M U Tresp

S Hammer

S Iseni

A Kupsch

K.-D Masur

Weltmann

J Winter
email: winter@inp-greifswald.de

H Tresp

M U Hammer

S Kupsch

A Schmidt-Bleker

K Wende

K-D Weltmann

S Reuter

Tracking plasma generated H 2 O 2 from gas into liquid phase and revealing its dominant impact on human skin cells

Keywords: hydrogen peroxide, human skin cells, gas phase, plasma jet, humidity, liquid phase

Tracking plasma generated H2O2 from gas into liquid phase and revealing its dominant impact on human skin cells

Joern Winter, H.

Introduction

Applications in plasma medicine require not only an understanding of the plasma sources and the generated plasma but also demand information on the active agent transition from the gas into the liquid phase and finally their interaction with the cells. However, this is a complex task since an interdisciplinary approach as well as extended experimental and theoretical research is necessary. Large efforts have been made in the diagnostics and control of atmospheric pressure plasmas intended for biomedical applications [START_REF] Von Woedtke | Plasmas for medicine[END_REF][START_REF] Laroussi | Low-temperature plasmas for medicine[END_REF][START_REF] Fridman | Applied plasma medicine Plasma Process[END_REF][START_REF] Kong | Plasma medicine: an introductory review New[END_REF]. Also, by the support of sophisticated theoretical models first insights into the complex nature of plasma cell and plasma liquid interactions were obtained [START_REF] Babaeva | Reactive fluxes delivered by dielectric barrier discharge filaments to slightly wounded skin[END_REF][START_REF] Tian | The interaction of atmospheric pressure plasma DBDs and jets with liquid covered tissues: fluxes of reactants to underlying cells[END_REF][START_REF] Bruggeman | Non-thermal plasmas in and in contact with liquids[END_REF][START_REF] Yusupov | Reactive molecular dynamics simulations of oxygen species in a liquid water layer of interest for plasma medicine[END_REF]. The aim of those investigations is to uncover work mechanisms of the plasma. This goes along with the identification of dominant species which are generated by the plasma in the gas and/or liquid phase and have a significant influence on the biological sample [START_REF] Graves | The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology[END_REF]. For prokaryotic cells Pavlovich et al and Shimizu et al found a correlation between the ozone concentration produced by a dielectric barrier discharge (DBD) operated in air and the inactivation efficacy of Escherichia coli [START_REF] Pavlovich | Ozone correlates with antibacterial effects from indirect air dielectric barrier discharge treatment of water[END_REF][START_REF] Shimizu | The dynamics of ozone generation and mode transition in air surface micro-discharge plasma at atmospheric pressure[END_REF]. The impact of ozone on bacteria is well known and many water disinfection facilities are based on DBDs [START_REF] Kogelschatz | Dielectric-barrier discharges. Principle and applications[END_REF][START_REF] Ehlbeck | Low temperature atmospheric pressure plasma sources for microbial decontamination[END_REF]. However, that does not automatically mean that ozone is the dominant species in plasma medicine, where usually eukaryotic cells are plasma treated. Kalghatgi et al showed that DBD treatment of mammalian breast epithelial cells differ completely from pure ozone treatment, although ozone is one of the dominant plasma generated species in the gas phase [START_REF] Kalghatgi | Damage in mammalian cells by non-thermal atmospheric pressure microsecond pulsed dielectric barrier discharge plasma is not mediated by ozone Plasma Process[END_REF]. They concluded that long living organic hydroperoxides mediate the DNA damaging effect of non-thermal plasma in mammalian cells. Haertel et al investigated the effect of DBD in argon and air on human skin cells (HaCaT keratinocytes) and observed that changes in integrin expression were related to intracellular reactive oxygen species (ROS) induction, while ozone has no influence [START_REF] Haertel | Differential influence of components resulting from atmospheric-pressure plasma on integrin expression of human HaCaT keratinocytes[END_REF]. Their study also discussed the dominant role of hydrogen peroxides (H 2 O 2 ). Two years before, Sato et al already found that H 2 O 2 is a key inactivation factor for HeLa cells treated by a pulsed corona discharge in air [START_REF] Sato | A key inactivation factor of HeLa cell viability by a plasma flow[END_REF]. For a radio frequency driven atmospheric pressure argon plasma jet, Winter et al observed that increased feed gas humidity results in an elevated inhibition of indirectly treated HaCaT cells [START_REF] Winter | Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells[END_REF]. Interestingly, the gas phase ozone concentration generated under these experimental conditions showed an opposing trend and was thus not considered as the dominant species responsible for the observed cell effect. It was pointed out in this work that H 2 O 2 in liquid phase increases with increasing feed gas humidity and a possible correlation with the cell response was discussed. Hydrogen peroxide was also identified as a central player in plasma-induced oxidative stress in human blood cells [START_REF] Bekeschus | Hydrogen peroxide: a central player in physical plasma-induced oxidative stress in human blood cells[END_REF].

H 2 O 2 is a quite important biologically relevant reactive species which can be detected throughout the human body. Peroxisomes and mitochondria show elevated levels as a by-product of mitochondrial respiration and β-oxidation of fatty acids, respectively [START_REF] Halliwell | Free Radicals in Biology and Medicine[END_REF][START_REF] Chance | Hydroperoxide metabolism in mammalian organs[END_REF][START_REF] De Groot | Hypoxia, reactive oxygen, and cell injury Free Radical[END_REF]. Hydrogen peroxide is also an important mediator of wound healing processes, as it is secreted by epithelial cells after injuries have occurred, thereby initiating inflammatory processes [START_REF] Niethammer | A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish[END_REF]. In a similar setting, it is an effective antimicrobial means of innate immune cells (macrophages, neutrophils) upon the uptake of pathogens [START_REF] Aderem | Mechanisms of phagocytosis in macrophages[END_REF][START_REF] Bogdan | Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity[END_REF][START_REF] Dahlgren | Respiratory burst in human neutrophils[END_REF].

However, its omnipresence strongly requires control mechanisms to level the intra and extracellular H 2 O 2 concentration. Many enzymes have evolved to that effect, the most efficient being catalase: one molecule of catalase readily transforms up to several million H 2 O 2 molecules into water (H 2 O) and oxygen (O 2 ) molecules every second [START_REF] Lisanti | Hydrogen peroxide fuels aging, inflammation, cancer metabolism and metastasis[END_REF]. Nevertheless, elevated levels or defective enzymes or detoxification pathways may cause serious damage to single cells or tissues, especially considering that it readily passes through cell membranes [START_REF] Halliwell | Free Radicals in Biology and Medicine[END_REF]. DNA damage has been described, from single base modifications to single and double strand breaks, as well as (per-) oxidation of (membrane) lipids (especially unsaturated lipids) and proteins leading to various cell changes, starting with mere modifications of cell shape and cytoskeleton to total loss of viability and disintegration of the cellular membrane [START_REF] Halliwell | Free Radicals in Biology and Medicine[END_REF][START_REF] Chance | Hydroperoxide metabolism in mammalian organs[END_REF][START_REF] Imlay | DNA damage and radical toxicity[END_REF][START_REF] Jaruga | Repair of products of oxidative DNA base damage in human cellsNucleic[END_REF][START_REF] Hinshaw | Cytoskeletal and morphologic impact of cellular oxidant injury[END_REF][START_REF] Simon | Hydrogen peroxide causes the fatal injury to human fibroblasts exposed to oxygen radicals[END_REF][START_REF] Weiss | Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cellsJ[END_REF]. Elevated levels of H 2 O 2 are also considered to play an important role in the skin depigmentation disease Vitiligo [START_REF] Hasse | Perturbed 6-tetrahydrobiopterin recycling via decreased dihydropteridine reductase in vitiligo: more evidence for H 2 O 2 stress[END_REF].

Even though H 2 O 2 is considered a mildly oxidizing species, it does not directly oxidize most biological molecules.

It rather acts through its conversion to the highly reactive products hydroxyl radical ( • OH) and hydroperoxyl radical (HO • 2 ), which can be formed by photodissociation of H 2 O, by the decay of peroxynitrite/peroxynitrous acid (O=NOO -/O=N-OOH) or by the Fenton reaction in the case of • OH [START_REF] Ueda | A comparison of scavenging abilities of antioxidants against hydroxyl radicals Arch[END_REF][START_REF] Hampton | Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis[END_REF].

The aim of the current study is to trace the H 2 O 2 molecule from its plasma based generation in the gas phase to the liquid phase and show that H 2 O 2 concentration correlates strongly with the inhibition of adherent human skin cells.

Materials and methods

Plasma jet

The plasma source, a cold atmospheric pressure plasma jet (kinpen, neoplas GmbH, Germany), used in this study is identical to the device used in the related study concerning the impact of feed gas humidity [START_REF] Winter | Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells[END_REF]. It consists of a centred rod electrode inside a ceramic capillary and a grounded ring electrode [START_REF] Weltmann | Atmospheric-pressure plasma sources: prospective tools for plasma medicine[END_REF][START_REF] Reuter | Detection of ozone in a MHz argon plasma bullet jet Plasma Sources[END_REF]. The ceramic capillary has an inner and outer diameter of 1.5 mm and 2.5 mm, respectively. When a sinusoidal voltage signal of 2 to 6 kV pp with a frequency of about 1 MHz is applied to the centred electrode, plasma is ignited within the core region of the jet and about a 10 mm long effluent becomes visible after ignition. The working gas was argon (Argon N50, Air Liquide) with a gas flow rate of 3 standard litres per minute (slm). In order to generate hydrogen peroxide by the plasma, the humidity concentration of the feed gas was changed by means of a bubbler setup exemplarily shown in figure 1. The feed gas humidification setting was similar to the setting used in [START_REF] Winter | Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells[END_REF]. The feed gas humidity was measured with a chilled mirror dew point hygrometer (DewMaster, EdgeTech, USA). For Fourier transformed infrared (FTIR) measurements the jet was equipped with a shielding gas device as reported in [START_REF] Schmidt-Bleker | Reactive species output of a plasma jet with shielding gas device-combination of FTIR absorption spectroscopy and gas phase modelling[END_REF]. For details see section 2.3. All other plasma treatment results are performed without a shielding gas device.

Detection OH in the plasma jet effluent

The detection of gas phase hydroxyl radical density under atmospheric conditions by laser-induced fluorescence (LIF) is still under investigation and requires significant calibration efforts to yield absolute densities [START_REF] Dilecce | Laser diagnostics of high-pressure discharges: laser induced fluorescence detection of OH in He/Ar-H 2 O dielectric barrier discharges Plasma[END_REF][START_REF] Verreycken | Absolute calibration of OH density in a nanosecond pulsed plasma filament in atmospheric pressure He-H 2 O: comparison of independent calibration methods[END_REF][START_REF] Voráč | Measurement of hydroxyl radical (OH) concentration in an argon RF plasma jet by laser-induced fluorescence Plasma Sources[END_REF][START_REF] Verreycken | Absolute OH density measurements in the effluent of a cold atmospheric-pressure Ar-H 2 O RF plasma jet in air Plasma Sources[END_REF]. However, relative hydroxyl radical density is already interesting since basic information about chemistry dynamics can be concluded. In this work, the relative density of hydroxyl radicals is measured for different feed gas humidity settings by LIF using the P1(4) transition of the OH(A,X)(1,0) at 283.452 nm and chosen for its good absorption efficiency and its well isolated spectral position from neighbouring lines. The excitation is performed by a frequency doubled dye laser (Cobra Stretch, Sirah Laser und Plasmatechnik GmbH, Germany) pumped with a 10 Hz pulsed Nd : Yag laser (SpectraPhysics, Inc., USA). The LIF signal is transmitted through a 10 nm FWHM bandpass filter centred at 308 nm. The detection is performed by a photomultiplier tube (R5916U51, Hamamatsu, Japan) connected to a digital oscilloscope (DPO 4104, Tektronix Ltd., United Kingdom). The laser beam diameter is adjusted by a 500 µm aperture and not focused. The laser spot is pointed on-axis on the plasma effluent at 1.0 mm distance from the nozzle. In order to ensure a linear LIF response to the laser energy and to avoid saturation effects, a laser energy scan is performed. Consequently, the laser energy is set to 120 µJ per pulse. The LIF intensity I LIF is known to be proportional to the laser energy I L [START_REF] Verreycken | Absolute calibration of OH density in a nanosecond pulsed plasma filament in atmospheric pressure He-H 2 O: comparison of independent calibration methods[END_REF] and can be simplified in our case by:

I LIF ∝ n OH i k i • n i • I L (1)
where n OH is the hydroxyl radical density, k i the quenching and vibrational energy transfer (VET) coefficient and n i is the density of a quencher species i which contributes to fluorescence losses. Argon from the feed gas, nitrogen and oxygen from the surrounding air and water from the admixture are the main quenchers of the fluorescence. However, it is a reasonable assumption to consider only water as a quencher since its density is a variable parameter, whereas argon, nitrogen and oxygen densities remain constant. The LIF signal is thus corrected from quenching and VET losses by using the following value reported in literature for water molecules, k H 2 O = 6.6 × 10 -10 cm 3 s -1 [START_REF] Williams | Collisional vibrational energy transfer of OH (A 2 + , v = 1)[END_REF].

Detection of H 2 O 2 in plasma afterglow

FTIR spectroscopy is a broadband absorption technique that enables the detection and quantification of molecules absorbing in the infrared spectral region. Since gas phase H 2 O 2 has significant absorption bands in the spectral range from 1200 to 1330 cm -1 FTIR spectroscopy is the method of choice to analyse H 2 O 2 [START_REF] Rothman | The HITRAN 2004 molecular spectroscopic database[END_REF]. In this study an FTIR spectrometer (Vertex 70v, Bruker Optics GmbH, Germany) was equipped with an additional white type multipass cell (A134G/QV, Bruker Optics GmbH, Germany), which provides a total absorption length of 19.2 m. The pressure in the multipass cell was reduced to 100 mbar by means of a vacuum pump in order to prolong the lifetime of the plasma generated species and to reduce the distracting influence of ambient water and carbon dioxide (CO 2 ). Due to this low-pressure setting the atmospheric pressure plasma jet could not be operated directly in the multipass cell. Thus, the jet was equipped with a pressurized air flushed shielding gas device with a nozzle diameter of 5 mm, similar to the one reported in [START_REF] Schmidt-Bleker | Reactive species output of a plasma jet with shielding gas device-combination of FTIR absorption spectroscopy and gas phase modelling[END_REF], and attached to an additional glass chamber as displayed in figure 1.

The gas from the shielding (5 slm compressed and dried air) and from the jet (3 slm argon) flows into the glass chamber and exhausts via an outlet. Hence, the pressure in the glass chamber is slightly higher than the ambient (1020-1030 mbar). Furthermore, the glass chamber was connected to the multipass cell by means of an adjustable needle valve so that a small fraction of the gas flow was sampled continuously by the FTIR system. The gas flow rate through the multipass cell is 1.5 slm. The gas mixing and particle residence time in the glass chamber was calculated by computational fluid dynamics simulations in a previous work for an identical geometry setting [START_REF] Schmidt-Bleker | Reactive species output of a plasma jet with shielding gas device-combination of FTIR absorption spectroscopy and gas phase modelling[END_REF]. It was shown that the majority of particles remain within the mixing cell for a few seconds before they enter the FTIR multipass cell. This means that highly reactive species have enough time to react and form long living products, like H 2 O 2 or O 3 , which were detected with the FTIR setup.

In order to investigate the influence of feed gas humidity on the produced species the argon feed gas was humidified with the bubbler setup as displayed in figure 1. Before the FTIR measurement was performed the plasma jet was switched off while the particular gas mixture was flushed through the jet and the multipass cell. After 10 min the background signal was obtained. Subsequently, the jet was switched on. Several FTIR spectra were obtained for at least 10 min to validate the constant absorption band intensity. This proves a steady state measurement of species concentration in the multipass cell. The obtained spectra were fitted using fitting tool software (QMACSoft 1.1.3, neoplas control GmbH, Germany), that is based on the Hitran database and on the Levenberg-Marquardt algorithm [START_REF] Rothman | The HITRAN 2004 molecular spectroscopic database[END_REF][START_REF] Zimmermann | QMACSoft-HT (computer program[END_REF]. This allows for the identification of absorption bands as well as the calculation of species densities by applying Beer-Lambert's law to the measured optical depths. This measurement and evaluation procedure was performed for all humidity settings, so that an individual background signal was obtained for every humidity condition.

By replacing the humidifier bubbler by an identical vessel filled with 150 ml of 10% H 2 O 2 solution, created by diluting a 30% H 2 O 2 stock solution (9.7 M, Merck, Germany) and leaving everything else unaltered, gas phase H 2 O 2 was generated without igniting the plasma. By varying the argon gas flow rate passing through the H 2 O 2 bubbler the amount of gas phase H 2 O 2 concentration was adjusted. The H 2 O 2 concentration was determined analogue to the previously described procedure.

Plasma treatment of RPMI solution

As in the previous study [START_REF] Winter | Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells[END_REF], a volume of 5 mL complete cell growth medium (Roswell Park Memorial Institute RPMI 1640 + 8% fetal calf serum + 1% penicillin/streptomycin solution) filled in a polystyrene Petri dish (60 mm diameter TPP, Trasadingen, Switzerland) was plasma treated. The treatment time was varied between 0 and 100 s. During that treatment time the plasma jet was constantly moved in a meandering pattern over the Petri dish by means of a xyzstepping motor. The distance between the nozzle and the liquid surface was 9 mm. During plasma treatment, the 3 slm argon gas flow rate leads to evaporation of the liquid. By measuring the remaining liquid volume after the longest treatment time (100 s), it was found that the initial 5 mL liquid volume misses 130 µl±10 µl. This evaporated liquid volume is only about 2.6% of the initial volume and is thus neglected in the H 2 O 2 concentration evaluation.

By means of a thermometer the liquid temperature was measured before and after 100 s plasma treatment and no relevant temperature increase was observed.

Detection of reactive species concentrations in liquid cell growth medium

Hydrogen peroxide was measured with the test stripe method described in [START_REF] Winter | Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells[END_REF]. According to this method three times 30 µl of the plasma treated RPMI medium were sampled with a pipette directly after plasma treatment. These sample volumes were put onto three H 2 O 2 test stripes (Merckoquant110011, Merck, Germany). After a waiting time of 60 s the test stripes were read out by a digital microscope camera (Conrad, Germany) in order to analyse their colour values. By comparing the obtained colour values with a calibration curve the H 2 O 2 concentration was determined. This procedure was conducted three times so that finally nine test stripes were evaluated for every feed gas humidity setting investigated.

The same test stripe method was utilized for the H 2 O 2 bubbler setup. For direct comparison the same H 2 O 2 bubbler setup was used as for the FTIR measurements. In this experiment, the cell growth medium was exposed to the working gas flow for 40, 60 and 80 s without plasma ignition. The gas flow and treatment procedure is identical to the plasma treatment using humidified feed gas. By comparing the results of both experiments the effect of plasma for H 2 O 2 production in liquid is investigated. The results are discussed in section 3.2.

Also the stability of plasma produced H 2 O 2 in RPMI was investigated. Therefore, three H 2 O 2 start concentrations of 0.8, 2.9 and 9.0 mg l -1 within a volume of 5 ml RPMI were produced via plasma treatment with three experimental settings, namely: dry argon gas and a treatment time of 20 or 180 s and with humidified argon (1660 ppm) and a treatment time of 80 s. Each setting was repeated three times. Subsequent to the plasma treatment the H 2 O 2 concentrations in each Petri dish were measured at different times after plasma treatment applying the test stripe method described above. We intentionally did not use commercial H 2 O 2 stock solution for the H 2 O 2 life time experiments, since in contrast to plasma treated H 2 O 2 , it contains stabilizers. This would distort the results of the life time measurements in the RPMI solution.

The measurement of low H 2 O 2 concentrations in the case of the dry feed gas for the purpose of cell viability comparison was performed by means of a homogeneous assay (Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, Invitrogen Ltd, USA). The assay was applied analogue to the method described in [START_REF] Winter | Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells[END_REF]. It is more sophisticated than the test stripe method but leads to a higher accuracy in H 2 O 2 concentration determination. This is important for the correlation evaluation described in section 2.6 since errors made in H 2 O 2 concentration measurement will directly affect the accuracy of the cell inhibition time determination (see figure 2).

The radical concentrations of hydroxyl ( • OH) and superoxide anion (O •- 2 ) in cell growth medium for different feed gas humidity settings and a plasma treatment time of 40 s were measured by electron paramagnetic resonance (EPR) spectroscopy. The protocol applied in this work is described in [START_REF] Tresp | Quantitative detection of plasma-generated radicals in liquids by electron paramagnetic resonance spectroscopy[END_REF]. Due to the short lifetime of these radicals in liquid spin traps, 5-tert-Butoxycarbonyl-5-methyl-1-pyrroline-Noxide (BMPO, Dojindo Laboratoire, Japan) and 5,5-dimethy-1-pyrroline-N-oxide (DMPO, Dojindo Laboratoire, Japan) were used. DMPO was solved in the RPMI cell culture medium to a concentration of 100mM. This spin trap cannot distinguish between hydroxyl radicals ( • OH) and superoxide anion radicals (O •- 2 ). BMPO on the other hand distinguishes between both species. However, until now we were not able to obtain an EPR signal from BMPO in RPMI. Thus, experiments with BMPO were conducted by solving 10mM of BMPO in Dulbecco's phosphate buffered saline (DPBS) solution instead of RPMI. Each EPR measurement was repeated three times.

Parallel to the above mentioned measurements the pH value of 40 s plasma treated RPMI solution was measured directly after plasma treatment using a pH meter (Sevenmulti M47, Mettler Toledo, Switzerland).

Relating H 2 O 2 concentration to cell viability

To answer the question, whether plasma generated H 2 O 2 correlates with the viability of indirectly treated adherent human keratinocytes (HaCaT cells), inhibition measurements are taken from figure 12 in [START_REF] Winter | Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells[END_REF]. This inhibition curve depicts how long a cell culture needs to be treated by the argon plasma jet at a certain feed gas humidity concentration in order to inhibit 50% of the cell metabolic activity. For instance, at completely dry conditions a plasma treatment duration of about 45 s is necessary to reach this inhibition level whereas at a significantly higher humidity concentration of 1250 ppm half the cells lose their viability already after a treatment time of 16 s. In order to relate H 2 O 2 measurements with these data the inhibition concentration of H 2 O 2 was determined. Therefore, the same HaCaT cell type was handled identically as in [START_REF] Winter | Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells[END_REF] with the difference that the cells were not covered with a plasma treated RPMI medium but with an RPMI medium containing different well defined H 2 O 2 concentrations. These concentrations were produced by diluting 30% H 2 O 2 stock solution (9.7 M, Merck, Germany) in RPMI. As a result, the H 2 O 2 concentration to inhibit 50% of HaCaT cells was determined as (21 ± 4)µM (solid horizontal line in figure 2, corresponding to (0.71 ± 0.14) mg l -1 ). The uncertainty interval (horizontal dashed lines) is the standard deviation of 8 measurements.

Figure 2 also shows the increase of the H 2 O 2 concentration in the RPMI cell growth medium for a plasma jet treatment with dry argon feed gas after different treatment times. From the intercept point of this curve with the solid horizontal line the half inhibition treatment time is deduced. At this time point of plasma treatment duration, the H 2 O 2 concentration due to plasma treatment reaches 21µM and is as high as the H 2 O 2 concentration to inhibit half of the HaCaT cells. Every plasma treatment that will produce this H 2 O 2 level after a certain treatment time will inhibit at least half of the HaCaT cells. Moreover, if the treatment time to inhibit half of the HaCaT cells by plasma is similar to the time in which the H 2 O 2 concentration due to the plasma treatment increased up to 21µM, then H 2 O 2 plays the major role in modulating cellular activities.

The method to estimate the uncertainty of the half inhibition treatment time is also indicated in figure 2. The lower and upper error is obtained by evaluating the intercept points of the H 2 O 2 inhibition concentration uncertainty and the error interval of the plasma generated H 2 O 2 measurement.

Results and discussion

Generation of H 2 O 2 in the gas phase

A FTIR spectrum of the exhaust argon jet plasma gas is presented in figure 3 for a feed gas humidity concentration of 1220 ppm together with the resulting fit curve. The most dominant absorption in this spectrum is given by H 2 O 2 . Furthermore, distinct absorption bands of ozone (O 3 ) were obtained. Both band structures, H 2 O 2 and O 3 , were fitted and their concentration in the multipass cell was calculated. For this particular humidity setting the concentration of H 2 O 2 and O 3 are 3.6 ppm and 2.0 ppm, respectively. Despite the long purging procedure and careful background acquisition CO 2 absorption bands originating from ambient impurities are detectable. The spectral range from 1350 to 1700 cm -1 is also influenced by ambient water absorption bands which influence the quantitative evaluation of other molecular absorption bands in that region. Even though nitrogen dioxide (NO 2 ) was also identified in the spectrum, it could not be evaluated quantitatively due to its small signal and the overlapping water bands.

Evaluating the H 2 O 2 absorption band for every adjusted feed gas humidity value leads to the H 2 O 2 concentration curve, which is displayed in figure 4(a). With increasing humidity in the argon feed gas the H 2 O 2 concentration in the multipass cell rises from almost zero for dry feed gas condition to a concentration of 5.3 ppm for a humidity setting of 1800 ppm. Additional to the measurement results in figure 4(a) the result of a simulation, based on the far field chemical reaction simulation reported in [START_REF] Schmidt-Bleker | Reactive species output of a plasma jet with shielding gas device-combination of FTIR absorption spectroscopy and gas phase modelling[END_REF], is plotted. From that simulation, relevant production and destruction reactions for H 2 O 2 were obtained. However, since the model aims to simulate particle densities in the far field of the jet without considering electron processes, it is difficult to include H 2 O 2 generation processes that occur in the plasma jets core, where electrons play a major role. With only H 2 O and Ar present in the core zone H 2 O 2 formation occurs by recombination of • OH radicals that are previously created by particle impact dissociation of water molecules according to [START_REF] Bruggeman | On OH production in water containing atmospheric pressure plasmas Plasma Sources[END_REF] H

2 O + P → • OH + • H + P (2)
and

• OH + • OH + M → H 2 O 2 + M, ( 3
)
where P is an energetic particle from the plasma (e.g. electron or excited argon atom) and M is a collision partner (e.g. argon). The dissociation process of equation ( 2) cannot directly be included into the simulation. Even so, by considering the almost linear slope of the measured relative • OH density (see figure 4(b)) and by varying the degree of dissociation of H 2 O as long as, under consideration of all included reactions, the simulated H 2 O 2 profile fits with the measured one, this shortcoming can be avoided. The experimental based simulation predicts a linear increase of the far field H 2 O 2 concentration with rising humidity. This result, together with positive simulation benchmarks on other experimental data [START_REF] Schmidt-Bleker | Reactive species output of a plasma jet with shielding gas device-combination of FTIR absorption spectroscopy and gas phase modelling[END_REF], indicates that the most relevant equations for describing the production and destruction of H 2 O 2 are considered within the simulation. The fact that a slight H 2 O 2 concentration is predicted for zero feed gas humidity, results from the model assumption of a 36% relative ambient humidity. H 2 O 2 cannot only be produced in the core region of the plasma. In the effluent plasma region ambient species like O 2 diffuse into the plasma zone and influence the plasma chemistry. Thus, atomic hydrogen (H • ) reacts with molecular oxygen to form hydroperoxyl radicals (HO • 2 )

• H + O 2 + M → HO • 2 + M. ( 4
)
The combination of two of these radicals

2HO • 2 + M → H 2 O 2 + O 2 +M (5)
leads to the production of H 2 O 2 . As discussed in Reuter et al the electron density and temperature in the effluent region is lower compared to the situation in the core plasma region [START_REF] Reuter | The influence of feed gas humidity versus ambient humidity on atmospheric pressure plasma jet-effluent chemistry and skin cell viability[END_REF]. Furthermore, the educts • OH and HO • 2 , important for the production of H 2 O 2 , strongly react with each other to form water and oxygen and are thus not available for H 2 O 2 production. Both effects make the effluent region less efficient for H 2 O 2 production as experimentally shown in [START_REF] Reuter | The influence of feed gas humidity versus ambient humidity on atmospheric pressure plasma jet-effluent chemistry and skin cell viability[END_REF]. It was shown by the help of the simulation that other reactions to form H 2 O 2 , for instance via the reaction of water and ozone, play only a minor role. Besides H 2 O 2 generation by the argon plasma jet, gas phase H 2 O 2 was alternatively created by bubbling a fraction of the argon feed gas through a reservoir of hydrogen peroxide solution. The amount of gas phase H 2 O 2 was adjusted by the ratio of the argon feed gas flow rate that passes the H 2 O 2 bubbler and the total argon flow rate of 3 slm. The H 2 O 2 concentration in the gas phase produced by this method is displayed in figure 5. The measured H 2 O 2 spectrum, on which the concentration determination is based, is also given in figure 5 (inlay) for different flow rates through the H 2 O 2 bubbler. When the gas flow rate through the bubbler is increased, more H 2 O 2 molecules are present in the gas phase and consequently the transmission of radiation in the FTIR multipass cell decreases. Evaluating these transmission curves yields a rising H 2 O 2 concentration curve, which is zero for no bubbling and 10.5 ppm when an argon flow rate of 2 slm passes through the H 2 O 2 bubbler. The produced H 2 O 2 concentrations are in the same range as the concentrations produced by the argon plasma jet for different feed gas humidity settings. An exponential growth function resembles the H 2 O 2 concentration curve quite well. This non-linear behaviour is due to the increased bubble size and turbulence within the H 2 O 2 bubbler with increasing gas flow rate, which leads to an increased interaction surface between the gas and liquid phases. This results in an increased amount of H 2 O 2 molecules in the gas passing through the bubbler. Together with the rising mixing ratio of this H 2 O 2 bubbled gas and the unbubbled gas, a disproportionate gas phase H 2 O 2 concentration is obtained. the liquid phase and initiates the production of an • OH radical within the liquid. Two of these radicals can recombine to form H 2 O 2 .

Transition of gas phase H

If the solubility process dominates, a correlation between H 2 O 2 production in the gas and liquid phase must exist. Therefore, the H 2 O 2 net production rate P g in s -1 in the gas phase is calculated from the measured concentrations c in ppm in the multipass cell according to

P g = c • 2.47 × 10 19 cm -3 • ( 1 + 2 ) • 16. 6 cm 3 s -1 slm -1 , ( 6
)
where 1 and 2 are the feed gas flow rate (3 slm) and the curtain gas flow rate (5 slm), respectively. The factor 16.6 is due to the unit conversion of standard litre per minute into the SI unit cm -3 s -1 . The calculation of H 2 O 2 net production rate P L in the liquid cell growth medium is done by

P L = A • V L • N A • 10 -3 M (7)
assuming a linear increase A (in mg l -1 s -1 ) of H 2 O 2 concentration with time. V L in L is the volume of the treated medium, N A is the Avogadro constant and M in g mol -1 is the molar mass of H 2 O 2 . According to figure 6, where the increase of H 2 O 2 concentration in RPMI is displayed for different feed gas humidity settings, the assumption of a linear H 2 O 2 increase with time is quite reasonable due to the good agreement of the measured values and the linear regression curves.

Figure 7 shows the result of the H 2 O 2 net production rate determinations of the gas and liquid phases for different feed gas humidity settings. Interestingly, both curves are in a remarkable agreement.

This means that the H 2 O 2 net production rate in the gas and liquid phases are identical for the investigated humidity conditions and implies that the presence of H 2 O 2 molecules in the gas phase directly influences the liquid phase H 2 O 2 concentration. Although this positive correlation is not proving that liquid phase H 2 O 2 molecules originate from gas phase H 2 O 2 , at least it is strong evidence that in our experiment solubility processes play a major role in generating H 2 O 2 in liquid.

In order to find further evidence for this indication an additional experiment was performed. If solubility processes determine the H 2 O 2 concentration in plasma treated cell growth medium, then a simple non-plasma argon gas flow through the jet nozzle, containing the same H 2 O 2 molecule density (in the multipass cell of the FTIR) as was generated by the humidified plasma jet, must result in the same liquid H 2 O 2 concentration. In a first approximation, it is assumed that the H 2 O 2 concentration above the liquid is identical for both the plasma generated and the bubbler produced H 2 O 2 . If plasma has a direct H 2 O 2 production effect in addition to solubility processes on the cell growth medium, then the H 2 O 2 concentration in the liquid will be higher for the plasma case than for the non-plasma case. As described in section 2, the non-plasma H 2 O 2 generation was performed by using a H 2 O 2 bubbler. The gas phase H 2 O 2 concentrations for the plasmaon case (humidified argon gas) and for the H 2 O 2 bubbler are taken from figures 4(a) and 5, respectively.

The results of this study are depicted in figure 8 for three different treatment times. With a rising H 2 O 2 amount in the gas phase, the liquid phase H 2 O 2 concentration increases. This general trend is independent whether the gas phase H 2 O 2 is created by the plasma jet or by a H 2 O 2 bubbler without any plasma. However, the resulting liquid phase H 2 O 2 concentration is slightly higher for the plasma case. Especially for the longest investigated treatment time of 80 s, more liquid phase H 2 O 2 is generated by the plasma jet than with the H 2 O 2 bubbler at comparable gas phase H 2 O 2 densities. Returning to the argumentation above, this discrepancy means that additional to the solubility of gas phase H 2 O 2 in the liquid the plasma itself has a direct contribution to the liquid phase H 2 O 2 amount. Thus, the hypothesis that entirely all hydrogen peroxide found in the liquid medium results from a solubility process of gas phase H 2 O 2 , is falsified. The amount of this direct plasma contribution, however, is smaller than the effect obtained by the H 2 O 2 bubbler. On average, more than 60 % of the liquid phase H 2 O 2 was generated without direct plasma effect. Thus, the solubility of gas phase H 2 O 2 plays an important role for the generation of liquid phase H 2 O 2 -at least in our experimental setup. The question, why the H 2 O 2 solubility process is so dominant, is answered when comparing Henry's constant for solubility of different species (see table 1). According to Henry's law the concentration of a species in aqueous solution is proportional to its partial pressure over the liquid [START_REF] Sander | Compilation of Henry's Law Constants for Inorganic and Organic Species of Potential Importance in Environmental Chemistry Version 3[END_REF]. The proportionality factor is Henry's constant. The higher the value of this constant, the better molecules dissolve into the liquid. Compared to argon or oxygen Henry's constant is more than 7 orders of magnitude higher for H 2 O 2 . In fact, H 2 O 2 has one of the highest Henry's constants compared with other inorganic species. Hence, a gas phase H 2 O 2 molecule in proximity to an aqueous solution will most likely enter it.

Stability of plasma generated H 2 O 2 in RPMI

In principle, to have an effect on adherent cells the H 2 O 2 molecule or its decomposition products must live long enough have an extremely short lifetime. In biological systems the hydroxyl radical has a lifetime in the nanosecond and the superoxide anion radical in the microsecond range [START_REF] Pryor | Oxy-radicals and related species: their formation, lifetimes, and reactions[END_REF]. Thus, to be effective they must be produced in direct proximity to the cell molecule to be attacked. Compared to this, the lifetime of plasma generated H 2 O 2 is quite long as the measured data in figure 9 depict. As described in section 2.5 three different H 2 O 2 starting concentrations were produced by plasma treatment. Afterwards the degradation of the plasma generated H 2 O 2 was determined. For the first half hour after plasma treatment the H 2 O 2 concentration stayed almost unchanged, independent of the concentration reached directly after the plasma treatment. However, after a sufficient period of time the concentration decreases. For the highest starting value the H 2 O 2 concentration in the RPMI medium decreases from 9 down to 1.5 mg l -1 after 2000 min. No H 2 O 2 concentration was measurable for the lower starting concentrations after that time. A possible reason for this decay is the decomposition of H 2 O 2 via the Fenton reaction, since the cell growth medium contains iron.

When performing this H 2 O 2 life time experiment with H 2 O 2 from stock solution that contains stabilizers, the concentration decrease is much lower. Within 120 h the H 2 O 2 concentration decreases from an initial concentration of 10 to 6.7 mg l -1 (data not shown). For long time experiments this different behaviour of stock solution H 2 O 2 and plasma generated H 2 O 2 must be considered.

H 2 O 2 correlates with inhibition of HaCaT cells

In figure 10, the half inhibition treatment time for different feed gas humidity settings is displayed for indirect plasma treatment (data taken from [START_REF] Winter | Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells[END_REF]) and for the case that the cell inhibition effect is based on H 2 O 2 only. The data for the latter is determined according to the procedure described in section 2.6. It is obvious that the effect of H 2 O 2 not only follows the cell viability decrease but is almost identical to the effect that originates from plasma treatment. This means that for our treatment conditions H 2 O 2 plays a dominant role for the inhibition of indirectly plasma treated HaCaT cells in RPMI cell growth medium. Whether this role has a direct H 2 O 2 impact on the cells or is due to the impact of its decomposition products cannot be answered with this data. Either way, the amount of H 2 O 2 produced within the liquid determines the viability of the cells. A similar effect of H 2 O 2 on viability of human thyroid epithelial cells (HTori-3) was found by Marinov et al using a water discharge setup [58,[START_REF] Marinov | Plasmas in contact with liquids and at the interfaces[END_REF].

Looking closer to the results in figure 10 the average time to reach the 50% inhibition level is slightly higher for the H 2 O 2 case, which would mean that besides the H 2 O 2 impact other agents must contribute to the cell inhibition. Owing to the large error bars the significance of this finding is debatable. However, to ensure the dominance of H 2 O 2 measurements of pH-value and of • OH and O •- 2 radical concentrations were also conducted.

Figure 11 shows the results for the pH-value. For different feed gas humidity settings no severe change in pH is observed and thus a contribution of pH to the cell inhibition is excluded. This was expected since the used cell growth medium is buffered. Only a general slightly elevated pH level is found when comparing the plasma treated values with the untreated control. This is due to a degassing effect [START_REF] Buxton | Rate constant for reaction of hydroxyl radicals with bicarbonate ions[END_REF]. During plasma treatment, the RPMI cell growth medium is strongly swirled by the feed gas flow and thus previously chemically bound CO 2 evaporates from the liquid. Since dissolved CO 2 can form carbonic acid within the liquid a lack of CO 2 leads to a more alkaline condition.

The radical concentrations for • OH and O •- 2 in a plasma treated DPBS medium are shown in figure 12. With rising feed gas humidity little change in the radical concentration is observed. At most a slight decrease is detected. The • OH and O •- 2 radical concentrations are between 0.2-0.3µM and 0.45-0.65µM, respectively. The observation that both concentrations do not increase with feed gas humidity implies two conclusions. Firstly, their production in the bulk medium is not directly or dominantly linked to the produced H 2 O 2 concentration which itself increases with increasing feed gas humidity. If a direct link would exist the radicals are expected to resemble the H 2 O 2 trend. Since this is not the case the radical production must be generated due to other plasma agents. Secondly, the bulk concentrations of • OH and O •- 2 do not contribute directly to the inhibition of the investigated adherent HaCaT cells. However, since the measured concentrations are volume averaged values it might well be that directly at the cells the radical concentrations are quite inhomogeneous. Thus, their concentration might be much higher close to the cells than what is measured in the bulk medium.

It has to be mentioned that the presented EPR measurements were not performed in an RPMI medium, but in a DPBS medium while the other measurements were performed in RPMI. As discussed in section 2.5 this was due to the lack of signal obtained with the BMPO spin trap in RPMI. The spin trap BMPO is to be preferred since with it • OH and O •- 2 radicals can be distinguished, whereas the spin trap DMPO only provides the sum of both radical densities. DMPO on the other hand was used in RPMI. In order to verify that the constant behaviour of both radicals with respect to the feed gas humidity is not only a DPBS specific observation the experiment was repeated in RPMI with a DMPO spin trap. The comparison of both measurements yielded that whether the sum of • OH and O •- 2 density is obtained with BMPO in DPBS or with DMPO in RPMI the trends of both curves are identical (data not shown). Even the absolute concentrations are in good agreement. The combined radical concentration of • OH and O •- 2 is only by a factor of less than two higher for the measurements of DMPO in RPMI. Thus, the results obtained in DPBS with the spin trap BMPO are transferable to the RPMI medium and are therefore relevant for the cell and H 2 O 2 measurements performed in RPMI. do not depend on the feed gas humidity and therefore do not correlate with the observed cell inhibition effect. As expected for the buffered cell growth medium no influence of pH value was detected. In the following it is discussed whether these findings can be generalized and what their impact for plasma medicine actually is.

Whenever H 2 O 2 is generated by plasma treatment in a liquid medium it will affect human skin cells (as was shown in this work) and other cell types within this medium (as described in the introduction). However, the implication that H 2 O 2 correlates in every case with an observed effect on cells and is therefore the only relevant agent in plasma medicine is not the correct conclusion. It was shown by Barton et al and Tresp et al with a similar plasma source that the viability of HaCaT cells, as well as of a monocyte cell line (THP-1), changes significantly when altering the nitrogen to oxygen surrounding gas ratio, while the H 2 O 2 concentration in the medium stayed almost unchanged [START_REF] Barton | Non-thermal atmospheric pressure plasma treatment of human cells: the effect of ambient conditions[END_REF][START_REF] Tresp | Effects of atmosphere composition and liquid type on plasma generated reactive species in biologically relevant solutions Plasma Med[END_REF]. Taking these results into account a more differentiated picture evolves. The strong role of H 2 O 2 concerning cell viability is observable especially when the plasma parameter variation leads to a severe change of H 2 O 2 concentration, as it is the case for feed gas humidity alteration. In other cases, where the resulting H 2 O 2 level is zero or invariant against parameter variation, other effects on top of the H 2 O 2 effect become visible. Hence, H 2 O 2 is a quite an important molecule in plasma medicine concerning cell activity but not the only one.

Conclusion

In this work, the pathway of hydrogen peroxide was followed from its generation in the gas phase by a humidified argon atmospheric pressure plasma jet into liquid cell culture medium. For this, FTIR and LIF spectroscopy were applied to measure the gas phase H 2 O 2 concentration and the relative

• OH radical density in dependence of the feed gas humidity level. By means of an adapted far field simulation the most important production and destruction mechanisms of H 2 O 2 were identified for the given experimental conditions. In the presented case, H 2 O 2 is mainly produced inside the core plasma zone by the dissociation of water molecules. The resulting • OH radicals recombine to form H 2 O 2 .

By comparing the H 2 O 2 net production rate in the gas and liquid phase a remarkable compliance was found. Moreover, by mimicking the H 2 O 2 generation of the plasma jet with the help of an H 2 O 2 bubbler it is concluded that the solubility of gas phase H 2 O 2 plays a major role in generating hydrogen peroxide in the liquid. This finding is explained by the high Henry's constant of H 2 O 2 compared to other present species.

The stability of this plasma generated and dissolved H 2 O 2 was measured in the cell growth medium to exceed half an hour without changing. After 40 h however, almost no H 2 O 2 was detected in the medium. Besides the physical and chemical focus on H 2 O 2 , its effect on the viability of adherent human skin cells was investigated. Pure plasma generated H 2 O 2 has the similar effect on the cell viability than indirect plasma treatment. This finding was ensured by analysing the pH-value and liquid-borne radical densities of • OH and O •- 2 by EPR spectroscopy. Neither of those agents showed a significant dependence on the feed gas humidity variation. Hence, no correlation with cell viability is expected for bulk concentrations of these agents. This leaves H 2 O 2 as the dominant species for our experimental conditions. Since experiments are known and discussed in this work where a variation of a biological marker does not correlate with H 2 O 2 it is concluded that H 2 O 2 is a quite important molecule in plasma medicine but not the only one.

Figure 1 .

 1 Figure 1. FTIR setup schematic for detecting H 2 O 2 in the plasma jet downstream using FTIR spectroscopy combined with a multipass cell. MFC: mass flow controller.

Figure 2 .

 2 Figure 2. Method to obtain the H 2 O 2 related inhibition time. The horizontal solid line depicts the H 2 O 2 concentration that inhibits 50% of adherent HaCaT cells. This value was determined experimentally. Square dots are the measurement values for plasma generated H 2 O 2 concentration in RPMI medium after different treatment times. The treatment time given by the interception point of both curves is the half inhibition treatment time, since the H 2 O 2 concentration in the medium reaches the concentration to inhibit 50% of HaCaT cells. The dashed lines represent the error intervals of both measurements. From these lines the lower and upper error of the half inhibition treatment time were deduced.

Figure 3 .

 3 Figure 3. FTIR spectrum in the far field of an atmospheric pressure argon plasma jet for a feed gas humidity of 1220 ppm measured in a multipass cell with an absorption length of 19.2 m and a pressure of 100 mbar. Absorption bands of ozone, hydrogen peroxide, nitrogen dioxide and carbon dioxide were detected. The fit was performed by using the Hitran database. For clarity every fifth measurement point is shown.

Figure 4 .

 4 Figure 4. Results of absorption experiments in the gas phase. (a) H 2 O 2 concentration in the multipass cell (far filed) and (b) relative LIF signal of the • OH radical in dependence on the feed gas humidity (measured in plasma effluent). The • OH LIF signal was fitted with a linear function whereas the dashed line in (a) is the simulation result.

Figure 5 .

 5 Figure 5. Gas phase H 2 O 2 concentration increases as a consequence of higher argon flow rate through an H 2 O 2 bubbler. No plasma is ignited in this experiment. As an inlay the FTIR transmission curves are given in the spectral range from 1200-1325 cm -1 for argon gas flow rates through the bubbler of 0.5, 1.0, 1.5 and 2.0 slm.

2 O 2

 22 into liquid phase H 2 O 2 can be generated in plasma treated liquids in different ways. Either it is created inside the liquid by plasma induced liquid chemistry processes or gas phase H 2 O 2 is dissolved in the liquid. In the first case plasma generated agents like energetic radiation or particles transfer energy from the gas to 6 This is the Accepted Manuscript version -post-print-of an article accepted for publication in Journal of Physics D: Applied Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the authors and IOP Publishing. This article may be found at doi:10.1088/0022-3727/47/28/285401. IOP Publishing is not responsible for any errors or omissions in this version of the manuscript or any version derived from it.

Figure 6 .

 6 Figure 6. Increase of H 2 O 2 concentration in 5 ml RPMI cell growth medium with plasma treatment time and for different feed gas humidity settings. The measurement was performed with colorimetric assays. The linear curves are results from linear regression.

Figure 7 .

 7 Figure 7. Net production rate of H 2 O 2 molecules in the gas and liquid phase for different feed gas humidity settings.

Figure 8 .

 8 Figure 8. Influence of gas phase H 2 O 2 on the H 2 O 2 concentration in liquid medium. The RPMI cell growth medium was treated (a) 80 s, (b) 60 s or (c) 40 s by the argon plasma jet (full symbols) and by a non-plasma condition, where an argon gas flow was enriched with H 2 O 2 molecules originating from a H 2 O 2 bubbler (line connected open symbols). The corresponding gas phase H 2 O 2 concentrations were obtained in the FTIR multipass cell (see figures 4(a) and 5) and do not represent the H 2 O 2 concentration above the RPMI solution.

Figure 9 .

 9 Figure 9. Stability of plasma produced H 2 O 2 in RPMI medium in dependence of the time after the plasma treatment has finished. Experimental settings were: (1) feed gas humidity: <20 ppm, treatment time: 40 s, (2) feed gas humidity: <20 ppm, treatment time: 180 s, (3) feed gas humidity: 1660 ppm, treatment time: 80 s.

2

 2

  This is the Accepted Manuscript version -post-print-of an article accepted for publication in Journal of Physics D: Applied Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the authors and IOP Publishing. This article may be found at doi:10.1088/0022-3727/47/28/285401. IOP Publishing is not responsible for any errors or omissions in this version of the manuscript or any version derived from it.

Figure 10 .

 10 Figure 10. Time to reach 50% cell inhibition for different feed gas humidity concentrations. The results of indirect plasma treatment (data taken from [17], treatment time of the medium: 40 s) and for the case that H 2 O 2 is the only agent agree remarkably well.

Figure 11 .

 11 Figure 11. pH-value measured in RPMI after 40 s plasma treatment time for different feed gas humidity settings. The squares are the measurements after plasma treatment. The dashed line depicts the pH-value for the untreated control.

Figure 12 .

 12 Figure 12. Radical concentrations of superoxide O •- 2 and hydroxyl • OH obtained by EPR spectroscopy in DPBS for different feed gas humidity concentrations. The plasma treatment time was 40 s.

2

 2

Table 1 .

 1 Henry's constants at standard conditions for inorganic species present in gas or liquid phase in the current study.

		Henry's constant
	Species	(mol l -1 Pa -1 )	Reference
	Oxygen (O 2 ) Argon (Ar)	1.2 × 10 -8 1.4 × 10 -8	[49] [50]
	6.5 × 10 -8 1.1 × 10 -7 4.0 × 10 -7 2.9 × 10 -4 2 ) 4.0 × 10 -2 Hydroperoxyl radical (HO • Nitrogen (N 2 ) Ozone (O 3 ) Nitrogen dioxide (NO 2 ) Hydroxyl radical ( • OH) Hydrogen peroxide (H 2 O 2 ) 8.3 × 10 -1	[51] [52] [53] [54] [55] [56]

Acknowledgments

This work is funded by German Federal Ministry of Education and Research (grant# 03Z2DN11 & 03Z2DN12). The authors thankfully acknowledge the valuable work of Liane Kantz, Sebastian Schlegel, Mattis Hänel, Sven Bordewick and Johannes von Saß.

This is the Accepted Manuscript version -post-print-of an article accepted for publication in Journal of Physics D: Applied Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the authors and IOP Publishing. This article may be found at doi:10.1088/0022-3727/47/28/285401. IOP Publishing is not responsible for any errors or omissions in this version of the manuscript or any version derived from it. This is the Accepted Manuscript version -post-print-of an article accepted for publication in Journal of Physics D: Applied Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the authors and IOP Publishing. This article may be found at doi:10.1088/0022-3727/47/28/285401. IOP Publishing is not responsible for any errors or omissions in this version of the manuscript or any version derived from it.