Joern Winter
email: winter@inp-greifswald.de

Kristian Wende
email: kristian.wende@inp-greifswald.de

Kai Masur

Sylvain Iséni

Mario Dünnbier

Malte U Hammer

Helena Tresp

Klaus-Dieter Weltmann

Stephan Reuter

S Iseni

K-D Weltmann

Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The past decade has seen the rise of atmospheric-pressure lowtemperature plasmas. These plasmas operated under ambient conditions find a wide range of applications, e.g. in surface treatment of polymers [START_REF] Sarra-Bournet | A study of atmospheric pressure plasma discharges for surface functionalization of PTFE used in biomedical applications[END_REF][START_REF] Bhoj | Plasma-polymer interactions in a dielectric barrier discharge[END_REF][START_REF] Fricke | Investigation of surface etching of poly(ether ether ketone) by atmospheric-pressure plasmas[END_REF], treatment of woven fabrics [START_REF] Rombola | On-line atmospheric pressure plasma (APP) treatment of polypropylene fabrics Czech[END_REF] and surface functionalization [START_REF] Yonson | Cell treatment and surface functionalization using a miniature atmospheric pressure glow discharge plasma torch[END_REF], and also as actuators for the control of air flow in aeronautics [START_REF] Moreau | Air flow control by non-thermal plasma actuators[END_REF]. Atmospheric-pressure plasmas are increasingly used for deposition of thin films [START_REF] Paulussen | Physical and chemical properties of hybrid barrier coatings obtained in an atmospheric pressure dielectric barrier discharge[END_REF] or generation of carbon nanotubes [START_REF] Kucerova | Carbon nanotubes synthesized by plasma enhanced CVD: preparation for measurements of their electrical properties Czech[END_REF]. Advantages are that large and expensive vacuum equipment can be omitted and high reactive species density can be achieved due to the high density 3 These authors contributed equally to this work. under ambient conditions. High focus is set on application of atmospheric-pressure plasmas in the medical sector. In this sector, a large scientific community has evolved investigating applications of wound treatment, biocompatible surfaces, or disinfection, inactivation of pathogens, sterilization of medical equipment as well as the inactivation of bacteria in biofilms, see e.g. [START_REF] Ehlbeck | Low temperature atmospheric pressure plasma sources for microbial decontamination[END_REF][START_REF] Kong | Plasma medicine: an introductory review New[END_REF][START_REF] Weltmann | Plasma processes and plasma sources in medicine Contrib[END_REF][START_REF] Marchal | Inactivation of Gram-positive biofilms by low-temperature plasma jet at atmospheric pressure[END_REF]. The high reactivity at low gas temperatures due to the strong non-equilibrium properties of these plasmas can be utilized to generate a reactive chemistry, usually available only at high temperatures, on temperature-sensitive surfaces such as human skin.

In particular in the medical sector, plasma sources are operated in ambient air, introducing humidity into the active plasma zone. Humidity plays an important role in plasma chemical processes since dissociation of water (H 2 O) opens a wide variety of subsequent plasma chemical reactions [START_REF] Fridman | Plasma Chemistry[END_REF]. This is especially true for atmosphericpressure plasmas operated in humid ambient air but also for noble gas driven atmospheric-pressure plasma jets [START_REF] Sakiyama | Plasma chemistry model of surface microdischarge in humid air and dynamics of reactive neutral species[END_REF][START_REF] Murakami | Chemical kinetics and reactive species in atmospheric pressure helium-oxygen plasmas with humid-air impurities Plasma Sources[END_REF][START_REF] Liu | Global model of low-temperature atmospheric-pressure He + H 2 O plasmas Plasma Sources[END_REF]. In the case of plasma jets, humidity is either introduced directly into the working gas as impurity or diffuses into the effluent when the jet is operated in a humid environment. In biomedical applications, changing humidity conditions not only has an influence on the plasma but also on the plasmatreated biological targets, such as cells or cell structures [START_REF] Kirkpatrick | Atmospheric pressure humid argon DBD plasma for the application of sterilization-measurement and simulation of hydrogen, oxygen, and hydrogen peroxide formation[END_REF]. The products originating from the water reaction pathways, for example hydroxyl (OH), atomic oxygen (O) and hydrogen peroxide (H 2 O 2 ) are potent to generate oxidative stress in biological samples. This is already known from decontamination experiments, where bacteria were plasmatreated under different humidity settings [START_REF] Hähnel | Influence of the air humidity on the reduction of bacillus spores in a defined environment at atmospheric pressure using a dielectric barrier surface discharge Plasma Process[END_REF]. For eukaryotic cells relevant for plasma medicine (e.g. human skin cells) no such data exist. Hence, the focus of this work is not only the investigation of feed gas humidity influence on the reactive component generation of an atmosphericpressure argon plasma jet by optical emission and absorption spectroscopy but also the observation of in vitro human keratinocyte behaviour to indirect plasma treatment. It is shown in this work that increasing feed gas humidity leads to a significant viability decrease in the investigated cell line. In order to get a first glimpse into the governing mechanisms, the H 2 O 2 concentration in the liquid growth medium is also monitored. The increase in H 2 O 2 concentration with feed gas humidity suggests a correlation between H 2 O 2 and cell viability.

In addition to the fundamental research aspects, it is shown that gas supply tubes made of polyurethane (PUR) are a major source of feed gas humidity and must be considered from the practical point of view. Moreover, the large relevance of feed gas humidity with respect to ambient humidity is emphasized in this paper.

Experimental setup

Plasma jet

The investigated plasma source is a cold atmospheric-pressure plasma jet (kinpen, neoplas GmbH, Greifswald, Germany). 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]. When a sinusoidal voltage signal of 2-6 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. Also an up to 10 mm long effluent becomes visible after ignition. The working gas is argon (Ar purity 99.999%) with a gas flow rate of 3 standard litres per minute (slm). Furthermore, the humidity concentration of the feed gas is changed with the setup described in detail in section 2.3.

Humidity measurements in gas supply tubes

Feed gas tubes made of PUR can absorb a significant amount of water when they are exposed to a humid environment. In operation, dry gas from the gas bottle becomes humidified while streaming through such previously humidified feed gas tubes. Hence, a significant number of water molecules are transported by the feed gas into the core plasma region of the investigated plasma jet. In order to determine the number of water molecules that originate from feed gas tubes the experimental setup in figure 1 was used. A 3 m long PUR tube (Norgren, Germany) with inner and outer diameters of 4 mm and 6 mm, respectively, was enclosed by a plastic box. The air atmosphere inside the box was set to a relative humidity level of 50% (corresponds to an absolute H 2 O concentration of 1.4%) by means of a humidified constant air flow rate of 1 slm. The measurement procedure had three phases. In the first phase dry argon gas at a flow rate of 1 slm was led through the 3 m tube for 12 h. Therefore, valves B were opened and valve A was closed. After this drying phase the second phase began in which valves B were closed and valve A was opened. Now, the dried argon filled tube was exposed to the humidified atmosphere in the box for another 12 h. Meanwhile the argon gas kept flowing through valve A. This provides unchanged dry conditions for the supply tubes. In the third phase, valves B were opened and valve A was closed again, so that dry argon gas flew through the humidified tube. The gas humidity was measured over time with a chilled mirror dew point hygrometer (DewMaster, EdgeTech, USA) with an uncertainty of about 2%. The humidity concentration detection limit of this hygrometer was 20 parts per million (ppm). For comparison, a 3 m long stainless steel pipe with the same dimensions as the PUR tube was analysed with the same procedure.

Feed gas humidification and optical emission spectroscopy

To investigate the effect feed gas humidity has on emission spectra and on biological samples the humidity of the feed gas was controlled.

Due to the difficulty in adjusting humidification by gas tubes, a controlled feed gas humidification was performed with a bubbler setup. Dry argon gas was split into two channels, a main gas channel and a channel which was guided through a water reservoir. Behind the bubbler, both argon gas streams were mixed and the resulting humidity was analysed by the chilled mirror dew point hygrometer already described in section 2.2. Afterwards the humidified feed gas was led into the plasma jet. By changing the argon flow rate through the bubbler in the range 0-0.5 slm the feed gas humidity was precisely adjusted between 20 and 1300 ppm. To provide a constant total argon flow rate of 3 slm the dry gas channel flow rate was appropriately reduced.

A sketch of the humidification setup in combination with the optical emission spectroscopy (OES) setup is displayed in figure 2. The OES was performed using a UV and a NIR sensitive spectrometer (AvaSpec-2048-USB2, Avantes) with a spectral resolution of 0.7 nm, respectively. The axially resolved emission spectra with a spatial resolution of 0.5 mm were obtained for different humidity concentrations. Particularly four spectral regions were evaluated in this work corresponding to the emission of argon atoms (Ar 4p-4s, 695-698 nm), hydroxyl radicals (OH A 2 + → X 2 , 305-313 nm), nitrogen molecules (N 2 C 3 u → B 3 g , 330-338 nm) and atomic oxygen (O 3p-3s, 775-778 nm). Integrating the respective emission intensities for different axial positions (0-10 mm, step size: 0.5 mm) and different feed gas humidity concentrations over the given spectral ranges and normalizing to the maximal value yields an emission intensity map for each species.

Furthermore, the plasma jet was equipped with a shielding device that enables the shielding of the effluent plasma from ambient species [START_REF] Reuter | Controlling the ambient air affected reactive species composition in the effluent of an argon plasma jet[END_REF]. In order to investigate whether feed gas humidity differentially influences the plasma composition than ambient humidity two experiments were performed using this shielded jet. Firstly, dry air as shielding gas and humidified argon with a humidity concentration of 490 ppm as feed gas were used. In this situation, the humidity concentration in the effluent is only determined by the feed gas humidity. Secondly, the opposite situation, namely humidified shielding gas with a humidity concentration of 20 500 ppm (corresponds to a relative humidity level of 72%) and dry feed gas, was investigated. As a measure for the impact of both situations the excited OH emission was analysed with the setup explained above.

Ozone net production rate

The ozone net production rate of the investigated plasma jet operated with 3 slm argon plus 1% oxygen was determined for different feed gas humidity concentrations using UV and IR absorption spectroscopy techniques identical to the setups reported in [START_REF] Reuter | Detection of ozone in a MHz argon plasma bullet jet Plasma Sources[END_REF][START_REF] Winter | Aspects of UV-absorption spectroscopy on ozone in effluents of plasma jets operated in air[END_REF]. For a comparison of both techniques, the measurement setup-independent net production rate was calculated, considering the velocity profile of the gas for UV absorption and the total flow rate for IR absorption. Details of the production rate calculation are also given in the two papers already mentioned above. The humidification of the feed gas was performed with the bubbler setup described in section 2.3.

Plasma treatment and hydrogen peroxide determination in RPMI solution

The plasma treatment was performed in a polystyrene Petri dish (53 mm diameter TPP, Trasdingen, CH) filled with 5 ml complete cell growth medium (Roswell Park Memorial Institute 1640-RPMI + 8% foetal calf serum +1% penicillin/streptomycin solution). In order to treat the liquid at different positions the plasma jet was mounted on an xy-table, and the plasma jet effluent was moved in a meandering pattern over the liquid for a constant treatment time of 40 s. The distance between the nozzle and the liquid surface was set to 9 mm. The H 2 O 2 concentration of the treated liquid was analysed using commercial H 2 O 2 test stripes (Merckoquant 110011, Merck) and via an Amplex Red homogeneous assay. Aliquots of 50 µl (Amplex Red assay) and 30 µl (test stripes) were taken immediately after 40 s plasma treatment time. The experiment was repeated three times for each humidity setting.

The H 2 O 2 test stripes were read out by a digital microscope camera (Conrad, Germany) in order to analyse their colour value. A calibration procedure was conducted prior to the experiments. Therefore, 13 different H 2 O 2 concentrations from 0 to 6 mg l Another method to detect H 2 O 2 in liquid is the usage of a homogeneous assay (Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, Invitrogen, Ltd). The kit uses the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine). After 40 s plasma treatment time 50 µl of the treated solution were transferred into a well of a 96-well plate. The well already contains a volume of 50 µl reaction buffer solution according to the protocol provided by the company. Furthermore, a standard curve with known H 2 O 2 concentrations was also prepared in the 96-well plate. The absorption and fluorescence signals at wavelengths of 560 nm and 590 nm, respectively, were obtained by means of a multiplate reader (Tecan infinite M200, Tecan Group Ltd, Switzerland). By comparing the results of the standard curve with the samples taken at different feed gas humidity levels the plasma treatment induced H 2 O 2 concentrations after 40 s treatment time were determined. By comparison with the test stripe method the Amplex Red assay is more sophisticated but also more sensitive. The H 2 O 2 test stripe lower detection limit is about 0.5 mg l -1 , whereas a significant lower detection limit of 0.2 ng l -1 is reported for the Amplex Red assay [START_REF] Zhu | A sensitive fluorimetric assay for pyruvate Anal[END_REF].

Cell viability assay

The viability of adherent human keratinocytes (HaCaT cells) was determined using the conversion of fluorescence dye resazurin by metabolic active cells into the highly fluorescent resorufin. Details of this assay are given in [START_REF] Reuter | From RONS to ROS: tailoring plasma jet treatment of skin cells[END_REF]. The cells were cultivated in the same medium that was used for the H 2 O 2 measurements.

After 40 s plasma treatment of 5 ml RPMI solution under the equivalent conditions as described for the H 2 O 2 measurements, the plasma-treated medium was immediately transferred into one row of wells of the cell culture plate and serially diluted with complete medium immediately (six parallel wells per treatment time) to obtain different treatment time equivalents. Analogous to the procedure described in [START_REF] Reuter | From RONS to ROS: tailoring plasma jet treatment of skin cells[END_REF] the cells were cultivated for a further 71 h and fluorescence intensity after resazurin addition (100 µM resazurin in complete RPMI w/o phenol red) was analysed by a Tecan M200 multiplate reader after 1 h of incubation. As a measure of viability the 50% inhibition value was determined for every investigated feed gas humidity level (example for 20 ppm see figure 4).

Results and discussion

Humidity originating from feed gas tubes

The humidity concentration in dependence on the gas tube flushing time is displayed in figure 5 for the two investigated materials, namely PUR and stainless steel. For the PUR tube an initial humidity concentration of more than 1000 ppm depicts a high porosity and storage capacity of this material for water molecules. With increasing flushing time a drying of the inner tube surface occurs. However, it takes more than 3 h of flushing time to decrease the feed gas humidity concentration of a 3 m long PUR tube under a level of 70 ppm, which from a practical point of view can be considered as dry. In contrast, the initial feed gas humidity concentration for the investigated metal tube of the same length is by one order of magnitude lower than for the polymeric tube. Already after a couple of minutes the humidity concentration is significantly lower than 70 ppm. A consequence of these findings is that not only dry but also stable humidity conditions in the feed gas can be easily achieved with metal tubes. However, metal tubes are expensive and by far not as practical as polymeric tubes. Thus, in the majority of cases, the usage of polymeric tubes can possibly not be avoided totally. In experiments that require stable humidity concentrations it is therefore important to consider polymeric feed gas tubes as a significant source of humidity. From a practical point of view the length of these tubes should be limited as far as possible in order to reduce the drying phase before the experiments.

Optical emission spectroscopy

The emission spectrum of the jet effluent at an axial position of 4 mm and for a feed gas humidity concentration of 350 ppm shows several emission bands and atomic lines in the ultraviolet (figure 6) and near infrared spectral range (figure 7). In the UV spectral range, emission bands of OH (A 2 + -X 2 ) and N 2 (C 3 u -B 3 g ) originate from the dissociation of feed gas water molecules and the diffusion of ambient nitrogen into the effluent [START_REF] Reuter | Controlling the ambient air affected reactive species composition in the effluent of an argon plasma jet[END_REF][START_REF] Reuter | From RONS to ROS: tailoring plasma jet treatment of skin cells[END_REF].

In the NIR spectral range, a set of atomic argon lines Ar(4p-4s) and the triplet of atomic oxygen lines O(3p 5 P-3s 5 S) around 777 nm were measured. The atomic oxygen can either originate from water dissociation alongside with the formation of hydrogen molecules or be generated by dissociation of ambient molecular oxygen that diffuses into the effluent. Focusing on the effluent no lines of atomic hydrogen were found. However, end-on OES measurements (not shown here) reveal the presence of atomic hydrogen (H α and H β lines) in the active plasma zone and thus confirm water dissociation at least in that region.

Since humidity concentration variation changes not only the plasma chemistry but also the excitation conditions, a strong influence on the emission intensity of the investigated species is expected. Furthermore, the density of molecular oxygen and nitrogen varies with axial distance whereby a spatial dependence on the emission intensity also occurs. Hence, the integral emission intensity was evaluated for both parameters. The result is presented in figure 8 for the four discussed species Ar, OH, N 2 and O. Every emission intensity map is normalized to the maximum value, respectively. The dots in the contour plot give the parameter settings at which the measurements were actually conducted. The intermediate values were calculated by 2D linear interpolation.

It has to be mentioned that careful interpretation is necessary when evaluating OES data.

Since emission intensities in figure 8 are integrated over band structures (OH, N 2 ) and over atomic lines (Ar, O) their resulting intensities cannot be directly compared.

However, by evaluating the respective dependences on the axial position and on the humidity concentration valuable information is obtained despite the lack of quantitative comparability.

With increasing humidity concentration in the feed gas the emission of argon and oxygen lines and the band emission of nitrogen decrease. Two different effects are responsible for this finding. Firstly, with increasing humidity concentration in the feed gas quenching of excited states by water molecules becomes more likely [START_REF] Bruggeman | Electronic quenching of OH(A) by water in atmospheric pressure plasmas and its influence on the gas temperature determination by OH(A-X) emission[END_REF]. This is illustrated by the quenching rate coefficients and decay rates given in table 1. For a humidity concentration of 1000 ppm (corresponding to a H 2 O density of 2.47 × 10 16 cm -3 at atmospheric pressure), the decay rate of N 2 (C 3 u , ν = 0) due to quenching by water molecules is in the same order of magnitude as the radiative decay rate and therefore plays an important role for depopulating the excited nitrogen state. Unfortunately, no data on the quenching rate coefficients by water vapour were found in the literature for the O(3p 5 P) and Ar(2p 2 ) states. However, assuming that the unknown coefficients are roughly comparable to the known quenching rate coefficients of O(3p 3 P) and Ar(1s 5 ) water molecules also play a major role in the depopulation of these excited atomic states.

Secondly, it is known that the admixture of molecular gases leads to a reduction in electron temperature [START_REF] Nikiforov | The influence of water vapor content on electrical and spectral properties of an atmospheric pressure plasma jet Plasma Sources[END_REF] and therefore affects the formation of photons and energetic species such as argon metastables Ar (1s 5 ) in the active plasma zone. These photons and also the metastable argon atoms with energies of 11.55 eV and a lifetime on the order of ten microseconds transport energy from the active plasma zone into the effluent and induce dissociation and excitation processes of other species [START_REF] Sands | Spatiotemporally resolved Ar (1s(5)) metastable measurements in a streamer-like He/Ar atmospheric pressure plasma jet[END_REF][START_REF] Bussiahn | Spatially and temporally resolved measurements of argon metastable atoms in the effluent of a cold atmospheric pressure plasma jet[END_REF]. If, for instance, the formation of Ar metastables is impeded by water molecules, Ar metastable induced excitation processes in the effluent are also impeded and the emission intensity deceases. Hence, the emission intensity of excited Ar, O and N 2 decreases with water vapour admixture.

The emission of OH (A 2 + , ν = 0) behaves differently from that of the other species. For small water admixtures an increase in OH emission is observed up to a humidity concentration level of about 400 ppm. This is similar to the results of Nikiforov et al on a DBD argon jet, who found an emission maximum of OH (A 2 + , ν = 0) at a water vapour concentration of 350 ppm. Presumably, the OH density increases with rising H 2 O admixture due to direct dissociation of water molecules by electrons. This effect would lead to a stronger emission signal provided that excitation mechanisms do not change significantly by small H 2 O admixtures. However, when increasing the humidity concentration beyond 400 ppm quenching of excited OH (see table 1) and the reduced electron temperature become relevant. Both counter the emission gain by increased OH density and a maximum evolves. Of course, no information on OH densities is given in this paper and therefore conclusions concerning OH densities are speculative. However, a rising OH density is a reasonable explanation for the formation of the observed maximum. Otherwise, it would mean that for increasing feed gas humidity up to 400 ppm more OH molecules become excited than for the dry condition despite de-excitation losses by increased quenching and reduced electron temperature.

Interesting results were also found by analysing the axial dependence of the emission signals. Except for nitrogen, the highest emission intensity was always found close to the nozzle. From there on, the intensity decreases towards longer nozzle distances. Nitrogen behaves differently because no significant nitrogen density is present close to the nozzle. Hence, no nitrogen excitation can occur in this region. With a longer nozzle distance, the diffusion of ambient nitrogen into the effluent drastically increases [START_REF] Reuter | Controlling the ambient air affected reactive species composition in the effluent of an argon plasma jet[END_REF][START_REF] Reuter | From RONS to ROS: tailoring plasma jet treatment of skin cells[END_REF] and a stronger emission intensity is obtained. However, since the excitation of nitrogen becomes less likely with increasing distance from the nozzle, an emission maximum is reached at an axial position of about 5.5 mm. For larger distances the emission signal decreases monotonically despite a still increasing nitrogen density.

Analogous to ambient nitrogen ambient oxygen also diffuses into the effluent and is potentially dissociated by 6 2 .39 ± 0.08 [START_REF] Morozov | Gas kinetic studies using a table-top set-up with electron beam excitation: quenching of molecular nitrogen emission by water vapour[END_REF] 1.75 ± 0.17 2.63 [28] OH (A 2 + , ν = 0) 4.25 [START_REF] Fallon | Potential energy curves for OH[END_REF] 5.66 ± 0.1 [START_REF] Mcdermid | Radiative lifetimes and quenching rate coefficients for directly excited rotational levels of OH (A 2 + , ν = 0)[END_REF] 0.139 [START_REF] Mcdermid | Radiative lifetimes and quenching rate coefficients for directly excited rotational levels of OH (A 2 + , ν = 0)[END_REF] 1 .40 ± 0.02 O(3p 3 P) 10.99 [START_REF] Kelleher | The new NIST Atomic Spectra Database[END_REF] 49± 3 [START_REF] Meier | H-atom and O-atom detection for combustion applications-study of quenching and laser photolysis effects[END_REF] 2 .69 ± 0.24 [START_REF] Meier | H-atom and O-atom detection for combustion applications-study of quenching and laser photolysis effects[END_REF] 12.1 ± 0.74 9.4 ± 1.5 [START_REF] Quickenden T I, Trotman | H 2 O quenching of the transitions H(n = 3 → n = 2) and O(3p 3 P → 3s 3 S 0 ) in pulse radiolytically excited water-vapor[END_REF] 2 .32 ± 0.37 O(3p 5 P) 10.74 [START_REF] Kelleher | The new NIST Atomic Spectra Database[END_REF] radiation or high energetic particles. However, the highest emission intensity is found in front of the jet nozzle. This indicates that, in addition to the atomic oxygen production in the jet effluent, atomic oxygen is also produced inside the active plasma zone. Analogous to the discussion of feed gas humidity originating from the gas supply tubes (see section 3.1), it is reasonable to assume that even for the dry condition, water molecules are still present in the feed gas in a concentration between 0 ppm and 20 ppm (the detection limit of hygrometer). Hence, atomic oxygen is generated from water dissociation in the active plasma zone and becomes excited by electron collisions. This explains the observed emission signal of atomic oxygen in front of the jet nozzle. OES was also used to indicate whether feed gas humidity has a larger effect on the OH emission than ambient air humidity. Figure 9 shows the integrated emission intensity of OH for the two investigated conditions. When dry feed gas and humidified shielding gas are used the axial intensity increases with distance to the nozzle. Similar to the discussion for nitrogen, the OH density continuously increases with distance. Since excitation is limited in the axial direction the emission profile forms a maximum. Identical to nitrogen emission, the highest emission signal was found at a distance of 5.5 mm. In contrast, when humidified feed gas and dry shielding gas are used the emission signal decreases steadily in the axial direction and no maximum evolves. Particularly interesting is the fact that although the feed gas humidity concentration in this case is by a factor of 42 lower compared with the humidity concentration in the wet shielding gas, a higher OH emission signal is detected (for z < 5 mm).

In conclusion, ambient air humidity influences the chemical and physical processes in the plasma effluent and must be considered especially for applications that require stable plasma conditions. In fact, much more important than controlling the ambient humidity is to consider the feed gas humidity since it affects the plasma substantially.

Ozone net production rate depends on feed gas humidity

In addition to other reactive species such as atomic oxygen or hydroxyl radicals, ozone (O 3 ) also plays a key role in plasma chemical processes. Under a condition where molecular oxygen density is significantly higher than the atomic oxygen density, which is usually the case for the effluent region of the plasma jet, ozone can be interpreted as an indicator for atomic oxygen since it is mainly produced by the following reaction:

O 2 + O + M → O 3 + M, ( 1
)
where M is an arbitrary collision partner, e.g. argon. Since the emission of excited atomic oxygen is linked to the humidity concentration in the feed gas it is reasonable to assume that the production of atomic oxygen and therefore the production of O 3 are also affected by feed gas humidity changes. The O 3 measurement result of the two applied absorption spectroscopic techniques (UV and IR laser absorption spectroscopy) is presented in figure 10. In order to compare both methods the ozone net production rate of the jet was calculated and plotted in this figure. Despite the different techniques remarkable agreement between both methods is ascertained. Generally, the highest O 3 net production rate of (3 . . . 4) × 10 17 s -1 is found for the driest condition. With increasing humidity concentration up to 200 ppm a steep decrease in ozone net production rate is obtained. For humidity concentrations larger than 200 ppm a decrease in ozone net production is still observed but is not as strong as for lower humidity concentrations. For more than 800 ppm the O 3 net production rate drops below 1 × 10 The decrease in ozone net production with increasing feed gas humidity means that a higher humidity level either inhibits the production and/or favours the destruction of ozone. Without a plasma chemical model or an O atom density measurement it is difficult to disentangle both mechanisms. However, since ozone is mainly formed by one single reaction (equation ( 1)), atomic oxygen acts as a bottleneck for the ozone production. If the atomic oxygen density is affected by the feed gas humidity, an increase in feed gas humidity will consequently influence the O 3 formation as well. A possible mechanism for the reduction of excited atomic oxygen density by water molecules is given by the following reactions [START_REF] Herron | Chemical kinetics database and predictive schemes for nonthermal humid air plasma chemistry: II. Neutral species reactions Plasma[END_REF]:

O( 1 D) + H 2 O → OH + OH, (2)
O( 1 S) + H 2 O → OH + OH. (3)
Both reduce the amount of atomic oxygen available for reaction [START_REF] Sarra-Bournet | A study of atmospheric pressure plasma discharges for surface functionalization of PTFE used in biomedical applications[END_REF]. This results in a lower ozone production with increasing water molecule admixture. However, not only a lower atomic oxygen concentration but also different humidity-depending destruction mechanisms reduce the ozone concentration and lead to the observed ozone behaviour of figure 10. One candidate is the reaction of ozone with OH in a cyclic process [START_REF] Skalny | Ozone generation in positive and negative corona discharge fed by humid oxygen and carbon dioxide[END_REF]:

O 3 + OH → HO 2 + O 2 (4)
O 3 + HO 2 → 2O 2 + OH, (5)
where a single OH molecule is able to destroy more than one ozone molecule. According to the discussion in section 3.2, the OH density most likely increases with humidity in order to explain the observed maximum in the OH emission signal. Hence, the ozone destruction cycle of equations ( 4) and ( 5) becomes prominent when the water concentration in the feed gas rises. Furthermore, reactions with hydrogen also lead to ozone decomposition [START_REF] Skalny | Ozone generation in positive and negative corona discharge fed by humid oxygen and carbon dioxide[END_REF]: Since hydrogen can only originate from water dissociation an increase in feed gas humidity will favour the generation of atomic hydrogen, which in turn will contribute to the ozone decomposition.

O 3 + H → O 2 + OH. ( 6
)

H 2 O 2 concentration in RPMI increases with feed gas humidity

The increase in feed gas humidity not only changes the optical emission spectrum and the ozone production of the jet but also affects the plasma-treated liquid. In figure 11 for the highest humidity setting and 2.0 × 10 -4 g h -1 for the driest feed gas condition are obtained. A similar value of 3 × 10 -4 g h -1 was obtained by Ikawa et al using a plasma jet driven in dry helium [START_REF] Ikawa | Effects of pH on bacterial inactivation in aqueous solutions due to low-temperature atmospheric pressure plasma application Plasma Process[END_REF]. Different mechanisms are known on how hydrogen peroxide is generated in liquids [START_REF] Locke | Review of the methods to form hydrogen peroxide in electrical discharge plasma with liquid water Plasma Sources[END_REF][START_REF] Oehmigen | Estimation of possible mechanisms of Escherichia coli inactivation by plasma treated sodium chloride solution Plasma Process[END_REF]. A basic pathway is the recombination of hydroxyl radicals in the liquid. These OH molecules are either generated directly inside the liquid by radiation, excited species or electrons from the plasma or are primarily produced in the plasma phase and recombine afterwards on the liquid surface to H 2 O 2 . Another possibility is that H 2 O 2 is produced in the gas phase and simply dissolved in the liquid. In that case the H 2 O 2 concentration in the gas phase must increase with increasing humidity level. Support for this theory is found in the model results of Liu et al where an increase in H 2 O 2 with humidity is predicted [START_REF] Liu | Global model of low-temperature atmospheric-pressure He + H 2 O plasmas Plasma Sources[END_REF]. However, this model is computed for helium, whereas in this work an argon plasma jet was used. Measurements of H 2 O 2 in the gas phase are necessary and planned for future work in order to identify a correlation between H 2 O 2 in the gas and liquid phases.

Cell viability after indirect plasma treatment is impeded with higher humidity

In figure 12, the influence of different feed gas humidity settings on the viability of human skin cells is presented. Cell viability detection utilized in this study relies on a cellular supply of reduction equivalents to reduce a tracer compound (resazurin, containing an N-oxide) enzymatically yielding a highly fluorescent reporter dye (resofurin). Hence, cells need to be both living and metabolically active to be detected, which allows the conclusion on their actual viability rather than their number only. Cell viability and cell division rates strongly depend on the general condition of the cells, which will be influenced by many factors, such as nutrition, availability of oxygen, and also on harming influences such as oxidative stress. Under this condition, cells face imbalance between reactive oxygen species (ROS) concentration and their ability to detoxify the reactive intermediates or to repair the resulting damage. Some ROS or their degradation products are known to be highly reactive with biomolecules such as proteins, lipids and DNA. In addition to a direct random modification of these molecules by covalent implementation of functional groups or altering their redox status, indirect effects can be transmitted via cellular redox sensors such as the different peroxiredoxins [START_REF] Cox | Mitochondrial peroxiredoxin involvement in antioxidant defence and redox signalling[END_REF]. In turn, these molecules activate cellular antioxidant defence mechanisms, which allow the cells to withstand a certain ROS excess. From figure 12 it is evident that the plasma generated by the feed gas with a content of humidity affects the cell viability even more than the plasma created from the dry feed gas. Considering the findings of the H 2 O 2 concentration measurements of section 3.4 a correlation between cell viability and H 2 O 2 concentration becomes obvious. This means when the H 2 O 2 concentration is low, the cell viability is high. With increasing H 2 O 2 concentration a decrease in cell viability is also observed. In conclusion, by applying the plasma source in our eukaryotic cell model the cells were exposed to ROS and, hence, to oxidative stress, which exceed the cells' capacity for detoxification and repair. Although H 2 O 2 plays an important role in the oxidative stress that affects the cell viability, other reactive species such as OH or O - 2 might also be involved in the observed effects. Cell viability could be influenced by mechanical stress or electric fields derived from the plasma source [START_REF] Legazpi | Kinetic analysis of hybridoma cells viability under mechanical shear stress with and without serum protection Bioprocess[END_REF][START_REF] Yano | Enhancement of proliferation activity of mammalian cells by intense burst sinusoidal electric fields[END_REF]. Since indirect plasma treatment was applied, these and other stress factors such as UV radiation were excluded for our experimental conditions.

Conclusion

In this paper, the significant influence of feed gas humidity introduced into an atmospheric-pressure argon plasma jet is demonstrated on the axially resolved optical emission spectrum of the jet effluent, the generation of hydrogen peroxide in a plasma-treated cell growth medium and on human skin cells charged with this treated medium. Optical emission spectroscopy reveals a strong dependence of present excited plasma species such as hydroxyl radicals, molecular nitrogen, argon and atomic oxygen on the feed gas humidity. Furthermore, it was demonstrated by this spectroscopic method that small feed gas humidity concentrations (below 1000 ppm) have a much larger effect on OH emission than high ambient humidity concentrations. From this finding it is concluded that whenever humid feed gas is applied, ambient humidity can be neglected for processes inside the effluent region. However, when the feed gas is dry, ambient humidity becomes important and must be considered in such a situation. The question whether used feed gas is dry or humid is complicated by the fact that polymeric supply tubes have a major contribution to the feed gas humidity. It is shown that a vast amount of water vapour diffuses through PUR tube walls and hence increases the feed gas humidity. Compared with metal tubes a ten times higher concentration was found for the polymeric tubes. Depending on the length of the feed gas tubes, a constantly changing feed gas humidity prevents stable plasma conditions for up to several hours. Furthermore, plasma treatment of the liquid cell growth medium with increasing feed gas humidity leads to an almost linear increase in H 2 O 2 concentration in the liquid. When human skin cells get challenged with this medium a strong correlation between cell viability and H 2 O 2 concentration is revealed, indicative of an oxidative stress response.

Figure 1 .

 1 Figure 1. Experimental setup for measuring the water concentration that originates from a previously humidified tube.

Figure 2 .

 2 Figure 2. Scheme of the optical emission spectroscopy setup and of gas humidification.

  -1 were produced by diluting 10M H 2 O 2 stock solution (340 g l -1 , Merck KGAA) in RPMI and H 2 O. The sum of the colour values of the red and green camera channel for different H 2 O 2 concentrations is plotted in figure 3. Regardless of whether the calibration was performed in RPMI or H 2 O almost the same linear calibration characteristic was observed. The linear approximation of the data allows the determination of an unknown H 2 O 2 concentration by measuring the colour change of a test stripe.

Figure 3 .

 3 Figure 3. H 2 O 2 test stripe calibration curve.

Figure 4 .

 4 Figure 4. Cell viability in dependence on the treatment time equivalent for a feed gas humidity level of below 20 ppm. The 50% value was determined via sigmoidal regression (here 41.7 s treatment time equivalent).

Figure 5 .

 5 Figure 5. Humidity concentration in argon feed gas (flow rate: 1 slm) in dependence on the flushing time through a 3 m long tube made of either PUR or stainless steel.

Figure 6 .

 6 Figure 6. UV emission spectrum at an axial position of 4 mm and for a feed gas humidity concentration of 350 ppm.

Figure 7 .

 7 Figure 7. NIR emission spectrum at an axial position of 4 mm and for a feed gas humidity concentration of 350 ppm.

Figure 8 .

 8 Figure 8. Integrated emission signals originating from Ar, OH, N 2 and O in dependence on the axial distance and feed gas humidity concentration. The dots in the contour plot give the position and humidity concentration where the measurements were conducted. The intermediate values were obtained by linear interpolation. Due to different integration intervals (wavelength integration ranges are given in parentheses below the species label), intensities of different species cannot be directly compared.

  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:10.1088/0022-3727/46/29/295401. IOP Publishing is not responsible for any errors or omissions in this version of the manuscript or any version derived from it.

Figure 9 .

 9 Figure 9. Comparison of the axial OH emission profiles for two conditions: firstly, dry shielding gas and humidified feed gas; secondly, humidified shielding gas and dry feed gas.

Figure 10 .

 10 Figure 10. Ozone net production rate in dependence on the feed gas humidity concentration. The measurements were made with UV and IR absorption techniques. humidity increase does not change the O 3 net production rate significantly.The decrease in ozone net production with increasing feed gas humidity means that a higher humidity level either inhibits the production and/or favours the destruction of ozone. Without a plasma chemical model or an O atom density measurement it is difficult to disentangle both mechanisms. However, since ozone is mainly formed by one single reaction (equation (1)), atomic oxygen acts as a bottleneck for the ozone production. If the atomic oxygen density is affected by the feed gas humidity, an increase in feed gas humidity will consequently influence the O 3 formation as well. A possible mechanism for the reduction of excited atomic oxygen density by water molecules is given by the following reactions[START_REF] Herron | Chemical kinetics database and predictive schemes for nonthermal humid air plasma chemistry: II. Neutral species reactions Plasma[END_REF]:

Figure 11 .

 11 Figure 11. H 2 O 2 concentration in a plasma-treated liquid increases with feed gas humidity. Merckoquant stripes and the Amplex Red assay were utilized for H 2 O 2 detection.

Figure 12 .

 12 Figure 12. Cell viability determined by resazurin assay in dependence on the feed gas humidity concentration. The cells were treated indirectly.

Table 1 .

 1 Quenching rate coefficients for different excited species with state energies E, when H 2 O is the quenching molecule. Furthermore, the decay rates due to radiative decay and due to quenching (for a humidity concentration of 1000 ppm) are given.

	Decay rate (10 7 s -1 )

u , ν = 0) 11.18

[START_REF] Benesch | Potential curves for observed states of N 2 below 11 eV[END_REF]

7.1 ± 0.7

[START_REF] Morozov | Gas kinetic studies using a table-top set-up with electron beam excitation: quenching of molecular nitrogen emission by water vapour[END_REF]

  17 s -1 . A further 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:10.1088/0022-3727/46/29/295401. IOP Publishing is not responsible for any errors or omissions in this version of the manuscript or any version derived from it.

Acknowledgments

This work is funded by the German Federal Ministry of Education and Research (Grant Nos 03Z2DN11 and 03Z2DN12). The authors gratefully acknowledge the precious work of Liane Kantz, Steffen Hahn, Sarah Kupsch, Sebastian Schlegel, Mattis Hänel and Sven Bordewick.

11

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:10.1088/0022-3727/46/29/295401. IOP Publishing is not responsible for any errors or omissions in this version of the manuscript or any version derived from it.


Sylvain Is Éni

Stephan Reuter

Klaus-Dieter Weltmann

NO 2 dynamics of an Ar/Air plasma jet investigated by in situ quantum cascade laser spectroscopy at atmospheric pressure

Keywords: quantum cascade laser absorption spectroscopy (QCLAS), atmospheric pressure argon plasma jet, reactive oxygen nitrogen species, atmospheric pressure diagnostic

In this work, quantum cascade laser (QCL) absorption spectroscopy was used to investigate the nitric dioxide (NO 2 ) production dynamics at ambient conditions. For the first time, QCL detection of NO 2 and NO was used at ambient conditions in order to remain close to the conditions that are present in the application of plasma jets in open atmosphere. For the investigations, the plasma jet was placed inside an open multi-pass cell. The detection limit of the setup was 20 ppb for NO 2 and similar for nitric oxide (NO). Since the effective production of NO was below the detection limit, further investigations of the NO density were performed with optical emission spectroscopy.

Introduction

The scientific and technological interest in quantum cascade lasers (QCLs) for plasma diagnostic has increased over the last few years [START_REF] Röpcke | Applications of quantum cascade lasers in plasma diagnostics: a review[END_REF][START_REF] Guaitella | Oxidation of NO into NO 2 by surface adsorbed O atoms Contrib[END_REF][START_REF] Marinov | Surface vibrational relaxation of N 2 studied by CO 2 titration with time-resolved quantum cascade laser absorption spectroscopy[END_REF][START_REF] Röpcke | Application of mid-infrared tuneable diode laser absorption spectroscopy to plasma diagnostics: a review Plasma Sources[END_REF][START_REF] Welzel | Quantum cascade laser absorption spectroscopy as a plasma diagnostic tool: an overview Sensors[END_REF]. These types of lasers emit monochromatic mid-infrared (mid-IR) radiation with appropriate power for absorption spectroscopy.

Laser absorption spectroscopy in the mid-IR spectral region of 3-20 µm has proven useful for diagnostics of infrared active molecules. Applications have included environmental monitoring [START_REF] Tuzson | Selective measurements of NO, NO 2 and NO y in the free troposphere using quantum cascade laser spectroscopy[END_REF], process monitoring [START_REF] Hübner | On time resolved gas temperature measurements in a pulsed dc plasma using quantum cascade laser absorption spectroscopy[END_REF] and plasma chemistry diagnostics [START_REF] Marinov | Surface vibrational relaxation of N 2 studied by CO 2 titration with time-resolved quantum cascade laser absorption spectroscopy[END_REF]. The technology is based on using a frequency tunable diode laser and determining a molecular fingerprint of the absorption spectrum based on Beer-Lambert's law [START_REF] Winter | Aspects of UV-absorption spectroscopy on ozone in effluents of plasma jets operated in air[END_REF]. Early on, lead salt lasers were used to generate the laser radiation [START_REF] Röpcke | Application of mid-infrared tuneable diode laser absorption spectroscopy to plasma diagnostics: a review Plasma Sources[END_REF]. The intricate use of the technology, especially the requirement for cryogenic cooling, made the search for new techniques a key issue. QCLs provide a new technology with the use of several tunnel barriers of varying thickness, resulting in quantum wells with different population probabilities and a resulting population inversion, from which lasing can be induced [START_REF] Faist | Quantum cascade laser: a new optical source in the mid-infrared Infrared[END_REF]. Compared with lead salt lasers distributed feedback (DFB) QCLs provide (i) continuous mode-hop free (MHF) wavelength tuning, (ii) increasingly high output powers, (iii) near room temperature operation, and in the case of cw-DFBQCL (iv) narrow linewidth radiation [START_REF] Röpcke | Applications of quantum cascade lasers in plasma diagnostics: a review[END_REF]. QCLs require a higher power input which, when pulsed, will increase the QCL diode's temperature considerably during one pulse. This will change the material's refractive index and thus result in an inherent frequency-down chirp [START_REF] Welzel | Quantum cascade laser absorption spectroscopy as a plasma diagnostic tool: an overview Sensors[END_REF]. This is applied to acquire entire absorption spectra during one laser pulse. The technology is known as intra pulse mode [START_REF] Normand | Fast, real-time spectrometer based on a pulsed quantum-cascade laser[END_REF].

In this paper, QCL mid-IR absorption spectroscopy is used for the first time to investigate NO x species produced by a radio frequency (RF) argon driven plasma jet operating at atmospheric conditions. This jet is suitable for biomedical applications [START_REF] Ehlbeck | Low temperature atmospheric pressure plasma sources for microbial decontamination[END_REF][START_REF] Weltmann | Antimicrobial treatment of heat sensitive products by miniaturized atmospheric pressure plasma jets (APPJs)[END_REF]. It is known that plasma treatment can induce biological responses [START_REF] Bundscherer | Impact of non-thermal plasma treatment on MAPK signaling pathways of human immune cell lines[END_REF] and anti-microbiological effects have been reported in recent literature [START_REF] Matthes | Pilot-study on the influence of carrier gas and plasma application (open resp. delimited) modifications on physical plasma and its antimicrobial effect against Pseudomonas aeruginosa and Staphylococcus aureus GMS Krankenhhyg Interdiszip 7 Doc02[END_REF][START_REF] Lademann | Comparison of the antiseptic efficacy of tissue-tolerable plasma and an octenidine hydrochloride-based wound antiseptic on human skin[END_REF]. One type of plasma component responsible for these effects is reactive oxygen and nitrogen species (RONS) by inducing oxidative stress to micro-organisms leading to microbial inactivation [START_REF] Matthes | Pilot-study on the influence of carrier gas and plasma application (open resp. delimited) modifications on physical plasma and its antimicrobial effect against Pseudomonas aeruginosa and Staphylococcus aureus GMS Krankenhhyg Interdiszip 7 Doc02[END_REF]. Previous studies have already reported the excellent anti-microbiological effects of argon plasma, which are mostly attributed to RONS [START_REF] Nosenko | Designing plasmas for chronic wound disinfection New[END_REF]. A study of the production of NO x species by plasma sources will help to disentangle fundamental processes in plasma medicine. By using mid-IR QCL absorption spectroscopy under atmospheric conditions, the NO 2 absorption profile and absolute NO 2 concentration in the ppb range were determined.

Experimental setup

Apparatus

The plasma source used for the investigations was an atmospheric pressure argon plasma jet (kinpen 09, neoplas GmbH, Germany) [START_REF] Reuter | Controlling the ambient air affected reactive species composition in the effluent of an argon plasma jet[END_REF] This plasma jet is a 1 MHz RF driven concentric needle electrode within a dielectric ceramic capillary of 1.6 mm inner diameter. A grounded ring electrode surrounds the outer side of the capillary. This type of plasma jet configuration is referred to in the literature as a DBD-like jet and more general information can be found in [START_REF] Lu | On atmospheric-pressure non-equilibrium plasma jets and plasma bullets[END_REF]. The electrical potential of the needle can be from 2 to 6 kV and the dissipated power within the plasma is known to be in the range 1.6-2.2 W. The kinpen generates an argon discharge with a spatial afterglow-also called effluent in this work-of about 10 mm outside of the ceramic capillary. The working gas (argon 99.999% purity) is fed through the discharge zone with a gas flow rate of 3 standard litres per minute (slm). To protect the plasma jet effluent from the surrounding atmosphere, a shielding gas curtain device, described and investigated in previous papers [START_REF] Reuter | From RONS to ROS: tailoring plasma jet treatment of skin cells[END_REF][START_REF] Reuter | Detection of ozone in a MHz argon plasma bullet jet Plasma Sources[END_REF], was used. As shielding gas pressurized air was employed.

The diagnostic of this plasma device has been performed by mid-IR QCL absorption spectroscopy. This spectroscopic technology has already been used on ozone, as presented by Reuter et al [START_REF] Reuter | Detection of ozone in a MHz argon plasma bullet jet Plasma Sources[END_REF] and also recently reviewed by Röpcke et al [START_REF] Röpcke | Applications of quantum cascade lasers in plasma diagnostics: a review[END_REF][START_REF] Röpcke | Application of mid-infrared tuneable diode laser absorption spectroscopy to plasma diagnostics: a review Plasma Sources[END_REF]. Figure 1 illustrates the complete diagnostic setup with the implementation of the kinpen and the gas shielding device which was used in this work to generate controlled and reproducible conditions [START_REF] Reuter | Controlling the ambient air affected reactive species composition in the effluent of an argon plasma jet[END_REF]. The measurement system is mainly based on the Q-MACS platform developed by Neoplas Control GmbH and adapted and optimized here in order to lead in situ diagnostics at atmospheric conditions. The mid-IR source is a nanosecond pulsed QCL from Alpes lasers SA which emits in the spectral range from 1607.72 to 1619.43 cm -1 . The laser is driven in the so-called intra-pulsed mode. The wavelength is tuned by temperature via a Peltier element which is directly in contact with the laser chip. A water cooling system is installed in order to increase the Peltier element efficiency and increase temperature stability. According to the manual, the laser operates in single mode emission. The wavelength fine tuning is realized by a current ramp which varies the temperature and allows for a sweeping of 0.8 cm -1 range within 1100 laser pulses. The laser beam, which comes directly from the chip, is not symmetric and the strong divergence is corrected by an off-axis parabolic gold coated mirror (PaM). It is then focused by a 1 m spherical Figure 1. Schematics of the spectroscopic experimental setup based on a QCL absorption diagnostic system with the implementation of the plasma jet. The system contains various reflective components such as planar mirror (PlM), off axis parabolic mirror (PaM), spherical mirror (SM), and a red laser diode as optical alignment for the IR beam. The plasma jet is inserted through the glass chamber wall in such a way that the plasma effluent of a few millimetres is blown into the chamber. The so-called far field is defined as post-effluent, where short living plasma species are not relevant and here considered as the volume of the chamber (3.6 L). mirror (SM) and directed through a 0.60 m length 'White' type multi-pass spectroscopic absorption cell. The number of passes within the multi-pass cell can be adjusted according to the requirements of the absorber densities. In the applied working conditions, 32 passes are achieved resulting in an optical path of 19.2 m within the multi-pass cell. The laser beam is then focused on a very fast mercury cadmium telluride detector via a PaM. Since mid-IR radiation is not directly visible, a red laser diode is installed and its beam pathway is overlapped with the mid-IR beam for easier optical adjustment through the multi-pass cell. Data acquisition is performed via a PC and processed by Q-MACSoft Monitor software [START_REF]neoplas control GmbH[END_REF]. Additionally, an optical fibre is inserted within the chamber and connected to a spectrometer (Avantes, AvaSpec, 25 µm slit, 0.7 nm resolution) in order to record optical emission spectroscopic information. The fibre is aligned with the plasma jet front side. A spectral range from 200 to 960 nm is observed.

Implementation and measurement of NO 2 produced by the plasma jet are possible under the following conditions. First, the plasma jet has to operate and be diagnosed as close to the operating conditions as possible, meaning that it has to be surrounded by air at atmospheric pressure. Therefore, a cylindrical glass chamber (3.6 L volume), 9 cm inner diameter and 57.5 cm length, which is positioned between the multi-pass cell's mirrors, was used. The two glass chamber exits are partially closed with glass but each side has a rectangular opening which the mid-IR reflection of the beam can go through without disturbance such as interferences. As shown in figure 1, the plasma jet is situated in the middle of the long side of the multi-pass cell. The plasma jet can qualitatively be separated in three distinguished regions: the core plasma region is within the dielectric tubing and is dominated by plasma processes. The effluent is the spatial afterglow of the plasma and exits the nozzle for a few millimetres; here plasma species are still present. The far field has no dominating plasma species and is the volume of the multi-pass cell in the present case. However, it is important to note that this type of multipass cell does not allow the absorption beam to go through the effluent. Indeed, the laser optical path is located within a sagittal plane to the plasma jet axis along the radial direction of the chamber. The 10 to 12 mm long plasma effluent is too short to be on the optical path. Moreover, this would induce steep density gradients and induce a non-homogeneous absorption medium. The plasma jet is fed with 3 standard litres per minute (slm) of argon (99.999%) and blows directly into the glass chamber. In order to avoid saturation of the inner atmosphere, a gas curtain all around the plasma jet body was used, which provides an additional 5 slm air flux within the chamber. More information about the purpose of the gas curtain can be found in [START_REF] Reuter | Controlling the ambient air affected reactive species composition in the effluent of an argon plasma jet[END_REF][START_REF] Rothman | The HITRAN 2008 molecular spectroscopic database[END_REF]. The reactive species are generated either within the plasma core or in the effluent and then blown directly into the chamber. Our working conditions allow us to presume a homogenous distribution of the species within the chamber. This assumption was partially checked by rotating the input or the plasma source by 90 • which changes its position regarding the two side outlets of the chamber.

Calibration procedure

In order to identify the spectral position of the laser radiation, a calibration process is required. For this, two gas reference cells of 15 cm long, filled with different gases, where used. Because of the complexity of the NO 2 absorption spectra, the spectral position cannot be easily identified. Therefore the second NH 3 filled reference cell is used because of its capability of absorbing mid-IR radiation in the close vicinity of NO 2 . With the absorption spectrum measured from the NH 3 -cell, the wavelength position can accurately be defined. The one with NO 2 is filled at 4.5 mbar and the other one with NH 3 at 10 mbar. Figure 2 shows the recorded absorption spectra of NO 2 and NH 3 from 1611.2 to 1612.4 cm -1 . The recorded spectra are compared to a simulation performed by Q-MACSoft-HT [START_REF]neoplas control GmbH[END_REF] software based on the HITRAN database [START_REF] Rothman | The HITRAN 2008 molecular spectroscopic database[END_REF]. From the graph it can be concluded that simulated and recorded spectra are in good agreement despite some mismatches. These mismatches can be explained by the fact that the reference cells are closed with MgF 2 windows whichdespite optical path optimization-are causing interferences, which appear in the spectra. However, by superposition of the simulated and the measured spectrum, excellent frequency correlation is observed. The calibration of the wavenumber scale is realized with a germanium (Ge) Fabry-Pérot cavity which generates a known interference signal with peaks of defined spectral distance. The absorption method is based on the Beer-Lambert law described by equation ( 1) for a simple ro-vibrational energy transition

T n = I T n I 0 = e -S n (T k )NLf pn . ( 1
)
With T n the transmittance, I T n the transmitted intensity, I 0 the intensity of the source, S n (T k ) the line strength in cm -1 /(molecule cm -2 ), N the density of absorbing molecules, L the absorption length in m and f p n the line shape of the n ro-vibrational transition including the broadening. The experimental conditions allow assuming pressure broadening as the major broadening effect of many orders of magnitude higher than Doppler and Stark broadening. Pressure broadening can be well described by a Lorentzian function. Therefore the Voigt function of the whole broadening can be simplified as a Lorentzian function L n (ν, γ n ),

L n (υ, γ ) = γ n π [(υ -υ 0 ) 2 + γ 2 n ] (2)
with

γ n = p atm η x γ n self -γ air + γ n air (3)
where γ n self and γ n air are the broadening coefficients of an n ro-vibrational transition, ν 0 is the centred spectral position of the transition n and η x is the density ratio between the selected molecule x and the total density. Then f p n is the results of a convolution between the natural line shape f (νν 0 ) and

L n (ν, γ n ) with f (ν -ν 0 )dν = 1: f p n (ν -ν 0 ) = f n (ν -ν 0 ) ⊗ L n (ν, γ n ) . ( 4
)
The absorption spectrum is then calculated by the sum of all the absorption lines,

T (ν) = n T n (ν). ( 5
)
It is also necessary to include the instrumental profile h(ν) in the simulation spectrum T s :

T s (ν) = T (ν) ⊗ h(ν). ( 6
)

Absolute densities determination

To determine the selected species density, the measured spectrum T m is first compared to T s in real time with Q-MACSoft Monitor software [START_REF]neoplas control GmbH[END_REF]. As temperature, pressure, absorption length and instrumental profile of the experiment are usually known one can run a fit routine with the concentration as a free parameter. Figure 3 depicts a recorded transmittance spectrum of NO 2 at atmospheric pressure generated by the plasma jet and the result of the Very good agreement is shown between both. Due to the very high spectral resolution, the measurement selectivity for the respective specie is considerably enhanced compared to other methods such as Fourier transform infrared spectroscopy (FTIR) or UV absorption. In the case of absorption overlap with other molecules, a significant difference will occur between the simulation and the measurement. As a consequence, this approach ensures a perfect and reliable detection of specific species. The recorded spectrum in figure 3 shows that the absorption process is occurring over the whole scan range, resulting from a global absorption. In other words, the inherent laser intensity I 0 cannot be measured in the presence of NO 2 . This is due to the pressure broadening which is dominating the broadening processes of the absorption lines. It is assumed that constant laser source intensity is not reasonable and must lead to significant measure uncertainty. The first investigations by Reuter et al were performed by normalizing the recorded spectrum to I 0 and fitting the baseline by a polynomial function. This polynomial was set as a baseline to initialize the fit routine. This approach, however, allows only short measurement times of a few tens of minutes until the light source intensity changes significantly. The signal-to-noise ratio was low and the typical detection limit was found to be around 300 ppb for the detection of O 3 . Additional issues have been found and linked to the non-uniform linearity of the detector which induces a shift of the signal intensity. This performance was not sufficient for the investigation of NO 2 . Therefore, a new method of measurements was developed as follows: First, a template baseline spectrum B temp at the intensity I 0 is recorded. Then, a transmission spectrum T m is acquired as shown in figure 3. The fit routine is executed to match the simulation spectrum T s and the measurement T m with B temp as an initial baseline parameter. The quality of the signal T m strongly affects the output result of the fit routine to match the simulation and associated spectrum. Once the optimum value of the concentration is reached, the position of the baseline is determined by the optimization of a first-order polynomial function as described by

B = a × B temp + b. ( 7
)
With a and b as two scalars adjusting the slope and the translation of the template baseline around the initial position. This approach has been validated by artificially reducing the intensity I 0 by 35% during experiment, where no change of the concentration was observed. This is due to the fact that the shape of the transmittance spectrum is the key to optimize b. It is observed that the parameter b of the polynomial has the strongest influence on the results and corresponds directly to a variation of the light source intensity. By this method, the typical noise deviation is measured to be around 15 ppb centred and a detection limit of 20 ppb is determined from the signal-to-noise ratio. Similar results have also been obtained for the detection of NO.

Working at atmospheric conditions is also intricate due to the abundance of water and carbon dioxide molecules within the chamber. These species efficiently absorb mid-IR radiation. Therefore, they may disturb the diagnostic of other species significantly. NO 2 and water absorption spectra overlap in many spectral ranges. The very narrow spectral radiation of the QCL has the advantage of selecting a range where the overlapping is negligible or well contained.

Results and discussion

Since water can strongly affect the NO (x) production (see e.g. Pipa et al [START_REF] Pipa | Controlling the NO production of an atmospheric pressure plasma jet[END_REF]), before each measurement, the gas flow is kept running for at least 2 h in order to reduce the residual humidity within the pipe. Recently, Winter et al also showed that humidity does influence the chemistry produced by this plasma jet [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 same procedure is applied for the gas shielding: pressured air is flowing for 12 h at least in order to dry the chamber. The plasma jet is switched on for 1 h before the first measurement in order to ensure stable conditions. While this is an overcautious procedure, it ensures that the results are independent of strong impurity influences. For each step of the varied parameter, the background spectrum is recorded for 5 min before and after the measurement and the measurement itself is performed for 5-6 min. One data sample is derived by recording and fitting an average spectrum every 5 s. The final density value is obtained from an average over the complete data set. The errors are stated as a standard deviation of the density. In addition the deviation of the baseline determined from the fit routine is also included in the final error estimation. However, in this study, the discharge fluctuations result on an error estimation which is in the order of 80 ppb.

Figure 4 presents the net production rate of NO 2 in the far field of the argon plasma jet as a function of air admixture to the feed gas. The NO 2 generation is strictly monotonically increasing and does not seem to reach a local maximum until 2.0% admixture of air. It was not possible to keep the plasma running at higher admixture, which is why the expected decrease due to a drop of the plasma efficiency cannot be observed. However, the points measured for 0.1% and 0.2% are quite interesting and can be ascertained only due to the sensitivity of the measurement technique. It can be clearly observed that admixing air to the argon feed gas induces a linear process of NO 2 production (at higher admixtures). The slight but observed deviation for 0.1% and 0.2% admixture can be explained by an effective dissociation process of molecular oxygen and nitrogen. An emission spectrum is presented on figure 5 and shows the emission bands of NO (A-X), the so-called γ system from 215 to 272 nm. It is also possible to observe the OH emission and some N 2 lines. As described in the experimental setup section, OES spectra are collected side on which has the advantage of obtaining information about the excitation on NO molecules within the plasma core, close to the needle electrode. During the experiment, NO (A-X) emission intensity appears to be dependent on the air admixture. Figure 6 shows the relative intensity of different NO (A-X) bands for several ratios of air admixture. As a result, we observe that the excitation process of NO (A-X) increases monotonically. In cases of low air admixture, a very drastic excitation of NO molecules is observed, especially at 0.1% and 0.2%. Moreover, Pipa et al [START_REF] Pipa | Controlling the NO production of an atmospheric pressure plasma jet[END_REF] were able to measure NO by absorption from a similar plasma jet at higher power for this type of discharge. The NO (A-X) emission dynamics depending on air admixture has an excellent agreement with the absolute density of NO. This is especially true for dry air admixtures. Nevertheless, the authors report that ozone was not detected, which should be attributed to the higher power input inducing a higher gas temperature in the reported case. According to their study, here it is assumed that the NO production dynamic follows in good approximation the excited NO species emission-however, a detailed study needs to follow. The QCL system was also set up to measure absolute NO molecule densities in the far field. The detection limit for NO was determined to be 20 ppb. However, no NO has been detected from this low power plasma jet. One reliable and evident reason is the high density of ozone, which is about two or three orders of magnitude higher than NO 2 . The main chemical loss of NO in the presence of O 3 is well known and the high reactivity of NO is also important to consider [START_REF] Van Gessel | Temperature and NO density measurements by LIF and OES on an atmospheric pressure plasma jet[END_REF]. It is very likely that the following equation is responsible for the transformation of NO into NO 2 :

O 3 + NO → NO 2 + O 2 . ( 8
)

Conclusion

In this work, for the first time, quantum cascade laser absorption was performed to determine NO 2 species production rate on a plasma jet operating in ambient conditions and pressure. The investigated rf argon plasma jet kinpen was placed in a multi-pass cell with open gas flux. For the measurements, an algorithm was developed to perform a fit of the background radiation. Due to pressure broadening, the observed transitions-in this case NO 2 (1611.3-1611.8 cm -1 )-are broadened so much that the values of zero absorption cannot be observed directly with the laser system. By performing a background measurement and by including a fitted polynomial of this background into the fit of the absorption signal with data taken from a spectra database, the absolute densities of NO 2 could be obtained. The quality of the gained evaluation was tested successfully. The accuracy of the measurements was determined to be down to 20 pbb for both NO and NO 2 molecules. However, tiny fluctuations of the plasma discharge increase the error estimations. This technique was used to investigate the far-field NO 2 production dynamics as a function of dry air admixture to the plasma jets feed gas. As described here, non-linear increase is observed at small admixtures whereas a linear increase takes place up to 0.3% admixed air.

From emission spectroscopic measurements, the NO dynamics were also investigated. As shown in [START_REF] Pipa | Controlling the NO production of an atmospheric pressure plasma jet[END_REF], the NO density correlates with the NO emission for low admixtures of dry air. These measurements were performed because, with the quantum cascade laser absorption, NO could not be detected within the multi-pass cell for the investigated plasma jet, which mainly produces oxygen derived species. The NO emission dynamic at air admixtures below 0.2% exhibits a non-linear increase, while at admixtures of greater than 0.4% this becomes an increasingly linear increase, which adds to the interpretation that dissociation of N 2 and O 2 at low admixtures dominate the NO and NO 2 dynamic.

The presented study yields a new way to investigate a plasma jet regarding its NO 2 production mechanisms at atmospheric conditions almost identical to those in applications such as plasma medicine.

Far-field measurements such as the ones shown in this study can be used to investigate the reactive species production processes at varying parameters and will help us to understand and control the reactive species component composition to tailor, e.g., biological responses to plasma treatment in plasma medicine.

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:10.1088/0022-3727/47/7/075203. IOP Publishing is not responsible for any errors or omissions in this version of the manuscript or any version derived from it.

Figure 2 .

 2 Figure 2. Absorption spectrum of NO 2 produced by the jet at atmospheric pressure plotted with the result from the real-time fit.

Figure 3 .

 3 Figure 3. Transmittance spectrum of NO 2 produced by the jet at atmospheric pressure plotted with the result from the real-time fit.

Figure 4 .

 4 Figure 4. Absolute concentration of NO 2 produced by the kinpen for different dry air admixtures.

Figure 5 .

 5 Figure 5. Emission spectrum, side on, from the argon plasma jet running with 3.0 slm and 0.3% admixture.

Figure 6 .

 6 Figure 6. Normalized integrated NO(A-X) bands emission for different dry air admixtures.

5

  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:10.1088/0022-3727/47/7/075203. IOP Publishing is not responsible for any errors or omissions in this version of the manuscript or any version derived from it.

Acknowledgments

The authors thank Dr Andrei V Pipa for the discussions about IR spectroscopy and Dipl. Ing. Henrik Zimmermann (neoplas control GmbH) and Dipl. Ing. Sven Glitsch for the technical support and their expertise about QCL. Very deep acknowledgements are addressed to Professor Dr Röpcke for his knowledge and previous works on QCL absorption spectroscopy. Dr Jan Schäfer and Dr Rüdiger Foest are also acknowledged for their contribution to the experimental settings. The authors gratefully acknowledge the funding by the Federal German Ministry of Education and Research (BMBF, grant number 03Z2DN12).

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:10.1088/0022-3727/47/7/075203. 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:10.1088/0022-3727/47/7/075203. IOP Publishing is not responsible for any errors or omissions in this version of the manuscript or any version derived from it.