Reactive oxygen species generated by cold atmospheric plasmas in

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Reactive oxygen species generated by cold atmospheric plasmas in aqueous solution: successful electrochemical monitoring in situ under a high voltage system Fanny GIRARD-SAHUN, Vasilica BADETS, Pauline LEFRANCOIS, Neso Sojic, Franck Clement, and Stephane Arbault Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01912 • Publication Date (Web): 07 Jun 2019 Downloaded from http://pubs.acs.org on June 7, 2019

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Analytical Chemistry

Reactive oxygen species generated by cold atmospheric plasmas in aqueous solution: successful electrochemical monitoring in situ under a high voltage system Fanny Girard-Sahuna,b, Vasilica Badetsb, Pauline Lefrançoisb, Neso Sojicb, Franck Clement*a and Stephane Arbault*b a b

UPPA, IPREM, CNRS UMR 5254, 2 avenue Président Angot, 64000 Pau, France Univ. Bordeaux, ISM, CNRS UMR 5255, INP Bordeaux, 33400 Talence, France

ABSTRACT: Many investigations are dedicated to the detection and quantification of Reactive Oxygen and Nitrogen Species (RONS), particularly when generated in liquids exposed to Cold Atmospheric Plasmas (CAPs). CAPs are partially ionized gases which can be obtained by applying a high electric field to a gas. A challenge is to get better insights on the plasma-liquid interactions in order to understand the induced effects on different targets (liquid, cells, tissues etc...). As RONS are bio-chemically reactive, the difficulty lies in finding efficient methods to get both dynamic and quantitative data. Herein, we developed an innovative setup aimed at performing an in situ electrochemical monitoring of redox species generated by CAPs in a physiological buffer (PBS, pH 7.4). The challenge was to apply millivolt-potential variations and measure nanoampere faradaic currents in presence of ionization waves generated by micropulsed electric fields of some 10 kV.cm-1 amplitude and ampere-transient currents. This was fulfilled by using dedicated working ultramicroelectrodes (Pt-black UMEs), and protect them as well as the reference and counter electrodes, within insulated-earthed containers. In this condition, we succeeded in performing both cyclic voltammetry and chronoamperometry in situ, with a resolution equivalent to working in a static solution (sub-nanoampere currents). Thus, we monitored the accumulation over time of species (H2O2, NO2-) generated by CAPs in PBS and observed the mean dynamic of RONS chemistry during and after plasma exposition, particularly through the detection of a short-living species.

Cold Atmospheric Plasmas (CAPs), and specifically the socalled “guided ionization waves”1,2 are increasingly used systems in very different fields ranging from polymer science, surface treatment and etching, and medical applications among others. They consist in a complex mixture of neutral atoms and molecules, electrons, charged particles (ions), excited atoms and molecules. To generate these partially ionized gases, a rare gas (e.g. helium, with the possibility to add mixtures of O2/N2) is subjected to a high electric field between two electrodes. Usual applied voltages are on the order of several kV, corresponding to local electric fields of several tens of kV.cm-1 3,4. When a liquid is exposed to such complex gases, many chemical reactions occur in solution due to the complex chemical composition of CAPs and their interaction with the surrounding air. In the context of biomedical applications (also known as “plasma biomedicine”)5, a particular attention is paid to reactive oxygen and nitrogen species (ROS/RNS or RONS as a general acronym) formed in liquids by CAPs, directly by dissolution from the gas phase or indirectly following side reactions. They are known to play an important role in biological and biochemical plasma-induced effects6,7. Indeed, the diverse reactive species (O3, ONOO-, etc.) and free radicals (NO°, O2°-, HO°, NO2°) produced in aqueous media are biochemically oxidizing, nitrating, nitrosating, etc. reactants inducing damages on all cell components (membranes, nucleus, mitochondria, proteins …). However, many questions remain about key mechanisms occurring on cell cultures and tissues

treated by CAPs, because of the complex mixture of reactive species generated in solution. Up to now, many studies demonstrated the presence of longlived chemical species in liquids treated by plasmas, kinetically stable until several hours after plasma treatment such as H2O2, NO2- and NO3- 5,8,9. Classically, these measurements are performed in liquids a posteriori to their exposure by CAPs and provide good insights on long-term chemistry remaining in solution. Moreover, it is well established that these long-lived species come from mother ROS/RNS produced in the early stages in the plasma and/or the liquid10–12. Several methods are described for the detection of reactive oxygen and nitrogen species in solution and most of them are based on an indirect detection by using different probes such as spin traps13, fluorescent6, colorimetric14 or biological probes15. These methods have however some limitations (semiquantitative results, auto-degradation of spin-adducts, damaging of biological probes, non-selectivity)13,15 which is problematic considering the wide variety of RONS produced by CAPs. Thus, even if these methods give a good assessment of RONS generation in liquids treated by plasmas, it is highly desired in the community to have more information about their nature and fate after production. In this note, we describe the development of a setup allowing to perform an in situ electrochemical monitoring of species produced in a physiological buffer during its exposure to CAPs.

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To do so, we had to adapt the usual analytical setup to overcome a certain number of technical constraints. The electrochemical detection consists in measuring nA currents compared to ampere currents involved in submicrosecond-pulsed plasmabased systems. Moreover, millivolt potential differences are applied to the working electrodes with respect to a reference while kilovolts are used to induce the ionization waves. Thus, such measurements close to a plasma setup appear a priori impossible. To overcome these severe limitations, we developed ultramicroelectrodes (UMEs) that are selective and sensitive for several RONS16,17 and integrated the electrochemical 3-electrode cell within well shielded and earthed containers. This rendered possible the direct in situ monitoring of RONS formed in solution, including H2O2 and NO2-, during a typical plasma experiment.

EXPERIMENTAL SECTION Reagents. PBS was from PAN BIOTECH (10 mM, pH 7.4, KCl 0.003 mol.L-1; KH2PO4 0.002 mol.L-1; NaCl 0.137 mol.L--1; Na2HPO4 0.01 mol.L-1, without Mg2+/Ca2+). Solutions of H2PtCl6, lead acetate and Fc(MeOH)2, H2O2 and NaNO2 were from Sigma Aldrich (France). Plasma treatment of physiological solutions. 6 mL of PBS solutions were exposed to plasmas in a homemade quartz cuvette (external diameter 44 mm, inner diameter 41 mm, depth 40 mm). A distance of 20 mm was fixed between the end tube of the plasma device and the solution (see Figure 1A characteristic distance “a” in Figure 1A). Moreover, the cuvette was placed over a metallic support connected to the ground in order to ensure a constant potential to the sample. Plasma setup. The plasma setup used in these experiments has been described previously12. A surrounding gas device allows to control the gaseous environment of the gas phase (plasma). We used helium as the working gas (1.67 slm rate) and the environment gas was composed of 100 % N2 (0.03 slm rate; Linde, 99.9995%), for a total gas flow of 1.7 slm. The helium

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flow rate was adjusted with adequate mass flow controllers (ELFLOW, Bronkhorst High-Tech) connected to a flow-bus. O2/N2 mixtures were always introduced around the helium gas and they were adjusted using another mass flow controller. The production of ionization waves was achieved using a pulsed high voltage power supply with rise and fall times of pulses in the order of 100 ns. The electrical parameters are: positive pulses of 7.5 kV in amplitude, 1 s in duration, 10 kHz repetition frequency and 1% duty cycle (ratio between the duration of the pulse and the signal period). Electrochemical experiments. We performed cyclic voltammetry using a three-electrode electrochemical cell composed of a platinized platinum UME as working electrode (a 25 µm-diameter Pt wire sealed in a glass capillary of 1 mm diameter, see photographs in Figure S1), an AgCl-coated-Ag wire as pseudo reference electrode (250 µm diameter) and a Pt wire as counter electrode (500 µm diameter). In these experiments, we used together two working electrodes (coupled with two counter electrodes and one reference) in order to perform different electrochemical measurements simultaneously or simply to have a duplicate. All electrochemical measurements were carried out using a bipotentiostat (BioLogic, VSP-300, EC-Lab software) equipped with low current modules kept in a home-made Faraday cage. In order to detect RONS generated in PBS during plasma exposure, cyclic voltammograms were achieved between -0.1 V and 0.9 V vs. Ag/AgCl, at 20 mV.s-1 scan rate12. Chronoamperometry was performed at either +0.3 V or +0.85 V vs. Ag/AgCl. Moreover, a magnetic stirring was necessary in order to increase convection of exposed solution from the plasma impact point in the PBS to the electrode surface (Figure 1).

Figure 1. Description of the setup combining a CAP source and the shielded electrochemical cell for in situ measurements. (A) Profile schematic view (not at scale) of the experimental setup including the plasma source equipped with a device providing a surrounding gaseous environment12, the cuvette containing the solution to be exposed (6 mL of PBS) and the two shielded containers for electrochemical measurements with 2 counter electrodes (CE 1 and CE 2) in the container 1, and 2 Pt working electrodes (WE 1 and WE 2) together with the reference electrode (Ag/AgCl, noted REF) in the container 2. Characteristic distances are: a = 20 mm; b = 7±1 mm; c = 12±1 mm; d = ~20 mm. (B) Top view showing the position of the electrodes in the containers (CE 1 and CE 2 in container 1; WE 1, WE 2 and REF in container 2) and in the quartz cuvette with respect to the plasma impact point and the magnetic stirrer (not at scale). (C) Picture of the experimental setup under operation (see the plasma wave generated in the tube, which propagates and reaches the solution).


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RESULTS AND DISCUSSION Development of the experimental setup based on a shielded electrochemical cell. The final goal of this work is to perform sensitive electrochemical measurements in a not compatible environment. The plasma setup used here is similar as the one previously described12. Ionization waves were generated by applying a pulsed electric field 𝑬 of high intensity (7.5 kV potential difference) in a He gas flow, within an external gas environment of 100 % N2 flown in a surrounding tube (see Figure 1). The goal of such a setup is to better control the nature and yields of RONS in the plasma phase and subsequently in the exposed liquid phase, herein a physiological PBS (pH 7.4). In the following studies, only 100% N2 environment was used since this condition provides us with high concentrations of dissolved RONS at minute time-scale exposure durations. RONS derive from nitrogen excited species (e.g. NO°, N2 (FPS)) and oxygen species (e.g. HO° and O) in the gaseous phase12 that were characterized by optical emission spectroscopy realized near the liquid surface (data not shown). As mentioned above, RONS lifetime in aerated PBS at room temperature and physiological pH ranges typically from nanoseconds (HO°), to hours (NO2-), but major ones (NO°, O2°-, H2O2, ONOO-) are stable in the second to minute timescale. Consequently, there is a need to detect and identify them in solution close from their source, i.e. the zone of interaction between the plasma and the solution (see scheme in Figure 1C). Electrochemical methods are particularly adequate to detect these reactive oxygen and nitrogen species17. However, the basic principle of a three electrode based-electrochemical cell is to apply a homogeneous and stable electric field (usually with a millivolt precision) between the WE and the REF. In the present experimental setup, a strong micro-pulsed electric field 𝑬, of 10-20 kV.cm-1 44,18 amplitude and transient currents of about 1 A, is propagated by the ionization wave and reach the solution where 𝑬 strongly dissipates. Thus, both electric systems appear incompatible in the same solution since the electric field of the plasma should polarize the electrodes directly, and possibly the potentiostat electrometer measuring the WE currents. This was indeed observed when placing the electrodes in the solution exposed to the plasma and trying to achieve any electrochemical measurements, the potentiostat was out of range for both its applied potential and detected currents (data not shown) and no experiment could be performed. Consequently, we searched for completely protecting the 3electrode setup from the surrounding electric field due to the CAP. The first approach consisted in placing the WE and REF electrodes in a shielded and earthed container made of a quartz glass capillary (few centimeters long, internal diameter 7-9 mm) covered by a conductive sheath connected to the ground of the building, and covered by an insulating layer of retractable polymer sheath for power cables. The quality of the earth grounding was found important for the efficiency and final sensitivity of experiments. Moreover, the container grounding should be different from the one of the plasma reactor to prevent from any induction of a potential difference and noise injection toward the electrochemical devices. Then, we decided to work with ultramicroelectrodes (UMEs) as WE for in situ detection. UMEs actually offer several advantages. Their limited physical size allows, as widely used

for measurements on living cells and tissues16, to position their surface with precision (sub-millimetric distance) from a source of redox species. Herein, UMEs were fabricated from a platinum microwire insulated by a millimetric glass capillary (Figure S1). Then such minimal final size permits their positioning at short distances from the tip of the shielded container, and from the REF electrode (few mm distance). Therefore, this system limits the distance on which the potential difference is applied and obviously minimizes the impact of any polarizing-surrounding electric field18,19. It even gives the possibility to insert two working electrodes next to the reference wire (Figure 1B) and achieve two different detections (see following paragraph). Eventually, as well known in the field of electroanalysis, UMEs provide steady-state responses and fast response time, making these types of electrode ideal for monitoring concentration variations of redox species, which is the final goal of the setup development described herein. Besides, the two counter electrodes CE were placed in a separated shielded container (Figure 1B). Indeed, this separation is essential to avoid any interferences between redox species generated at both working and counter electrodes. Finally, the position of the electrode containers compared with the plasma spot was adapted (characteristic distance “d” in Figure 1A) and was maintained in the quartz cuvette using dedicated apertures. The final working setup is imaged in Figure 1C. In situ electrochemical measurements during plasma exposure. The immunity of the electrochemical cell to the tension at the plasma generator and to electric field transported by ionization waves was assessed first by cyclic voltammetry (CV). Because of its working principle, CV should provide an information on any overpotential or added electrochemical currents due to the plasma-solution interaction. Voltammograms were then first acquired in a PBS solution, directly into the quartz cuvette where the plasma will be applied. This allowed to check the stability of the baseline response for the two WE used in parallel. Then, the electrochemical response of a common redox probe, ferrocene-dimethanol (Fc(MeOH)2, 3 mM in 10 mM PBS, pH 7.4) was tested. Neat quasi steady-state voltammograms were observed for the oxidation of Fc(MeOH)2, (cycles between 0 and +0.4 V vs Ag/AgCl at low scan rate). During continuous cycling, the plasma setup was turned on and the solution exposed to the ionized gas flow. We observed a small spike of current just when the plasma became generated but no specific shift of the CV current. Similarly, in the following we did not observe any clear shift of the potentials vs the REF since CVs could superimpose before/after plasma was ON, as shown in Figure 2. Only the CVs detected in the first minutes of the experiments were compared since other species (RONS) could be produced in solution and detected later on.


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Following, chronoamperometry was used to assess in more details the sensitivity of the in situ measurements, and then to detect the accumulation of redox species, namely RONS, in PBS over time. As can be observed in Figure 3, the plasma onset induced a small current artefact and no increase of the noise or of the background current itself. We confirmed here the setup efficiency and our ability to perform very sensitive measurements under plasma exposure since the resolution in current was a few hundred picoamperes. As two working electrodes are available, we performed simultaneously two measurements at two different potentials (bi-potentiostat mode) to possibly observe different redox species. With respect to our previous studies performed in a post-treatment study with the same types of platinized UMEs,12 we used the following potentials for detection: +0.3V at one working electrode (WE 1) to detect selectively hydrogen peroxide and +0.85 V applied to the second one (WE 2) to detect supplementary RNS such as nitrite. Chronoamperometric responses at +0.3 V and +0.85 V are depicted in Figure 3.

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Chrono-amperometric experiments provide different kinetic information: 1) when plasma is turned ON, a latent period of 1-2 minutes is visible, before the current starts to increase (Figure 3); this delay is necessary for the concentration of species dissolving from the gas phase and created in solution to reach a level superior to the detection limit of our sensors. For platinized UMEs, this limit is close to 100nM for hydrogen peroxide and other RONS detectable in these conditions. Besides, we estimate that the travel time of such small, fast diffusing RONS under solution stirring is about a few seconds to the UME surface in the container (about 10 mm distance from the plasma impact point); 2) both 𝑰 +𝟎.𝟑 𝑽 and 𝑰 +𝟎.𝟖𝟓 𝑽 currents increase continuously and linearly until plasma was OFF. This current evolution reveals firstly that some redox species are indeed oxidized at these potentials and secondly that they accumulate (concentration rise) in PBS. Moreover, the current difference 𝑰 +𝟎.𝟖𝟓 𝑽 ― 𝑰 +𝟎.𝟑 𝑽 increases over time, indicating that at least one species is oxidized at +0.85 V in addition to the ones oxidized at +0.3 V; 3) both 𝑰 +𝟎.𝟑 𝑽 and 𝑰 +𝟎.𝟖𝟓 𝑽 continue to increase after plasma is OFF, (only few nA during about 2 minutes), probably because of chemical reactions occurring before a real stabilization of the solution composition. This point is very interesting since it highlights a post-plasma chemical reactivity during few minutes. Based on our previous work, we attributed 𝑰 +𝟎.𝟑 𝑽 and 𝑰 +𝟎.𝟖𝟓 𝑽 variations to (at least) H2O2 and both H2O2 and NO2- detections, respectively. In order to characterize these species more precisely, CVs were realized in situ, during PBS plasma exposure, between -0.1 and +0.9V, i.e. in a range of potentials that comprises at least the oxidations of H2O2 and NO2-, and possibly of nitric oxide NO°. The voltammetric behavior of PBS was first recorded before and just after the start of plasma exposure (Figure 4A). A first observation is that all signals on Figure 4A are relatively free of noise (only forward part of each scan is shown to improve readability of superimposed responses), similarly to the experiment in Fc(MeOH)2 solution described above (Figure 2). There is no resolution change when plasma is OFF/ON, which is a technical breakthrough provided by the setup described here. Secondly, from the first minutes of plasma exposure, oxidative current increases in agreement with the measurements performed by chrono-amperometry. Two oxidation waves are clearly visible after 10 minutes: the first one between +0.05 V and +0.35 V, and a second one between +0.65 V and +0.85 V. These waves are characteristic of H2O2 and NO2- irreversible oxidations (same waves as in vitro experiments, see Figure S2 in supporting information), and their amplitudes increasing as a function of time is linked to the species accumulation in solution. As the current is directly proportional to the concentration, we evaluated the concentration of both H2O2 and NO2- as a function of time, by using calibration curves of H2O2 and NO2- prepared in PBS (10 mM, pH 7.4) and detected with the same platinized UMEs.

Time (min) Figure 3. Chronoamperometric measurements performed in PBS with 2 WEs in the shielded configuration, before, during and after plasma exposure. WE 1 is set at +0.3 V and WE 2 at +0.85 V vs. Ag/AgCl.


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As mentioned above, some other RONS may be detected in the potential window used here. We showed previously that the oxidation wave of NO° is detected in PBS with such platinized electrodes between +0.4 V and +0.65V vs Ag/AgCl. No distinct wave was actually observed in PBS exposed to plasma, showing that though nitric oxide may be produced in the gas phase, its presence in solution was not detected (meaning at a sufficiently high concentration for an analysis by CV). However, we noticed on the voltammograms of Figure 4A, the rise of a voltammetric current near 0 V, which is additional to the slightly negative current at this potential in PBS only or in H2O2 solutions. Such voltammetric wave corresponds to a species that is never observed in chemically stable solutions after the plasma exposures (typically 30 min. after). This response is potentially due to superoxide anion oxidation, which requires low energy to be oxidized19,20 and is one of the major unstable species produced by cold atmospheric plasmas11,20. Superoxide should evolve into hydrogen peroxide, detected here in solution, or combine with nitric oxide to form peroxynitrite, which also evolves into nitrite and nitrate at pH 7.4. This specific response and its dynamic will be studied in more details in a future work, including by varying the surrounding gas composition to decipher better on its relation with the plasma chemistry. Nevertheless, it already demonstrates that several species can be detected directly and followed over time by in situ electrochemistry, including at least one unstable species during its production.

CONCLUSION

Figure 4. (A) Cyclic voltammograms realized in PBS solution during plasma exposure and under magnetic stirring; Scan rate 20 mV.s-1. (B) Quantification of H2O2 and NO2- as a function of time, according to CVs depicted on (A). Concentrations were calculated thanks to calibration curves obtained in pure solutions of both species in PBS (Figure S2).

The calculated concentration for both H2O2 and NO2- were plotted as a function of time (Figure 4B). In agreement with chronoamperometric measurements, there is a latent period before the concentration of both H2O2 and NO2- rise after plasma ON. This delay is higher than the one observed in Figure 3 (about 10 minutes here), simply because the sensitivity of cyclic voltammetry is lower than the one of chronoamperometry at low concentrations. Then, concentrations increase linearly until plasma was turned OFF. After 30 minutes of plasma exposure and just before plasma OFF, concentrations reach approximately 600 µM for both H2O2 and NO2-. Following plasma OFF, the NO2- concentration is quite stable (final concentration: 700 µM), while the one of H2O2 continues to increase within 10 minutes after plasma is OFF (≈ 850 µM). These values are coherent with those measured post-plasma exposure (in an ex-situ stationary experiment, data not shown). The evolution of concentrations is perfectly consistent with current evolutions depicted on Figure 3. The quasi-linear increase of 𝑰 +𝟎.𝟑 𝑽 and 𝑰 +𝟎.𝟖𝟓 𝑽 correlates with a concentration increase of H2O2 and NO2- into the PBS, while the current evolution after plasma OFF is probably due to a post-plasma chemistry in solution during several minutes before stabilizing (because there is no more contribution of H2O2 and NO2-, brought by the ionization waves).

In this work, we developed an innovative electrically-shielded setup allowing to perform in situ electrochemical measurements in PBS exposed to ionization waves (CAPs). Two common electrochemical techniques (cyclic voltammetry and chronoamperometry) have been successfully performed in solution while a high voltage (7.5 kV) is applied locally to generate a plasma in atmospheric conditions. By using specific microelectrodes, we monitored in situ in a physiological solution the formation of redox species such as H2O2 and NO2-. Experiments depicted different kinetics of appearance of redox species, linked to plasma ignition or extinction. We observed a post-plasma evolution of their concentration in solution as well as a transient reactive oxygen or nitrogen species, possibly O2°-. The next step will thus to take benefit of the advantages of in situ detection to decipher on the formation of certain short-lived RONS produced by plasmas. In addition, by decreasing further the dimensions of the electrodes in combination and of the shielded containers, we could achieve a spatial mapping in the solution of the species concentrations. Otherwise, such perfectly isolated electrochemical cells should find applications in other analytical fields where high electric or electromagnetic fields are used to generate physico-chemical processes probed by electrochemistry.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author Dr Stéphane Arbault, email : [email protected] Tel.: +33540008939 ; Dr Franck Clément, email: [email protected] Tel.: +33559 (407657) / (574190)

Author Contributions


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The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work was financially supported by the Centre National de la Recherche Scientifique (CNRS), the French Ministry of Research (MESR) and the Agence Nationale de la Recherche (ANR, PLASMAREGEN project, no. ANR-14-CE16-0007-01).

Present addresses The current affiliation of F. Girard-Sahun, V. Badets and P. Lefrançois are, respectively: Chemistry Department, University of Antwerp, Campus Drie Eiken Universiteitsplein 1, Belgium. University of Strasbourg, Chemistry Institute, UMR CNRS 7177, 4 rue Blaise Pascal, CS 90032, 67081 Strasbourg cedex, France. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Description of UME fabrication protocol and H2O2/NO2calibration curves by cyclic voltammetry.

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Reactive oxygen species generated by cold atmospheric plasmas in

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