access products are nitrite (NO2-) and nitrate (NO3-). Both can be determined via

colorimetric reactions. For NO2- the well-known Griess assay can be used which is based on the

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reaction with sulfanilic acid and N-(1-naphthyl)-ethylene diamine hydrochloride via azo sulfanilic acid

to a magenta colored azo dye whose absorption at 525 nm can be measured [39]. For NO3- detection

either 2,6-dimethyl phenol [21, 40] can be applied or it can be converted enzymatically or by the use

of vanadium (III) chloride to NO2- and also measured via Griess assay [41-44]. More advanced

methods for the detection of nitrite and nitrate are ion chromatography (IC) [45] or HPLC [13, 22, 28].

Pavlovich et al. [46, 47] successfully detected nitrite and nitrate by UV-visible absorbance

spectroscopy.

In 2013 the group of Bruggeman published the first liquid-chemistry model-approach in the field of

plasma medicine [45]. With their publication they highlighted the importance of the synergetic effect

of experimental and theoretical work for getting a better understanding of the complex chemistry in

plasma treated liquids. In the following years the amount of literature regarding plasma-liquid models

increased (see also chapter 4.): the groups of Kushner [48-50], Hamaguchi [51], Kong [52] and

Graves/Shannon [53] are now also working on that topic. Chen et al. [52] did not include reactive

nitrogen species in their model up to now, but they extent their model to plasma-tissue interaction.

Beside the reactive oxygen and nitrogen species (ROS, RNS/RONS), which were experimentally

determined in plasma treated liquids H2O2, OH, O2-, 1O2, NO2-, NO3-, NO the models [45, 48-51,

53] include also ROS/RONS which were previously discussed [54] to be assumable present in plasma

treated liquids such as peroxynitrite (ONOO-) or peroxynitrous acid (ONOOH) and nitrogen dioxide

(NO2) radicals. There are groups working on an experimental verification for the presence of

peroxynitrite in plasma treated liquids [28, 55]. Up to now only the model of the Bruggeman

group [45] also includes chloride ions (Cl-, Cl2-, Cl3-) so that the modeled liquid in this case could be

also saline solution.

2.2. Saline solution

Species determined after plasma treatment of simple saline solutions are the same as detected in

plasma treated water, mainly hydrogen peroxide, nitrite and nitrate [21, 40-42, 45, 54, 56, 57]. Usually

physiological sodium chloride solution (0.85-0.9% NaCl) is used. Up to now only Tresp et al. [41]

also measured the related oxygen free radicals, hydroxyl and superoxide anion, in sodium chloride
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solution. The group of Rumbach et al. performed some calculation based on the pH measurements and

existing knowledge, especially from the electrolysis field, to estimate the formation of sodium

hydroxide (NaOH), nitric acid (HNO3), nitrogen dioxide radical (NO2) as well as hydrogen

peroxide [58].

However, in the different studies different kinds of plasma sources were used. The group of von

Woedtke et al. [21, 40, 54, 57] used dielectric barrier discharges (DBD) whereas Rumbach et al. [58],

van Gils et al. [45] and Tresp et al. [41] investigated different plasma jets. In general it can be stated

that the same species, although in different concentrations and also trends, will be formed. Several

publications indicated that these plasma activated physiological saline solutions are effective in the

inactivation of microorganisms [16, 17, 20-22, 40, 54, 59-64].

The group of Graves et al. [46, 47, 65] investigated ozone (O3) by the use of indigo dye. Although

the applicability of this assay for ozone in the presence of hydroxyl radicals and other ROS is

discussed in literature, they deduced from their high gaseous O3 concentration that it is possible to

detect ozone dissolved from the gas phase in the liquid with this assay.

2.3. Buffered saline solution

The next step in complexity will be the addition of a buffer system to the saline solution, so that the

pH kept constant during the plasma treatment. Most common solution is phosphate buffered saline

(PBS) which includes a buffer system based on disodium phosphate (Na2HPO42-) and potassium

dihydrogen phosphate (KH2PO4-) yielding a pH of 7.4. These additional ingredients can also have an

impact on the generated species or on their concentrations as it was discussed by Tresp et al. [41].

Identical to non-buffered saline solution also in the buffered ones hydrogen peroxide , nitrite and

nitrate were found after plasma treatment [21, 40, 41, 43, 44, 61, 66-68]. Some groups also

investigated hydroxyl radicals, superoxide anion radicals, singlet oxygen and peroxynitrite. For

colorimetric detection of OH, Arjunan et al. [66] used 3-p-(hydroxyphenyl fluorescein (HPF) in

combination with mannitol or DMSO as hydroxyl radical scavengers, whereas Takamatsu et al. [67]

applied terephthalic acid for hydroxyl radical detection. Electron paramagnetic resonance

measurements of OH and O2- in plasma treated PBS solution were published up to now by
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Tresp/Jablonowski, only [24, 32, 41, 42, 69]. Dobrynin et al. [70] used a different buffer, namely Tris-

buffered saline (TBS). They transferred the well-known H2O2-fluorescence assay, Amplex UltraRed,

for detection of superoxide anion radicals. The Amplex UltraRed assay is an improved version of the

Amplex Red assay. This assay is based on the reaction of the non-fluorescent 10-acetyl-3,7-

dihydroxyphenoxazine with hydrogen peroxide in presence of horseradish peroxidase (HRP) to the

fluorescent resorufine (3H-phenoxazinone,7-hydroxy). The transfer of this assay for superoxide

anion radicals is based on the combination with SOD. They expected all present O2- molecules will

react with SOD to increase the final hydrogen peroxide concentration [70]. In the same study they also

reported the presence of nitric monoxide measured by the fluorescent dye 4,5-diaminofluorescein

(DAF-2). As both NO and O2- are the precursors of peroxynitrite, they also detected ONOO- by the

use of 2,7-Dichlorodihydrofluorescein (DCDHF). This reagent is similar to 2,7-

dichlorodihydrofluorescein diacetate (DCFH-DA), which the group of Machala was investigating for

peroxynitrite detection [55]. Singlet oxygen sensor green is also a fluorescent dye and can be used for
1
O2 measurements, as the group of Fridman showed qualitatively [67, 70]. Arjunan et al. [66] applied

for superoxide anion radical detection Tempo-9AC in combination with ascorbic acid as O2--

scavenger and tried to detect singlet oxygen by the use of trans-1(2-methoxyvinyl)pyrene (tMVP)

together with sodium azide as 1O2-scavenger. Unfortunately, they did not see any increase in

fluorescence although they expect it being present.

Wende et al. [43] investigated in their study also hydrogen peroxide, nitrite and nitrate, but in

addition they used tyrosine nitration for the detection of superoxide anion radicals or peroxynitrite. L-

tyrosine will be nitrated to 3-nitrotyrosine when ONOO- is present in the solution. Since O2- will

almost immediately react with nitric oxide to peroxynitrite, they combined the tyrosine detection with

the addition of a nitric donor to detect superoxide anion radicals.

2.4. Cell culture media

Only a few groups investigated the generation of reactive species in plasma-treated cell culture

media. Arjunan et al. [66] measured H2O2, OH, ONOO-, O2-, 1O2 and NO2- in serum free Dulbeccos

modified Eagles medium (DMEM). The same medium was also investigated by Adachi et al. [39] and
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Hoentsch et al. [71]. In the study of Hoentsch et al. an oxygen microsensor was used to determine O2

content after plasma treatment in DMEM.

Some other studies investigated serum free Rosewell Park Memorial Institute (RPMI) 1640 cell

culture medium [24, 32, 41]. In these studies hydroxyl radicals and superoxide anion radicals as well

as hydrogen peroxide were determined. Tresp et al. [41] additionally measured the nitrite

concentration after plasma treatment in RPMI 1640.

The cell culture medium RPMI 1640 with addition of serum and antibiotics was analyzed with regard

to OH and O2- as well as H2O2 in the studies of Winter et al. [23, 69]. Bekeschus et al. [72] as well as

Lazovic et al. [60] investigated human peripheral blood mononuclear cells (PBMC) in plasma treated

RPMI 1640 and DMEM media, respectively. By the application of Amplex UltraRed and Measure-

IT Nitrite assay, Bekeschus et al. [72] observed hydrogen peroxide and nitrite concentrations in the

plasma treated RPMI 1640 medium. The Measure-IT Nitrite assay is considered to be more sensitive

than colorimetric assays based on Griess reagent. It is based on the reaction of 2,3-Diaminonaphtalene

(DAN) with nitrite under acidic conditions to 1-(H)-naphthotriazole, which is fluorescent. The formed

compound is terminated by sodium hydroxide to enhance the fluorescent signal [73].

Thi et al. [74] performed the Griess assay for nitrite in tissue lysates from artificial wounds of an

animal model, furthermore they also measured the general ROS concentration in this tissue lysates by

DCHF-DA assay.

From the best of the authors knowledge, the only study investigating an bacterial growth medium

was performed by Liu et al. [13]. They analyzed a mixture of sterile water and the bacterial growth

medium Luria-Bertani culture medium. In the mixture they measured a decrease of pH, the other

species, hydrogen peroxide, nitrite and nitrate, determined in this study were formed after plasma

treatment of pure sterile water.

Summarizing this chapter that gives an overview about detected reactive species in different liquids

after plasma treatment, it can be stated that the detection of hydrogen peroxide, nitrite and nitrate as

well as pH measurements are now established as basic parameters to characterize results of plasma-

liquid interaction in general. These species are considered to be representative for more or less

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complex reactions chains with the participation of other reactive oxygen and nitrogen species which

are induced in liquids by plasma treatment.

Hydrogen peroxide is taken as a general parameter for ROS generation whereas nitrite and nitrate

are representative parameters for RNS/RONS generation. Characterization of pH is essential to

characterize the reactive conditions in the liquid phase. Because of its stability they are easy to detect

by common techniques like color forming reactions/photometry or ion chromatography. Qualitative as

well as quantitative detection of pH, hydrogen peroxide, nitrite and nitrate allows basic conclusions on

main reactive processes in plasma-liquid interaction and is therefore representative for basic plasma-

source characterization with regard to its biological activity.

As most important precursors of hydrogen peroxide as well as nitrite and nitrate, hydroxyl radicals

(OH), and superoxide anion radicals (O2-), singlet oxygen (1O2), and nitric oxide (NO) were detected

in different plasma treated liquids. Because of its high reactivity and thus limited stability and life

span, detection of these species is much more challenging. It has to be stated that these ROS and

RNS/RONS were identified in general both in all liquids including water, buffered and non-buffered

saline solutions and cell culture media but also after treatments with different plasma sources using

different working gases.

Consequently, it is hypothesized that the pathways of generation of reactive species in liquids after

treatment with atmospheric pressure plasmas can be generalized at least partially.

Table 1 gives an overview of the publications in the field of plasma medicine relevant plasma-liquid

interaction of the past five years with regard to the reactive species detected in different liquids (water,

saline solution, buffered saline solution, and cell culture medium) after plasma treatment using helium,

argon, or air as working gas, respectively.

Table 1: Overview of the measured species in water, saline solution, buffered saline solution and cell culture medium for

helium, argon or air as working gas.

helium Argon air

species water saline buffered cell water saline buffered cell water saline buffered cell

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solution saline culture solution saline culture solution saline culture

solution medium solution medium solution medium

[13, 14,

[23, 39, 16, 20, 21, [21, 40, [21, 44,

[17, 19, [22, 24, [41, 43, 41, 69, 71, 40, 75, 46, 57, 77, 46, 61, 66,

H2O2 59] [68] 62] [41, 58] 67] 72] 76] 78] 67, 70] [13, 66]

[17, 27,

33, 35, [22, 24, [24, 41,



OH 79] 36] [41, 67] [24, 41] 69] [14, 76] [66, 67] [66]

ONOOH [54, 55,

/ONOO- [43] 76] [66, 70] [66]

[17, 34, [24, 41, [24, 41,

O2- 35, 80] [24] [41] 43, 69] 69] [14] [66, 70] [66]

[61, 66,
1
O2 [17] [22] [67] [14] 70] [66]

[13, 21, [21, 40, [21, 44,

[41, 43, [39, 41, 40, 47, 65, 46, 57, 77, 46, 66,
-
NO2 [68] [22] [41] 67] 72, 74] 76] 78] 67] [66]

[13, 20, [21, 40,

[41, 43, 21, 40, 47, 46, 57, 77, [21, 44,
-
NO3 [22] [41] 67] [41] 65, 76] 78] 46, 67]

O3 [75, 76] [46] [46]

NO2 [58] [67] [76]



NO [33] [22] [67, 70]

HNO2 [75]

HNO3 [58]

O2 [81]

H [33]

ROS [82] [39, 74] [83] [83, 84]

3. Identified and assumed formation pathways from experiments

For a better control of plasma treatments and as a consequence for the design of plasma sources for

medical applications adapted for specific application needs it is quite helpful to gain knowledge about

the chemical pathways triggered in the liquids due to plasma treatment and its immediately resulting

biological effects.

13
Since the plasma-liquid interaction research in the field of plasma medicine primarily originated

from groups investigating the antimicrobial effectivity of atmospheric pressure plasma, the first

assumptions on biological relevant chemical mechanisms in plasma treated liquids came from these

groups [40, 59, 85].

Chen et al. [85] pointed out the importance of pH decrease for the inactivation of Escherichia coli

(E.coli). This was further verified by the studies of Ikawa et al. [59] and Oehmigen et al. [40]. Both

measured the amount of NOx- species in plasma treated saline solutions and compared the observed pH

change and accordingly the corresponding H+ concentration with the detected nitrites and nitrates,

respectively. In the study of Ikawa et al. the total NOx- amount was taken into account whereas

Oehmigen et al. took into account the nitrate concentration, only. Both groups obtained good

agreement and stated a key role of generation of nitrate and nitrate and its corresponding acids,

respectively, for acidification of non-buffered liquids by plasma treatment. Ikawa et al. [59] suggested

the origin of the nitrogen species from the plasma/gas phase of the helium plasma jet used, being

generated there by the interaction of the plasma plume with the ambient air. By variation of the pH in

the liquid between 6.5 and 3.7 using a buffer, they found a critical pH around 4.7 being necessary for

inactivation of bacteria. The addition of the enzyme superoxide dismutase (SOD) in their system

showed an inhibition of bacteria inactivation and consequently an involvement of superoxide anion

radicals was suggested. The obtained critical pH of 4.7 is close to the acid dissociation constant (pKa)

of the equilibrium reaction between the superoxide anion radical O2- and the hydroperoxyl radical

(HO2) which is 4.8. They concluded that at pH lower than that pKa, instead of O2- mainly its

protonated form HO2 is present. HO2 can cross or enter membranes in contrast to O2-. To identify the

origin of O2-, Ikawa et al. [34, 35, 80] studied different plasma setups and found that the presence of

molecular oxygen in the gas phase enhanced the superoxide anion output, whereas the absence of O2

and a direct interaction of the plasma plume with the liquid surface produced more hydroxyl radicals.

However, they found that an addition of air to the feed gas resulted in a higher efficacy in bacteria

inactivation compared to O2 admixture [59]. Hence, they assumed that also RNS are necessary,

probably via peroxynitrite.

14
The necessity of this combination of ROS and RNS/RONS for antimicrobial effectivity was also

stated by Oehmigen et al. [40, 54]. They could also show that in buffered systems only very reduced

bactericidal effects occurred. In their system using a surface dielectric barrier discharge (surface-DBD)

with atmospheric air as working gas a decrease of pH lower than 3 was reached within 30 minutes and

the determined nitrate concentration for these treatment parameters agreed well with the obtained pH,

i.e. acidification was considered to be a result of nitric acid generation. This was furthermore verified

by a comparative nitric oxide gas treatment of sodium chloride solution. Furthermore, they studied the

antimicrobial effectivity of either acidic environment or addition of nitrite and nitrate salts as well as

hydrogen peroxide to sodium chloride solution and found not as good inactivation as due to plasma

treated sodium chloride solution. Consequently, they assumed a synergistic effect of reduced pH and

other active, but short-lived and thus hardly to detect reactive plasma components. In a following

study [54] they focused more on possible formation mechanisms of peroxynitrite which is well-known

for its high antimicrobial effectivity and was assumed to be present in plasma treated liquids as it was

concluded by means of theoretical considerations. Since they could show that also with a delay of

30 minutes between plasma treatment of the liquid and addition of the bacteria an albeit lower but still

detectable antibacterial effect occurred, they concluded that a more or less stable chemical

modification of the liquids should be the main reason for its antimicrobial impact. Consequently, the

active species seem to be mid-stable and perhaps induce secondary and tertiary post-treatment

reactions. Due to the presence of NOx- species and H2O2 they focused on the possible reactions

triggered by these species. Several of the possible reactions will have somehow and somewhere

peroxynitrite (ONOO-) or peroxynitrous acid (ONOOH) as transient products (see Figure 1). As a first

but still doubtful verification of the presence of these substances they detected ONOOH via absorption

spectroscopy in 30 minutes treated water and sodium chloride solution.

15
Figure 1 Assumption of chemical reactions in plasma treated liquids [54]

Lukes et al. [28] also investigated the peroxynitrite chemistry in plasma treated liquids. On the one

hand they obtained the kinetics of hydrogen peroxide and nitrite and nitrate, assuming that these

substances are the precursors of peroxynitrite in plasma treated water and on the other hand they used

phenol as chemical probe for ONOO-/ONOOH decay products, namely hydroxyl and nitrogen dioxide

radicals. This experimentally obtained kinetics of phenol degradation and generation of its reaction

products have been compared with a model system of H2O2 and HNO2 at defined pH and gave a good

agreement. In their E.coli inactivation tests they found a similar trend but an offset between model-

chemistry and plasma activated water (PAW) the PAW is more efficient than just hydrogen peroxide

and nitrous acid-containing liquids.

However, all three groups ascertain both ROS and RNS being essential for antimicrobial effects. As

shown in Table 1 in most of the studies the stable RNS and ROS hydrogen peroxide, nitrite and nitrate

were detected. Consequently, it can be assumed that in probably all these solutions also ONOO-

/ONOOH were present, at least transient.

The group of Graves [46, 47, 65] investigated a surface-DBD in air, too, with different power

modes, low and high power, and determined the pH, as well as H2O2, O3, NO2- and NO3-

concentrations. Additionally, bacteria inactivation was investigated. They found that with what they

called the low power mode, liquid chemistry was dominated by ozone whereas for the high power

mode hydrogen peroxide and nitric oxide species were dominating [46].

16
Jablonowski et al [24] investigated the impact of argon plasma jet-generated vacuum ultraviolet

(VUV) radiation on its antimicrobial activity as well as the VUV absorption by liquid layers. With a

special designed experimental setup the absorption of VUV radiation by liquids of different

complexity (water, buffered saline solutions, cell culture media) as well as the reactive species

formation were determined and compared with the effects achieved with the plasma jet treatment of

the liquids. Using EPR spectroscopy, hydroxyl and superoxide anion radicals were measured. Also

hydrogen peroxide generation was observed both after argon plasma jet and VUV only treatment. This

study reveals the impact of the VUV radiation for the generation of these ROS, at least for this specific

argon-fed plasma source. Due to the required energy for the cleavage of the O-H bonds of the water

molecule, 5.01 eV, it could be clearly stated in this study that the plasma jet provides this energy by

the argon continuum, which was emitted around 126 nm. The dissociation of water should lead mainly

to the formation of OH. As the reaction of two hydroxyl radicals to hydrogen peroxide is well known,

they investigated also the behavior of this more stable ROS, resulting in well-fitting trends of both

species. It could be demonstrated that vacuum ultraviolet radiation of the argon plasma jet contributes

partially but not completely to the formation of H2O2, since e.g. 180 s complete plasma jet treatment

produces 350 mmol hydrogen peroxide, whereas the plasma jets VUV radiation only generates

1.5 mmol H2O2 in the same liquid [24]. By comparison of different complex solutions regarding the

amounts of OH and O2-, respectively, formed due to plasma jets VUV radiation, it was found that

these species seemed to be more or less exclusively formed due to dissociation of water molecules: no

remarkable difference in generated oxygen free radicals was detectable between water, a buffered

saline solution and a cell culture medium.

A detailed study on the effect of humidified feed gas in an argon plasma jet was published by Winter

et al. [23, 69]. They observed the reaction pathway of hydrogen peroxide from the plasma/gas phase

via the liquid phase to mammalian cells. The varying parameter was only the feed gas humidity. Due

to the presence of water in the feed gas the hydroxyl radical as well as hydrogen peroxide

concentrations in the plasma/gas phase increased with increasing amount of water, whereas the ozone

concentration was decreasing. In the liquid phase the hydrogen peroxide concentration also increased

linearly with the feed gas humidity which led to the expected decrease in cell viability [23]. A follow-

17
up study of Winter et al. [69] was focused on the origin and the pathway of hydrogen peroxide

generation from the plasma/gas phase to the liquid phase. Hence, measurements of the precursors of

hydrogen peroxide, hydroxyl and superoxide anion/hydroperoxyl radicals, were performed both in the

plasma/gas as well as in the liquid phase. The gas phase measurements were compared with a

chemical model of the far field, i.e. the gas phase region separated from the plasma jet. Resulting from

this comparison they could clearly identify the gaseous generation and destruction pathways of ROS.

The production rates of the gaseous and the liquid hydrogen peroxide were almost identical.

Interestingly, the measurements of liquid phase precursor of H2O2, OH and O2-, did not show a

dependency on the feed gas humidity and consequently no similarity to the trend of the aqueous

hydrogen peroxide concentration. Consequently, the liquid hydrogen peroxide origin was assumed to

be in the gas phase. By bubbling a fraction of the argon feed gas through a reservoir of hydrogen

peroxide solution the gaseous hydrogen peroxide concentration was artificially produced without

plasma to mimic the H2O2 formation for different plasma treatment times. This experiment shows

good agreement of the aqueous H2O2 amount both after plasma treatment and artificial gaseous

hydrogen peroxide application. Hence, due to the results and the relatively high Henry coefficient they

conclude that in this specific case of humidified feed gas hydrogen peroxide is mainly produced in the

plasma/gas phase and dissolved in the liquid instead of being formed inside the liquid [69].

Bekeschus et al. [86] detected metastable nitrogen in the gas phase of an argon plasma jet, which is a

high energetic species. They assumed in their study with regard to the effect of a nitrogen-surrounded

argon plasma jet on human immune cells, that these metastable nitrogen may cause dissociation of

water molecules on the liquid surface resulting in hydrogen peroxide generation.

Wende et al. [43] studied the effect of different molecular admixtures (water, air, oxygen) to the feed

gas of an argon plasma jet to trigger the plasma induced chemistry in the liquids. Therefore, they used

as first test the cells as a biological sensor, obtaining their viability under the varying conditions in

combination with either Fe2+ or catalase. By Fe2+ addition, Fenton reaction should be introduced if

hydrogen peroxide is present leading to generation of hydroxyl radicals. By increasing Fe2+

concentration they observed increasing cell viability, which is in contrary to the expected behavior.

They assumed that cell culture medium ingredients (e.g. glucose) could be responsible for scavenging

18
of the Fenton reaction-formed OH. By addition of catalase, they tested if other reactive species are

responsible for plasma-caused effects on cell viability. Catalase addition resulted after the pure argon

or humidified argon plasma treatment in a complete inhibition of the plasma effect on cell viability,

whereas for the treatments using admixed air (Ar/air) or oxygen (Ar/O2) catalase had no impact on the

plasma effect. Similar to Winter et al. [23, 69] they identified for humidified feed gas hydrogen

peroxide as main species acting on cell viability. By comparison of a gas phase model of this argon

plasma jet for the four different conditions (pure and humidified argon feed gas without and with

catalase in the liquid phase) they assumed that because of the increasing amount of reactive nitrogen

species in the Ar/air condition, the Ar/O2 case resulted in higher amounts of atomic oxygen, singlet

oxygen and ozone. Therefore, they analyzed the treated liquids regarding H2O2, NO2- and NO3-. Ar and

Ar/H2O treatments produced detectable concentrations of hydrogen peroxide but for Ar/air and Ar/O2

no H2O2 could be measured as well not after addition of scavengers for possible interfering species.

For these two conditions also neither nitrate nor nitrite was detected. This gives first hints that the

gaseous nitric oxide species for this argon plasma jet are not dissolved in the treated liquid, at least not

in form of nitrate and nitrite but possibly peroxynitrite chemistry is triggered in the liquid. Due to their

measurement of peroxynitrite and superoxide anion radicals they could deduce that RNS do not have a

remarkably impact on cell viability in case of Ar/O2 but for Ar/air treatment. For the Ar/O2 case they

conclude that singlet oxygen, ozone or atomic oxygen might be relevant species due to the addition of

different scavengers. Since they also tested the effect of the distance between plasma jet and liquid

surface and compared that with a gas phase model, they could exclude ozone. By comparison with

another argon plasma jet giving similar ozone and singlet oxygen concentrations in the gas phase but

not the same biological effects for the same conditions, they expect atomic oxygen being the effective

species or its precursor. Atomic oxygen is expected to form hypochlorite in presence of chloride ions.

Hypochlorite is known to be strongly antimicrobial and could decompose hydrogen peroxide, what

they observed for the Ar/O2 case in presence of chloride. However, to the best knowledge of the

authors, direct hypochlorite detection was not realized yet in plasma-treated sodium chloride-

containing liquids.

19
In a recent publication of Hnsch et al. [78] an alternative origin of hydroxyl radicals are suggested

for a surface dielectric barrier discharge in air above sodium chloride solution. A dissociation of water

by VUV radiation could be excluded with this plasma source and under these experimental conditions.

But, since they found hydrogen peroxide as stable ROS end product, they suggest a production of

hydroxyl radicals via photolysis of aqueous nitrate and nitrite. This assumption based on literature on

photochemistry [83]. Hnsch et al. [78] investigated in their study also hydrogen peroxide, nitrite and

nitrate concentrations in plasma activated sodium chloride solution dependent on storage time after

plasma treatment. Interestingly, they found a difference dependent on the plasma activation time. After

4 minutes plasma treatment they found a higher amount of nitrite than hydrogen peroxide immediately

after the plasma treatment. This relationship remains for post-treatment storage times of 30 minutes up

to 90 minutes. For 5 minutes plasma treatment, this trend shifts. Immediately after the treatment, the

nitrite concentration was higher than the H2O2 concentration, similar to the 4 minutes treatment case.

In contrast to that, for all post-treatment times of the 5 minutes plasma treatment this relationship

reversed in favor of hydrogen peroxide. As they investigated these two conditions for their

antimicrobial activity and found for both an immediate inactivation of bacteria but only for the

5 minutes plasma treatment time also a long-term antibacterial activity. Hence, they assumed that the

short-term activity mainly caused by nitrogen species and the long-term activity of the plasma

activated sodium chloride solution is mainly caused by hydrogen peroxide. Furthermore, they also

included the formation of peroxynitrite, supported by higher amounts of H2O2 in their considerations

regarding the long-term effect of plasma treated sodium chloride solution.

4. Relevant chemical pathways identified by means of modelling

As a first plasma-liquid model approach in the field of plasma medicine van Gils et al. [45] assumed

mainly three different constant fluxes from an argon plasma jet via the plasma/gas-liquid interphase to

the liquid bulk being relevant. One is the flux of hydroxyl radicals, which is originally a combination

of two, namely the flux of vacuum ultraviolet radiation leading by contact with water molecules to

OH generation and directly plasma-born OH radicals. The second is also a reactive oxygen species

20
flux, namely that of ozone, which is a prominent ROS in the gas phase. The third flux they included is

that of nitric oxide species (H)NOx. They used as starting concentrations for their model literature

values of plasma/gas phase amounts determined for a similar plasma source and evaluated the

behavior during and after plasma treatment of up to 60 s in 100 L liquid volume. The model

calculations resulted in the generation of different RNS and ROS during plasma treatment. Hydroxyl

and nitric oxide radicals, hydrogen peroxide and ozone increased linearly with the treatment time in

the first 10-5 s. The hydroxyl radical reached a kind of a temporary plateau at approximately 1 s,

afterwards it starts to decrease. Hydrogen peroxide increased linearly over the whole plasma treatment

period. Nitric oxide was increasing strongly with the treatment time, but different to H2O2 it reached

also a plateau after 10 s. Starting after 10-5 s also HNO2 and N2O3 and after 10-4 s and 1 ms HO2 and

ONOO- were formed, respectively. Due to the model the expected pH after treatment should be around

4, the concentrations of the hydroxyl radical were 310-12 M and for the hydroperoxyl radical 410-
15
M. Ozone and peroxynitrite were both in the order of almost 10-6 M, whereas NO2- ended up at

approximately 10-5M and the other species NO3-, N2O3, HNO2, NO and H2O2 reached values between

10-4 and 10-3 M. These concentrations remained constant in the liquid in the first 60 seconds after

treatment according to the model for all species besides the radicals, OH, HO2, and NO, and ozone.

Albeit these four species decreased post treatment, this happened not simultaneously for all four. The

two oxygen free radicals were already below 10-16 M after 5 s; ozone reached this concentration

threshold after 10 s. At this time point NO just started to decrease, reaching a concentration of 2 nM

after 54 s. In their model-approach they also compared the calculated data of nitrite, nitrate and

hydrogen peroxide. By this comparison they could verify that the applied fluid chemistry is a good

estimation for the concentrations of the chemical species in the bulk liquid.

Norberg et al. [48] modeled a helium plasma jet interacting with a liquid-covered tissue, with the

focus on the difference between contact or non-contact modes of the plasma jet with a 200 m thick

water layer. Three different setups were calculated in this model, one non-contact mode and two

contact modes: contact after 35 ns and spreading for 15 ns or contact after 20 ns and spreading for

30 ns. For all three cases H3O+, O2-, O3-, NO3-, ONOO-, O3, OH, H2O2, and HO2 were investigated in

the liquid. The trends for the neutral species in the contact modes were identical although the densities

21
and the starting points were different. As shorter the distance between the nozzle and the liquid

interface was, as sooner the plasma had contact to the liquid. So, for the same treatment time different

contact times occurred, which were directly linked to the resulting reactive species concentrations in

the solution. The hydroxyl radical density increased really fast to its maximum, but it also immediately

started to decrease again. The velocity of the formation reaction was albeit faster than that of the

decomposition. Hydrogen peroxide increased slower but reached a constant density level during the

modeled time frame. Similar to OH, also HO2 increased quite fast but the decrease was much slower

than for hydroxyl radicals. The aqueous ozone density increased remarkably later and also really slow

compared to the other neutral species in the contact modes and no decay was observed, at least not in

the investigated time frame. The non-contact mode behaved completely different there. Ozone and

hydroxyl radicals increased in the same manner, but other than ozone OH also immediately decayed

again, whereas O3 remained constant. Hydrogen peroxide and hydroperoxyl radicals appeared later;

also here the radical was not stable whereas the H2O2 density remained more or less constant. The

charged species in the contact mode had again the same behavior and were different both in the

resulting densities and the starting point for the two contact times. In both contact modes O2- and

H3O+ differed in the raising time only. Nitrate and peroxynitrite not even differed in that parameter

and again here the two contact modes also varied in the final density and the starting point only. In the

non-contact mode the trend for O2- and H3O+ was the same as well as its density. NO3- and ONOO-

were also quite similar in trend but appeared later than the oxygen species. Consequently, these

models highlighted the resulting differences in chemical species if the plasma is in contact with the

liquid or not [48-50].

Hamaguchi et al. [51] performed a numerical simulation of reaction diffusion equation and a global

one to investigate the impact of plasma born electrons entering the water. As one result of this global

model they could identify hydrogen peroxide as one central species and the essential role of dissolved

oxygen from the air for its generation. In the reaction-diffusion model they investigated the density of

reactive oxygen species over the depth of the liquid for two different time points, 510-5 s and 110-3 s.

The main outcome of this investigation was that in the bulk liquid the active ROS species is mainly

22
hydrogen peroxide, whereas the hydroxyl radicals are generated in a thin layer closed to the gas-liquid

boundary.

In the model of Chen et al. [52] this boundary region was separated in two small areas between the

plasma/gas and liquid bulk. So they divided the whole system in four parts, with the plasma/gas bulk

as the upper one followed by the gas reaction region, the liquid absorption area and the liquid bulk.

Between the gas reaction and the liquid absorption regions a boundary with constrained conditions,

such as a pressure balance and the Henry law was situated. The plasma/gas bulk model - a fluid model

- was linked to the mass transfer model for the gas-liquid interface - the reaction-penetration model -

which was also linked to the diffusion-reaction model for the liquid bulk. In the liquid bulk they

modeled 19 hydrogen and oxygen species with 84 reactions. The thickness of the boundary region was

not fixed in their model, since this region is dominated by water vapor. Hence a formation of a

helium/oxygen/water plasma afterglow in the gas region was probable. They also included in their

model the generation of water clusters due to the water content in the boundary region. If this water

content was about 1%, the formation of water clusters was prominent so that they became the

dominating species, but if 1% water was exceeded the afterglow behaved like a He/H2O plasma. In the

reaction-penetration model they treated the upper part as a reactive gas film and the lower part as a

liquid absorption film, both together being a kind of a bridge between the gas bulk and the liquid bulk.

Chen et al. [52] compared the downstream gas chemistry they gained for their model with the

downstream on a liquid bulk. In the gaseous downstream, in contrast to typical plasma chemistry

models, they found that plasma species itself are less involved and therefore less important. Most

important species determined in this gas downstream are neutral oxygen species, namely atomic

oxygen, singlet oxygen, and ozone. Furthermore they highlighted the sensitivity of O and 1O2 on the

distance between plasma and target. For distances greater than 10 mm the O3 produced by He/O2

plasmas will act as reactive species, only. They obtained that downstream on a liquid bulk changes the

acting species. O and 1O2 have only penetration depth of much less than 1 m. From this model they

found that atomic oxygen and singlet oxygen from the gas phase are therefore converted via water

species to e.g. H2O2, O2-, HO2, OH, and O3. Due to Henrys law the concentration of O3 in the liquid

is lower than in the gas phase. In presence of plasma it will further be decreased by the loss reaction

23
with plasma generated radicals such as H, OH, O2- as well as by H2O2 and O-. However, since ozone

and hydroxyl radicals have also limited penetration depth of approximately 5-6 m and hydrogen

peroxide, superoxide anion and hydroperoxyl radicals have penetration depth of 1.3, 1.0 and 0.25 mm,

respectively, these three species were assumed to be most relevant in their system [52]. Due to the

boundary region they could deduce two main species, O2- and H2O2, whose concentration in the liquid

bulk is strongly dependent on its boundary concentration close to the liquid surface.

Lindsay et al. [87] also modeled the interface region between plasma/gas and liquid phase. They

investigated a streamer discharge in air interacting with water. Besides ROS they also included RNS in

their model. In their study they obtained the impact of the temperature gradient in the interface, which

occurs due to convective cooling and water evaporation. Remarkably, the water vapor concentration

gradient and the temperature gradient in the boundary region will change the chemistry. Furthermore

they found a local domination of the hydroxyl radical over other species at the place of interaction of

the plasma with the liquid, since there is the origin of the aqueous OH radical. In the center of the

interaction the concentration of hydroxyl radicals was 3-4 orders of magnitude higher than in the

interface and nine orders of magnitude higher than in the bulk. During the treatment, the reaction of

nitrogen dioxide radicals with hydroxyl radicals was the main production pathway of peroxynitrous

acid. In the post treatment time, their model resulted in a more or less exclusive formation of ONOOH

by the reaction of nitrite with hydrogen peroxide [87]. The main outcome of this model were the two

different formation regions, the interface, which is the dominant during treatment, and the liquid bulk,

where stable species were formed also post treatment.

5. Summary

The aim of this review paper is to summarize the state of the art in research of plasma medicine-

related plasma-liquid interaction primarily from an analytical point of view and to try to to answer

mainly two questions:

1. What species are generated in liquid phases by atmospheric pressure plasma treatment?

2. What pathways of generation of these species are known or assumed?

24
Examination of the past five years publications on plasma-liquid interaction in the field of plasma

medicine, it became visible that from the experimental side only very few studies were performed

for helium plasma sources which are mainly focused on treatment of water. With argon and air as

working gas some more studies have been performed yet. In general, there is a lack of detection of

several species, e.g. nitrogen dioxide, nitric oxide, and hydrogen radicals as well as peroxynitrite,

singlet oxygen and ozone. This situation is mainly caused by a lack of specific and selective

detection methods, in particular for peroxynitrite. This absence of proper methods is more

pronounced for the detection of species at the gas/plasma-liquid interface, especially for spatially

and temporally observation of the short-lived transient species. In the ideal case such a detection

should be not only time and space resolved but also without need of chemical sensors such as dyes

or scavengers to follow the complex reaction kinetics in the liquid phase after atmospheric pressure

plasma treatment. From the modeling aspect there is a lack of plasma-liquid models for more

complex liquids like cell culture media.

In the experimental studies the detected species do not differ generally neither dependent on the

working gas helium, argon or air nor dependent on the treated liquid (water, buffered and non-buffered

saline solutions, cell culture media). Aside from variations in quantity dependent on the specific

treatment conditions, the stable end-products which were detected in the plasma-treated liquids were

more or less always the same: hydrogen peroxide as well as nitrite and nitrite, respectively. In non-

buffered systems an acidification was observed.

Hydrogen peroxide is taken as a general parameter for ROS generation whereas nitrite and nitrate

are representative parameters for RNS/RONS generation. Measurement of pH is essential to

characterize the reactive conditions in the liquid phase. Consequently, these species are considered to

be representative for more or less complex reactions chains with the participation of other reactive

oxygen and nitrogen species which are induced in liquids by plasma treatment.

Based on these observations it is hypothesized that the basic pathways of generation of reactive

species in liquids after treatment with atmospheric pressure plasmas can be generalized. Consequently,

qualitative as well as quantitative detection of pH, hydrogen peroxide, nitrite and nitrate should allow

25
basic conclusions on main reactive processes in plasma-liquid interaction and are therefore

representative for basic plasma-source characterization with regard to its biological activity.

However, it is quite evident that these stable and easy to detect species are mostly not generated

directly by plasma-liquid interaction, but rather occur post treatment from secondary and tertiary

reactions of short-lived and often quite reactive precursor species inside the liquid phase.

Peroxynitrite has been identified by several groups to play a crucial role for plasma-caused biological

effects in the liquid environment of cells, above all for antibacterial plasma effects.

Unfortunately, peroxynitrite and all other assumed short-lived and reactive species are difficult to

measure, because many of the techniques available yet are based on color-forming reactions, which

usually require to be performed after treatment. However, the detection of the transient species is

possible by some techniques using chemical probes such as phenol for the HPLC or spin traps for EPR

measurements.

In general, the combination of experimental data and theoretical considerations including modelling

approaches is the best and possibly only way to get some more insight in the much complex processes

of plasma-liquid interaction.

In some of the studies similarities could be found in both the hypothesized and experimentally

verified formation pathways. The studies of Winter et al. [23, 69] could identify the generation of

hydrogen peroxide in the humidified argon plasma/gas phase and its transfer into the liquid by

dissolution. Similar to these studies, in the models of Chen et al. [52] and Norberg et al. [48] also

found a close relation of the H2O2 concentration in the liquid bulk with that in the plasma/gas-liquid

boundary region which was assumed as a region with high humidity.

The model of Chen et al. [52] for the downstream gas chemistry identified atomic oxygen, singlet

oxygen and ozone as prominent species, the same was found in the gas phase model used in the study

of Wende et al. [43]. However, Wende et al. have found some hints that atomic oxygen is the main

precursor of other highly reactive oxygen species. This precursor role was also assumed by Chen et al.,

if a liquid bulk is implemented in the downstream model.

In their study on the impact of plasma jet VUV on liquids, Jablonowski et al. [88] verified the

photochemical contribution of argon plasma jet-emitted VUV radiation in the formation of hydroxyl

26
radicals in the liquid via dissociation of water. The same was also reported by Norberg et al. [48] for

contact as well as for the non-contact case of plasma with thin liquid layers. Jablonowski et al [24]

also assumed a locally high concentration of hydroxyl radicals at the point of plasma treatment which

was also found in the model of Lindsay et al. [87]. As the model of Lindsay additionally includes RNS

they found identical generation pathways as were suggested by experimental results. Lukes et al. [28]

investigated the post treatment formation of peroxynitrite (ONOO-) by reaction of nitrite with

hydrogen peroxide. This reaction was also found by Lindsay et al. [53] to be the peroxynitrite

production pathway after treatment, whereas during treatment the short-lived radicals, nitrogen dioxide

and hydroxyl radicals were found to be dominant. Oehmigen et al. [40, 54] also suggested the

formation of ONOO- via superoxide anion and nitric oxide radicals. The important role of HO2 and

O2- as main acting species was experimentally verified by the studies of Ikawa [34, 35, 59, 80] and by

modeling by Chen et al. [52].

The detection of ozone in the liquid phase is quite complicated due to the poor selectivity of the

common detection methods. However, from modeling studies [52] the impact of O3 during plasma

treatment of liquid seems to be not prominent in contrast to the gas phase. Consequently, for an

inactivation of microorganisms on dry surfaces it could be of higher importance as it was shown by

Pavlovich et al. [65]. This needs to be determined by further experiments.

In liquids containing chloride (e.g. saline solutions as well as cell culture media) the generation of

the biologically active hypochlorite has to be taken into consideration resulting from interaction with

reactive oxygen species. Similar to peroxynitrite, a specific and selective detection method is needed

to find out if these substances play a key role in plasma-caused biological affects transmitted via liquid

phases or not.

6. Conclusion

During the last five years mechanisms of plasma-induced change of liquid chemistry have drawn

huge attention in basic research in plasma medicine because liquid phase processes have been

identified to be the main key to understand detailed mechanisms of atmospheric pressure plasma
27
effects on living systems. Moreover, plasma-activated liquids are considered to be useful for several

applications also in the medical field.

The crucial role of reactive oxygen and nitrogen species (ROS, RNS/RONS) in these processes is no

longer in question [6]. Because these ROS and RNS/RONS are the same as occur in regular

physiological and pathophysiological processes in living organisms, the huge and established field of

redox biology is now open to interpret and to explain but also to control and to tailor biological effects

of atmospheric pressure plasmas. This is one of the main preconditions for further development of

clinical plasma medicine on a sound scientific basis.

Now, a big challenge of basic research in plasma medicine is the identification of the plasma-

generated active species responsible for specific biological effects. The main problem is not only the

high reactivity and consequently limited stability of these species. In conventional chemical and

pharmaceutical analytics, in most cases single substances have to be identified and/or quantified in

mixtures with more or less stable concentrations of the ingredients. In the case of plasma-treated

liquids, a complex and dynamic interactive reaction process is triggered where the analytical

procedures itself can influence this process and consequently distort the results. Therefore, innovative

and sophisticated analytics possibly at least partially beyond the classical chemical and pharmaceutical

techniques have to found to solve these problems.

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Physics of Plasmas (accepted)

34