Reaction mechanism for atmospheric pressure plasma treatment of cysteine in solution

Mechanisms for the cold atmospheric plasma (CAP) treatment of cells in solution are needed for more optimum design of plasma devices for wound healing, cancer treatment, and bacterial inactivation. However, the complexity of organic molecules on cell membranes makes understanding mechanisms that result in modifications (i.e. oxidation) of such compounds difficult. As a surrogate to these systems, a reaction mechanism for the oxidation of cysteine in CAP activated water was developed and implemented in a 0-dimensional (plug-flow) global plasma chemistry model with the capability of addressing plasma-liquid interactions. Reaction rate coefficients for organic reactions in water were estimated based on available data in the literature or by analogy to gas-phase reactions. The mechanism was validated by comparison to experimental mass-spectrometry data for COST-jets sustained in He/O2, He/H2O and He/N2/O2 mixtures treating cysteine in water. Results from the model were used to determine the consequences of changing COST-jet operating parameters, such as distance from the substrate and inlet gas composition, on cysteine oxidation product formation. Results indicate that operating parameters can be adjusted to select for desired cysteine oxidation products, including nitrosylated products.


Introduction
Cold atmospheric pressure plasmas (CAPs) have potential applications in plasma medicine including cancer treatment [1,2], chronic wound healing [3], disinfection [4,5], and medical device decontamination [6]. A variety of wellcharacterized CAP devices have been used for plasma medical applications, including dielectric barrier discharges, atmospheric pressure plasma jets, and plasma needles [7][8][9][10]. These devices are either 'touching', in which the plasma is in direct contact with the target, or 'non-touching' in which reactive species generated in the plasma transport across a nonpowered region to reach the target. Plasma jet devices for biomedical applications are generally sustained in either argon or helium with additives or impurities (i.e. air, oxygen, water) that result in generation of reactive oxygen and nitrogen species (RONS) in the plasma.
The RONS produced in these plasma jets include some of the same RONS involved in biological processes in living organisms where they play an active role in redox biology [11]. RONS, generated by CAP or a living system, are known to affect proteins [12] particularly proteins with the thiol (-SH)-containing amino acid cysteine which is prone to oxidation and nitrosylation [13,14]. The low molecular weight protein glutathione (GSH), for example, is the major cellular cysteine-containing protein participating in cellular redox reactions [15]. Crucial biological processes involve the thiol redox state. The inflammatory extracellular signaling protein high mobility group box 1 (HMGB1), for example, contains three redox-sensitive cysteine residues [16]. Depending on the dynamic redox state of each of the cysteine residues, the protein fulfills different functions. The consequences of plasma treatment of proteins in vivo could be, to a certain extent, transported or preserved via thiol group oxidation [17]. Oxidative modifications of biomolecules have the potential to modulate and control downstream physiological processes [18].
Cystine (RSSR), a commonly found product after plasma treatment, contains a disulfide bridge, a key structural element essential for protein structure and function [19]. Cystine is a sensitive sensor of the redox state of a cell [19] which can be further oxidized or nitrosylated. These molecules can lead to bioactive derivates that have anti-proliferative effects on cancer cells [20], playing a role in the nitric oxide pathway with implications for wound healing, cancer biology, and cardiac functioning [21][22][23]. A range of products, including nonstable transient products, can be found after plasma treatment [14,21]. Bioactive reactive sulfur species (e.g. cyst(e)ine sulfoxides and cysteine-S-sulfonate) have the potential to influence cellular redox balance [20,24].
Given the large number of in-solution RONS produced by CAP, and the many reactions that may occur with living cells, isolating specific reaction pathways is challenging [25,26]. The amino acid cysteine has been used in studies of CAP interacting with water-based solutions as a surrogate for biological models, due to its amino, carboxyl, and thiol groups that are common in biological macromolecules, and cell membranes in particular [21,[27][28][29], and the biological relevance of the molecule, as discussed above. Developing mechanisms for in-solution interactions of RONS with simpler amino acids provides a baseline for understanding such interactions with living cells.
Developing an in-solution reaction mechanism for CAP produced RONS interactions with cysteine will benefit from a combined experimental and computational approach. Cysteine interactions with RONS have been modeled by Lackmann et al [30] using molecular dynamics (MD) simulations. The MD simulations included interactions of reactants that may be produced by two plasma jet sources, the kINPen sustained in argon [31] and the European Cooperation for Science and Technology (COST)-jet sustained in helium/argon mixtures [32]. Cysteine oxidation was initiated by OH aq or NO aq at the thiol (-SH) site on the cysteine molecule. (The subscript 'aq' denotes an in-solution species, to differentiate from their gas phase analogues). Interactions with H 2 O 2aq , O 2aq , and O 3aq were also addressed, but were found to be less important than interactions with OH aq and NO aq on the short timescales that could be addressed with MD (⩽10 ps). A detailed mechanism was produced, which included branching ratios of cysteine oxidation products formed after interactions of cysteine or cysteine intermediate products with RONS. However, due to the computational limitations of MD, longer timescale interactions on the order of seconds or minutes could not be resolved.
Computational models with the ability to address timescales consistent with experimental treatment times (seconds to minutes) would aid in further understanding of plasmaassisted modifications of organic molecules in solutions. Recent measurements have shown that atomic O aq is one of the most abundant ROS produced by CAP sources sustained in mixtures containing oxygen impurities, and so its interactions with organic molecules should also be considered [33,34].
In this paper, we discuss results from computational and experimental investigations in which we propose a reaction mechanism for the COST-jet treatment of cysteine in water solutions. A 0-dimensional (0D) global plasma chemistry model was used in a plug-flow mode to simulate the gas-phase reactive species densities and fluxes that come into contact with ambient air in the gap between the reactor outlet and continue to the liquid surface. The gas-phase fluxes impinge on a 1 mm thick solution of cysteine in water. A liquid reaction mechanism that accounts for transport across the gasliquid interface and liquid-phase reactions was coupled to the gas phase plasma-chemistry model, providing densities of in-liquid species after plasma treatment. The reaction mechanism was validated with experimental mass spectroscopy (MS) measurements from the He/O 2 and He/H 2 O COSTjet treatment of cysteine in a water solution. Experiments were also performed for solutions treated by the COSTjet sustained in He/N 2 /O 2 to produce nitrosylated cysteine products, in addition to oxidation products. The results from the model were used to investigate reaction pathways and mechanisms responsible for the production of cysteine oxidation products as a function of plasma operating parameters including distance between the reactor outlet and the liquid surface, oxygen inlet flow rate, and nitrogen inlet flow rate. Results indicate that some degree of control over the formation of specific products can be achieved by leveraging these plasma parameters.
The experimental methods used to investigate He/N 2 /O 2 COST-jet treatment of cysteine in solution are described in section 2. A description of the model and the cysteine reaction mechanism are in sections 3 and 4. A comparison of the model results against experimental data for validation of the reaction mechanism is contained in section 5. Plasma and liquid properties produced by the model for base-case conditions are in section 6. Results, including comparison of the model with experimental mass spectroscopy measurements are discussed in section 7. Conclusions are in section 8.

Plasma source
The experiments utilize the atmospheric pressure COST reference microplasma jet (the COST-jet), which has been previously described in detail by Golda et al [32]. The COST-jet is a capacitively coupled plasma that operates at a frequency of 13.56 MHz. The source has two 30 mm long electrodes positioned 1 mm apart, resulting in an active plasma region measuring 1 mm × 1 mm × 30 mm. Helium was used as the primary inlet gas. Experiments used for validation of the model kept a total gas flow of 1 slm for the two gas mixtures of He/H 2 O = 99.75/0.25 and He/O 2 = 99.4/0.6. For the He/H 2 O mixture, dry helium was passed through a bubbler before being mixed with additional dry helium downstream to produce the desired mole fractions. To reduce impurities, the helium and oxygen (research grade ARC3 PurityPlus 5.0) were delivered through stainless steel tubing. The nitrogen containing gas mixture for the base case was He/N 2 /O 2 = 99.0/0.8/0.2. The power deposition was kept constant at 750 ± 10 mW and was continuously monitored with integrated current and voltage probes.

Sample treatment
To investigate the consequences of plasma treatment of biologically important molecules, the amino acid cysteine was used as a surrogate. The L-form of cysteine was sourced from Alfa Aeasar (L-Cysteine A10435, ⩾98%) and dissolved in high performance liquid chromatography (HPLC) grade water to a final concentration of 100 µg ml −1 . One milliliter of these solutions was then transferred to 12-well plates producing a solution depth of 1 mm and exposed to the COST-jet effluent at a gap distance of 4 mm. For each gas mixture, control samples were also taken where the cysteine solution was only exposed to the respective gas flow without igniting the plasma for a duration equal to the maximum treatment time used. All experiments were performed in triplicate.

Mass spectroscopy
The chemical changes that occurred in cysteine due to plasma treatment were experimentally analyzed using mass spectroscopy (MS). An autosampler vial was filled with 100 µl of each 1 ml sample. A UPLC-MS/MS method was used, utilizing a Thermo Vanquish LC instrument (Thermo Fisher Scientific) coupled to a Thermo Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) with a heated electrospray ionization source. Chromatographic separation was accomplished with a Waters BEH Amide column (2.1 × 100 mm, 1.8 µM) that was maintained at 45 • C. A linear gradient of mobile phase A (H 2 O + 0.1% FA [formic acid]) and mobile phase B (MeCN + 0.1% FA) was used: 0-0.1 min (99% B, 0.4 ml min −1 ), 0.1-7 min (99%-30% B, 0.4 ml min −1 ), 7-10 min (99% B, 0.4 ml min −1 ). Samples were analyzed as 2 µl injections in positive ion mode (spray voltage 3.5 kV, vaporizer temperature 350 • C, ion transfer tube temperature • C, sheath gas 50 a.u., sweep gas 1 a.u., and aux gas 10 a.u.) with a mass range of m/z 60-1000. Mass spectrometer 1 (MS1) data were collected with a resolving power of 60 000 and an automatic gain control target of 1 × 10 −6 . Mass spectrometer 2 (MS2) data were collected with a resolving power of 30 000, cycle time of 0.6 s, and stepped higher-energy collision dissociation energy of (30, 50, 150 eV). The raw data were imported into Skyline, an open-source MS analysis software package [35], for peak integration and selection using targeted data processing.

Description of the model
GlobalKin is a 0D plasma chemistry model that includes the capability to address plasma-liquid interactions across an interface. Detailed descriptions of GlobalKin and methods used to address plasma-liquid interactions can be found in [36,37] and so are discussed only briefly here. In short, species densities in the gas phase are determined according to continuity equations that account for sources and losses due to electron impact and heavy particle reactions, diffusion to the walls, and gas flow. The electron energy equation is solved to provide a reactor averaged or position dependent electron temperature. Electron energy distributions are determined by solving the stationary Boltzmann equation over a range of E/N values. Rate coefficients obtained from these distribution functions are stored in a look-up table as functions of electron temperature. The look-up tables are interpolated throughout the execution of the model to provide electron impact reaction rate and transport coefficients at the current electron temperature. The table is periodically updated to reflect changes in gas composition.
Gas flow is approximated using a plug-flow approach. The plug-flow approximation is valid for subsonic flow systems having constant pressure with there being little diffusion in the flow direction. In the plug-flow approach, gas flows down the length of the reactor as a slug of gas having cross-sectional area A = WD where W is the electrode width and D is the distance between the electrodes. Transport perpendicular to the flow direction to the walls of the reactor is addressed by ambipolar diffusion for charged particles and fundamental mode free diffusion for neutral particles. Integration in time is converted to integration in space along the flow direction by translating As such, there is not a spatial mesh. Values are recorded after the spatial location changes by fixed increments, here about every 70 µm. The speed of the plug is a function of the inlet flow rate (sccm), gas-density, and the reactor cross-sectional area. The flow speed is adjusted with changes in total gas number density due to gas heating or reactions to maintain a constant pressure. When using plug-flow, input power (W cm −3 ) is specified as a function of reactor position in the flow direction. In this investigation, the specified power density is constant along the length of the reactor with the exception of a 1 mm ramp-up distance at the beginning of the reactor and 2 mm ramp-down distance at the end of the reactor. The power density profile is normalized such that the integral over the entire reactor volume of power density equals the total desired input power. Although the COST-jet operates with a radio frequency power supply, the power along the axis is not time varying-the power is the average over the RF period.
GlobalKin includes a liquid module that accounts for the interaction of the gas-phase plasma in contact with a liquid layer. All gas-phase species have solvated liquid counterparts. A separate liquid-phase reaction mechanism is included for these solvated species. The gas-phase species and liquid-phase species interact only at the gas-liquid interface. Transport is limited to diffusion into or out of the liquid layer. Electrons and heavy charged species solvate into the liquid with unity probability where most positive ions charge exchange with water produce to H 2 O + aq which then rapidly converts to H 3 O + aq . Penning ionization or dissociative excitation transfer occurs with excited states of He incident onto the liquid. That said, the plume of the COST-jet onto the liquid is nearly charge free (by design) and excited states of He are largely quenched prior to reaching the liquid. As a result, the dynamics of charge particles (or Penning processes) due to their fluxes from the gas phase onto the liquid are not particularly important in this investigation.
Neutral gas-phase species diffuse into and out of the liquid with a probability determined by the Henry's law equilibrium constant h i for that species. The probability that a neutral gasphase species will solvate into the liquid can be thought of as a sticking coefficient at the liquid interface, S i,l , where n i,l is the density of species i in the liquid and n i,g is the density of species i in the gas. If the gas-phase and liquid phase are in equilibrium, then n i,l = n i,g × h i . Species will solvate from the gas phase into the liquid as long as n i,l ni,g < h i . When n i,l ni,g > h i the liquid is super-saturated and net transport is from the liquid into the gas-phase. The Henry's law constants used in this work are shown in table 1. Liquid species densities are determined by a species continuity equation that is similar to that used for the gas-phase. Sources and losses due to liquid phase reactions, solvation, and evaporation are accounted for in the liquid species density calculation. Several observations of plasma-jet induced vortexes in liquid have been reported, for example Stancampiano et al [38]. Such vortexing is not resolved in the global model. Creating these vortexes typically requires a deep solution, whereas the solution used in the experiment is only 1 mm thick. An important component of the forces creating vortexes is electrohydrodynamic forces which are not important for the COST-jet as the plume is largely neutral. As a result, we do not expect there to be significant vortexing for our conditions. If vortexing did occur, then there would be more mixing in the solution which would actually better align with the global model.

Reaction mechanism
The gas-phase mechanism for a He/O 2 /N 2 atmospheric pressure plasma has been discussed in detail in previous works Electron energy is primarily lost through inelastic collisions that produce electronically, rotationally, or vibrationally excited states of O 2 , N 2 , and As is common in modeling of complex plasma chemistry, excitation to several excited states is lumped into producing a single state, here denoted with * , which is usually the lowest lying state with the longest lifetime. This practice acknowledges that higher excited states will relax or be quenched on times shorter than those of interest. Typically, only the lowest lying, metastable states survive in the plume to reach the liquid. Every gas-phase species has a liquid-phase counterpart. The liquid-phase mechanism has been discussed previously in [36,41]. Neutral species from the gas-phase solvate into the liquid-phase as described in the previous section. Reactive neutrals can combine in the liquid to form more stable molecules (e.g. OH aq , H 2 O 2aq , O 3aq , HNO 3aq , NO 3aq ). Solvated RONS and RONS produced by plasma-assisted liquid reactions react with organic molecules in the liquid to produce oxidated and nitrosylated organic products.
Intermediate product formation

Additional reactions
a Reaction rate coefficients have units of cm 3 s −1 . b Reaction rate is approximated from an analogous gas-phase reaction with a long chain alkane from the NIST database [44] or from analogous reactions in solution from the NIST solutions [45] database and adjusted to match experimental measurements.
Cysteine is an organic compound containing a carboxyl group (-COOH), an amine group (-NH 2 ), and a thiol group (-SH). (See figure 1.) The cysteine reaction mechanism includes 17 cysteine-derived species and 40 liquid-phase reactions of RONS with cysteine. The reaction mechanism and reaction rate coefficients used in this work are shown in table 3. Only reactions with the thiol group of the cysteine molecule are considered in this work. The sulfur atom in the thiol group is highly reactive in comparison to the other parts of the cysteine molecule, leading to preferential interaction of RONS with the thiol site [21,29]. The presence of the carboxyl group leads to resonance stabilization of the cysteine molecule which contributes to a reduction in reactivity at sites other than the thiol site [42].
Similar to the gas-phase oxidation of long-chain alkanes [43], the liquid-phase oxidation of the cysteine thiol site can be considered a three-step mechanism. Oxidation begins with abstraction of the H atom from the thiol site by O aq , O − aq , OH aq , OH − aq , or HO 2aq to form a thiol radical site. Reactions of RONS at the thiol radical site result in short-lived intermediate products that can undergo rearrangement or subsequent reactions with RONS to form stable oxidation products. In this work an oxidation product is considered stable when its lifetime is long enough to enable experimental detection [30]. We consider cysteine sulfenic acid (Cys-SO 2 H), cysteine sulfonic acid (Cys-SO 3 H), cysteine-S-sulfonate (Cys-SSO − 3 ), cystine (Cys-S-S-Cys), and nitrosylated cysteine (Cys-SNO) to be stable compounds. The structures of these molecules are shown in figure 1. (Since Cys-species only exist in solution, the 'aq' suffix will not be used in the following text).
The reaction mechanism of RONS with cysteine shown in table 3 was initially based on MD simulations by Lackmann et al [30]. Reaction rate coefficients for oxidations of organic molecules in solution are largely unavailable and so have been estimated based on analogous gas-phase reactions of RONS with alkanes taken from the National Institute of Science and Technology (NIST) Chemical Kinetics Database [44]. In the few cases where they are available, reaction rate coefficients of RONS with the native cysteine molecule were taken from the NIST Solutions database [45]. Reaction rate coefficients have been adjusted to account for differences in the interactions of molecules in the liquid-phase rather than the gasphase and to achieve consistency with experiments. In general, gas-phase reaction rate coefficients produced shorter times for cysteine depletion and oxidation in the liquid-phase than observed experimentally. These smaller rate coefficients in large part reflect transport limits in the liquid compared to the gas phase.

Validation of the reaction mechanism
Stapelmann et al [46] tracked several COST-jet produced ROS from the gas-phase to the liquid phase to predict their impact on the modification of cysteine molecules in a water solution.  fluxes to the liquid. The stable cysteine oxidation products considered were cysteine-S-sulfonate (Cys-SSO 3 H), cysteine sulfenic acid (Cys-SO 2 H), and cysteine sulfonic acid (Cys-SO 3 H). The combination product cystine (Cys-S-S-Cys) was also measured. The authors found significant differences in the reaction pathways between treating the solution with the COST-jet sustained in He/O 2 and He/H 2 O. Treatment with the COST-jet using the He/O 2 mixture resulted in preferential formation of irreversible oxidation products (i.e. Cys-SO 3 H) while treatment by the COST-jet using the He/H 2 O mixture favored reversible oxidation products (i.e. Cys-SO 2 H). The authors attributed the difference in product formation to the origin of the ROS responsible for the modification. ROS known in redox biology, like OH in the He/H 2 O mixture, caused more reversible oxidation products while ROS unknown to nature, i.e. O in the He/O 2 mixture, caused irreversible reaction products.

He/O 2 and He/H 2 O COST-jet treatment
Results from the Stapelmann et al study [46] have been reproduced in this section to provide validation for the reaction mechanism discussed in section 4. The MS-produced peak intensity measurements for cysteine-derived oxidation products formed over a five-minute treatment period by exposure to the effluent from COST-jets sustained in He/O 2 and He/H 2 O which are shown alongside model-produced densities in figures 2 and 3. Values have been normalized to one to provide a more direct comparison of the trends in product formation over time between the experiments and the model. The model uses the same reaction mechanism as discussed in section 4. Only the gas composition has been adjusted to match the gas composition used in the experiments.
Overall, trends predicted by the model are in good agreement with the measured values. For treatment of cysteine using the COST-jet sustained in He/O 2 (figure 2), cysteine is significantly depleted after five minutes of plasma treatment as H is abstracted from the thiol group by ROS (O aq , OH aq , HO 2,aq ) to form thiol radical sites. Thiol radical sites are converted to intermediate oxidation products by subsequent reactions with ROS. Intermediate products can then be converted to stable oxidation products by reactions with RONS or with reactions involving O 2aq . The reaction mechanism relating the formation pathways of the cysteine oxidation products to the fluences of plasma-produced RONS is discussed in section 7.
Cys-S-SO 3 H and Cys-SO 3 H (irreversible oxidation products) are preferentially produced after 2-3 min of plasma treatment, which is consistent with the results by Bhoj and Kushner [39]. The model also accurately predicts the trend in generation of Cys-SO 2 H, which forms over the first minute of plasma treatment, then is depleted over the next four minutes as Cys-SO 2 H is converted to Cys-SO 3 H by reactions with solvated ROS.
When exposed to the COST-jet sustained in He/H 2 O, Cys-SO 3 H and Cys-S-SO 3 H are also formed, though neither are Hydrogen is abstracted from the thiol (-SH) site of the cysteine molecule to create a thiol radical site. In the absence of significant densities of ROS, and especially at early treatment times, two thiol sites will react to form Cys-S-S-Cys.

Base case plasma and liquid species properties
The reactor configuration used for the results in this section is based on the experimental setup discussed in section 2. The COST-jet sustained in He/O 2 has been extensively characterized [32]. Typical O atom densities for 0.2-1.0% O 2 admixtures have been reported to be between 1 and 3 × 10 15 cm −3 while O 3 densities increase from 1 × 10 14 cm −3 to 1 × 10 15 cm −3 as the percent of O 2 increases [47][48][49]. The COST-jet is most often operated in a power range between a few hundred milliwatts and a few watts, which yields electron densities on the order of a few times 10 10 cm −3 [50].
The model considers the radio-frequency (RF) powered, 1 atm COST-jet operated in He/N 2 /O 2 mixtures with a constant power of 750 mW. The base case is 1010 sccm of a feed gas consisting of He/N 2 /O 2 = 99/0.8/0.2 with a 1 ppm water impurity. The gas flows through the top of the reactor and moves vertically down the length of the reactor (3 cm) and across the gap between the reactor and liquid (4 mm). The gap between the powered and grounded electrode is 1 mm and the width of the electrodes is 1 mm, yielding a plasma with a cross-sectional area of 1 mm 2 . The plasma is assumed to be uniform over the electrode cross-sectional area. There is a 4 mm gap between the reactor outlet and the liquid surface in which the reactor effluent comes into contact with ambient air. In this plug flow approach, the entrainment of ambient air into the effluent is approximated by adding humid air (N 2 /O 2 /H 2 O = 79/20/1) with a flow rate of 10 sccm into the mixture as the gas exits the discharge channel.
The treated liquid is a 1 mm thick solution of water containing 100 µg ml −1 cysteine (5 × 10 17 cm −3 cysteine molecules). The liquid initially has a pH = 7 and is initially saturated with atmospheric gases.

Plasma properties
The densities of gas phase species predicted by the model for the base case conditions are shown in figure 4. The average electron density in the plasma channel predicted by the model is 3 × 10 10 cm −3 and the average electron temperature is 2.2 eV. (Here, electron temperature is 2/3 of the average electron energy, obtained from the electron energy distribution that in most cases is non-Maxwellian.) These values are consistent with experimentally measured values for the COST-jet sustained in He/O 2 [50]. RONS including O, O 3 , NO, OH, and HO 2 are produced in the powered region and exit the reactor at 3.0 cm to mix with ambient air in the gap above the liquid. Atomic oxygen is formed predominantly through electron impact dissociation of molecular oxygen or through dissociative excitation transfer to O 2 or excited state O 2 from excited state N 2 . The predicted atomic oxygen density (4 × 10 15 cm −3 ) is consistent with experimentally reported values of atomic oxygen measured in the He/O 2 COST-jet [48,49].

Liquid RONS production
Densities of aqueous species produced during five minutes of treatment by the COST-jet sustained in He/O 2 /N 2 are shown in figure 5. Plasma produced RONS solvate into the liquid according to their Henry's Law equilibrium constants (i.e. species having higher Henry's law constants will solvate more readily than those with lower Henry's law constants). Solvation is primarily responsible for the production of reactive aqueous neutrals such as OH aq , NO aq , and O aq . Solvation and subsequent saturation of OH aq , NO aq , and O aq occur quickly (about 2 ms) after liquid exposure to the plasma effluent while other species such as H 2 O 2aq , O 3aq , and HO 2aq require longer exposure (0.5 s-1 min) to saturate. Species, that as a result of aqueous reactions, become supersaturated will desolvate into the gas phase. In addition to solvation, H 2 O 2aq   H 2 O 2aq and O 3aq are considered long-lived species as they are relatively stable (thermal decay occurs, but on timescales longer than the studied treatment time). Long-lived species can produce more reactive products over their lifetimes, either in reactions with other RONS or by slow-decay processes.
(H)NO x species are some of the most abundant plasmaproduced RONS that solvate into the liquid. The dominant gas phase RONS are NO (5 × 10 14 cm −3 ) and HNO 2 ( The increase in H 3 O + aq results in a decrease of the modelpredicted pH from 7 to 3.9 over the course of the five-minute treatment for the COST-jet sustained in He/O 2 /N 2 , whereas mixtures not containing N 2 tend to produce smaller changes in pH [51,52]. Cysteine is stable within the pH range studied in this work and so pH does not play a direct role in the modification of the cysteine molecule [53]. However, the pH tolerances of the target molecule and surrounding system should be taken into consideration when designing plasma devices for biological treatments.

Formation of cysteine oxidation products
In this section, the model-predicted densities of cysteine oxidation products after plasma treatment will be discussed while varying the gap distance between the reactor exit and the liquid and the inlet gas composition. A comparison between model results and experimental mass spectroscopy measurements will be presented to motivate the addition of N 2 to the gas inlet.

Base case modifications to native cysteine
Plasma jets in general, and the COST-jet in particular, potentially offer precise control over RONS delivered to a target by varying the inlet gas composition [54,55]. For example, addition of N 2 to the COST-jet sustained in He/O 2 increases the flux of plasma-produced (H)NO x species reaching the liquid while decreasing the inventory of O 3 formed in the plasma. However, the effects of plasma-produced (H)NO x on the modification of biological targets in plasma treated solutions is still unclear. Experiments described in section 3 were performed to help illuminate the consequences of N 2 addition to the inlet gas on cysteine oxidation product formation. MS was performed and used for further validation of the reaction mechanism for the tri-gas conditions. Normalized model-produced densities of cysteine and several oxidation products for the base case discussed in section 6 are shown in figure 6 alongside normalized MS peak intensity measurements from experiments also performed under the base case conditions in figure 6.
The model-produced aqueous number densities of cysteine and its stable derivatives are shown in figure 7 as a function of plasma treatment time. The model predicts major cysteine depletion occurs after 10-20 s of plasma treatment. The model is initialized with the only organic molecule in the water being cysteine while the experiments naturally include cystine or other unstable products that may revert back to cysteine as a result of plasma treatment. This likely explains the differences in the severity of depletion of the native cysteine between the model and the experiments. Cystine (Cys-S-S-Cys) and Cys-SSO 3 H are the first stable products to form with significant number densities (>10 8 cm −3 ).
Short-lived O and OH solvate into the liquid and their inwater densities reach saturation within a few ms. These saturation values are not a classical Henry's law equilibrium but rather an equilibrium between the rates of solvation of these species (a source) and their reaction with the organic molecules in solution (a sink). These ROS abstract H atoms from the cysteine thiol (-SH) group to form thiol radicals. Cystine initially forms from two thiol radicals, as densities of RONS, including negative ions and long-lived species such as H 2 O 2aq , O 3aq , HO 2aq , and NO xaq increase by solvation or through aqueous reactions; Cys + OH aq → Cys − S • + H 2 O aq (16) Cys Within the first few milliseconds of plasma treatment, Cys-SSO 3 H also becomes a major product. Several mechanisms for the formation of Cys-SSO 3 H from cysteine have been proposed [56,57]. The formation of Cys-SSO 3 H may occur through cleavage of the SH group by radical attack and subsequent oxidations by SH-derived species (e.g. SO − 2 aq , SO − 3 aq ) at the radical site, or through the cystine oxidation-reduction cycle. The mechanism for Cys-SSO 3 H formation in this work is based on that proposed by Wende et al [25]. Cys-SSO 3 H is represented in the mechanism as Cys-SSO − 3 , which is conventional in oxidation-reduction chemistry. We assume that Cys-SSO − 3 and Cys-SSO 3 H are quickly reversible in solution and use the two representations interchangeably from this point forward. The   Cys After about 4 ms, HSO − 3 aq forms with high enough number density (a few times 10 10 cm −3 ) to produce Cys-SSO 3 H through reaction with either cysteine or Cys-S-S-Cys. Cys-SSO 3 H is the primary stable oxidation product for the first 20 s of plasma treatment, after which production of sulfonic acid (Cys-SO 3 H) dominates.
While Cys-SSO 3 H and cystine are being formed, the first oxidation state of the thiol radical, sulfenic acid (Cys-SOH), is formed through a direct reaction involving OH aq or a series of indirect reactions involving both OH aq and O 3aq . The density of O 3aq saturates about ten times more slowly than OH, which saturates within 2 ms, as aqueous reactions contribute to the accumulation of O 3aq . O 3 has a saturated aqueous density of around 1 × 10 14 cm −3 while OH has a saturated aqueous density of around 7 × 10 13 cm −3 . A density of 2 × 10 8 cm −3 Cys-SOH forms by the time the density of OH aq has reached saturation, after which the rate of formation of Cys-SOH briefly matches the rate of accumulation of O 3aq (from 2-20 ms).
At early treatment times (<a few ms), before saturation of most other RONS, the direct pathway to Cys-SOH formation (equation (26)) is likely dominant. Direct OH aq addition to cysteine is slow compared to the formation of the Cys-SOH 2 intermediate and most other competing reactions that also consume OH aq . At later times, when most other RONS are saturated, the indirect pathway for Cys-SOH formation is likely dominant, Cys + OH aq → Cys − SOH 2 (27) Cys − SOH 2 + O 3aq → Cys − SOH + O 2aq + OH aq . (28) Cys-SOH forms continuously for the first 1.5 min of treatment time before depletion starts to occur faster than formation as Cys-SOH is converted to more stable, higher oxidation products.
Conversion from Cys-SOH to sulfinic acid (Cys-SO 2 H) requires the addition of OH aq to Cys-SOH to form the intermediate product Cys-S(OH) 2 After 20 s of plasma treatment, Cys-SO 3 H becomes the predominant stable oxidation product over Cys-SSO 3 H. The model-predicted density of Cys-SO 3 H after 5 min of plasma treatment is 2.4 × 10 16 cm −3 and the density of Cys-SSO 3 H after 5 min of plasma treatment is 5.2 × 10 14 cm −3 . Cys-SO 3 H is not an easily reversible product. In living organisms, formation of Cys-SO 3 H is irreversible, though formation of Cys-SO 2 H and Cys-SOH can be reversed [27,58]. This reversibility of lower oxidation states may contribute to lower densities of Cys-SOH and Cys-SO 2 H in the experiments, especially at longer treatment times and under anaerobic conditions.
The oxidation of cysteine appears to be limited both by the step-wise nature of its oxidation pathways and by the hydrogen abstraction step. The hydrogen abstraction step is dependent on the solvation of fluxes of gas phase O and OH into the liquid. Cysteine oxidation product formation sensitivity to RONS production will be discussed in the following subsections.

Air gap
The distance between the reactor outlet and the liquid surface can be used as a control parameter to tune the flux of RONS reaching the liquid. The gap is adjusted in the model by extending the length over which plug flow is calculated after power is turned off at the end of the 3 cm electrode length. To emulate ambient air mixing, air is introduced to the plasma effluent at 3 cm with a flow rate that is 1% of the total inlet flowrate. Increasing the gap increases the amount of mixing between the COST-jet's effluent and the ambient air. The fluence of gas phase species onto the liquid and number densities of aqueous cysteine oxidation products after five minutes of treatment with the COST-jet sustained in the He/N 2 /O 2 mixture are shown in figure 8. The densities of the aqueous species are normalized by their values for a gap of 10 mm to emphasize the trends with gap.
As the gap increases, the final number density of the cysteine increases (from 1 × 10 13 cm −3 when the gap is 1 mm to 1 × 10 16 cm −3 when the gap is 10 mm). This corresponds to a reduction in cysteine depletion from nearly 100% with a 1 mm gap to 49% for a gap of 10 mm. As the gap increases, air entrainment into the plasma effluent also increases. The fluence of plasma-produced short lived RONS onto the liquid decreases as reactive species are converted into more stable species in the air gap. The fluence of short-lived RONS such as O that are primarily produced in the plasma are more sensitive to changes in the gap. There are less dramatic decreases in aqueous number densities as the gap increases for RONS that can be replenished by aqueous reactions, such as O 3aq and OH aq . Increasing the gap from 1 mm to 10 mm results in a five times reduction in saturated O aq density from 3.5 × 10 11 cm −3 to 7 × 10 10 cm −3 , whereas the reduction in OH aq is only two times, from 8.6 × 10 13 cm −3 to 3.6 × 10 13 cm −3 . The O 3aq density is transient, and does not reach saturation, but has maximum values at the end of treatment time (3.6 × 10 14 cm −3 for a 1 mm gap and 2 × 10 14 cm −3 for a 10 mm gap). O 3 solvates into the liquid, but O 3aq can also be generated by aqueous reaction (equation (12)). O 3aq is consumed by reaction with intermediate cysteine oxidation products (reactions (13), (15), and (22) in table 3) or by charge exchange with negative ions (equation (38)) faster than it is produced by reactions with aqueous ROS for the first 6-7 s of plasma treatment time, The rate of consumption of O 3aq gradually decreases over the remaining treatment time (as intermediate cysteine products are converted to higher oxidation states) while the rate of production due to aqueous reactions is nearly constant (as O aq and O 2aq saturate). At the end of the plasma treatment time, the rate of O 3aq production by aqueous reaction is nearly double the rate of O 3aq consumption. With the gas phase density of O 3 above the water being 5 × 10 14 cm −3 , and a Henry's law constant of 0.3, the saturated aqueous density is 1.5 × 10 14 cm −3 . So the in liquid production of O 3aq produces slightly super-saturated conditions that result in the desolvation of O 3aq . Trends in oxidation resulting from varying gap can be extended to understanding the hierarchy of RONS' importance in the production of cysteine oxidation products. As the gap increases from 1 mm to 10 mm, the final number density of Cys-SSO 3 H increases from 4 × 10 14 cm −3 to 8 × 10 14 cm −3 , Cys-SO 2 H increases from 5 × 10 13 cm −3 to 9 × 10 13 cm −3 , and Cys-SNO increases from 1 × 10 13 cm −3 to 2 × 10 13 cm −3 . The final number density of Cys-SO 3 H decreases from 3 × 10 16 cm −3 to 1 × 10 16 cm −3 . Cys-SSO 3 H formation requires O aq or OH aq for H abstraction, but subsequent reaction steps are driven by O 2aq . There is little change in the O 2aq density as gap distance increases due to its saturation, indicating that a reduction in short-lived aqueous ROS favors Cys-SSO 3 H formation.
While not shown in figure 8, as it is not considered a stable product, the final number density of Cys-SOH also increases from 5 × 10 15 cm −3 at 1 mm gap to 2 × 10 16 cm −3 at 10 mm gap, though more Cys-SOH is produced in the 1 mm case than in the 10 mm case for the first 110 s of plasma treatment. The formation of Cys-SOH enables pathways to formation of higher order oxidation products. Cys-SOH formation is dependent on OH aq and O 3aq (equations (27)-(29)) while its conversion to Cys-SO 2 H requires O aq , Oaq , OH aq , or O 2aq (equations (30)- (34)). Further conversion to Cys-SO 3 H (equations (35)-(38)) requires O aq , OH aq , O 2aq , or H 2 O 2aq . H 2 O 2aq is a long-lived aqueous species whose saturated number density decreases from 1 × 10 16 cm −3 to 7 × 10 15 cm −3 as the gap increases from 1 mm to 10 mm.
As the fluences of short-lived ROS onto the liquid decrease with increasing gap, the formation of Cys-SOH and Cys-SO 2 H, the first and second oxidation states, are not particularly sensitive to reduction of O aq . However, as Cys-SO 2 H fails to be converted to Cys-SO 3 H at larger gaps, formation of the third oxidation state does become sensitive to O aq concentration, H 2 O 2aq concentration, or both. Based on gas phase analogues and agreement with experiment, the reaction rate coefficient of H 2 O 2aq with Cys-SO 2 H (equation (37)) is 3-6 orders of magnitude smaller than reactions of Cys-SO 2 H with other ROS (O aq , OH aq ). As a result, Cys-SO 3 H formation is likely most sensitive to the O aq density. However, as Cys-SO 3 H is the most abundant oxidation product under all conditions, fluences of O on the order of a few times 10 18 cm −2 are sufficient to promote full oxidation. Further studies are needed to decouple the effects of short-lived and long-lived species on the formation of the Cys-SO 3 H.
Cys-SNO formation is dependent on the presence of NO aq . As the gap increases from 1 mm to 10 mm, there is a small increase in the saturated density of NO aq from 4 × 10 12 cm −3 to 5 × 10 12 cm −3 though the fluence of NO decreases (from 4 × 10 17 cm −2 at 1 mm to 2 × 10 17 cm −2 at 10 mm). Higher air entrainment in the larger gap leads to conversion of NO to HNO x in reactions with H and a third body. HNO x hydrolyzes after solvation to regenerate NO aq (equations (13) and (14)), which contributes to the slight rise in NO aq density as the air gap increases. As the increase in NO aq concentration between the smaller and larger gap is not large, it is more likely that as the gap increases, the reduction in O, OH, and NO fluences, and the resulting aqueous ROS concentrations, contribute to a decrease in competition for reactions at the thiol radical site and a higher rate of formation of Cys-SNO. These trends suggest that increasing the air gap could be a control mechanism if nitrosylated cysteine is the desired product.
Cystine is the only stable product that is not significantly affected by changing the gap. Cystine forms quickly after initial H abstraction from the thiol group (equations (15)-(17)), which indicates that the flux of RONS from the plasma to the liquid is sufficient to initiate cysteine oxidation for all gaps. This result suggests that fluences of RONS on the order of 10 18 cm −2 are greater than needed to promote H abstraction, though the percentage depletion of the initial cysteine molecule may decrease at larger gaps. As such, gap size can be used as a control parameter to select for desired cysteine oxidation products. However, if complete conversion of native cysteine to oxidized products is desired, gap size may not be the most effective operating parameter.

Oxygen inlet flowrate
To determine the consequences of ROS fluxes produced by the He/N 2 /O 2 COST-jet on the formation of cysteine oxidation products, the inlet mole fraction of oxygen was varied while keeping the inlet mole fraction of nitrogen (f N2 = 0.8%) and the total inlet flow rate (1010 sccm) constant. The gas-phase densities of a selection of RONS as a function of position in the reactor (and gap) are shown in figure 9 for oxygen inlet flow rates from 1-30 sccm, corresponding to O 2 mole fractions of 0.1%-3%.
As oxygen inlet flow rate increases from 1 sccm (f O2 = 0.1%) to 8 sccm (f O2 = 0.8%) there is an increase in gas-phase RONS production. O and NO have maximum densities of 5.6 × 10 15 cm −3 and 6 × 10 14 cm −3 for f O2 = 0.1%. For O 2 inlet mole fractions higher than 0.1%, the densities of O and NO begin to decrease as O is consumed by three-body reactions in favor of O 3 production. There is a monotonic increase in O 3 production as O 2 inlet flow rate increases. As the level of water impurity is kept constant, the amount of OH produced in the gas-phase is not greatly affected.
The smallest density of O is produced when the inlet oxygen mole fraction is f O2 = 0.1% and the smallest density of NO is produced when f O2 = 3%. At low flow rates under low power conditions where the fractional dissociation of O 2 is low, there is simply not enough O 2 in the inlet to produce high densities of O. At high inlet O 2 fractions, the consumption of O by three-body processes competes with the consumption of O by reactive nitrogen species to form NO. To maximize both O and NO production in the gas phase, the inlet O 2 fraction must be high enough to produce sufficient O by dissociation, but low enough so that losses of O to O 3 are not dominant.
Although the highest density of O is produced in the gasphase when the O 2 inlet mole fraction is f O2 = 0.8%, the highest density of O aq (2 × 10 11 cm −3 ) occurs when the O 2 inlet fraction is f O2 = 0.2%. For these conditions, O 2 mole fractions higher than 0.2% result in significant consumption of O in favor of O 3 production in the air gap. The highest densities of OH aq (6.4 × 10 13 cm −3 ) and H 2 O 2aq (1.1 × 10 16 cm −3 ) are also produced at 0.2% inlet O 2 . Losses of NO are not as large as loss of O in the air gap and so the highest NO aq density (5.5 × 10 12 cm −3 ) occurs at f O2 = 0.8%, when the gas-phase NO production is highest.
Fluences after five minutes of plasma treatment of select RONS that are responsible for the formation of cysteine oxidation products are shown in figure 10(a) for different inlet O 2 fractions. The densities of cysteine and its oxidation products after five-minutes of plasma treatment are shown in figure 10(b). As O 2 inlet fraction increases, depletion of cysteine decreases from over 99% at the lowest O 2 fraction to about 84% at the highest O 2 fraction. Low O 2 inlet fractions yield higher fluences of RONS (O aq , OH aq , HO 2aq ) that contribute to hydrogen abstraction from  With an increase in O 2 inlet fraction, the fluence of O 3 onto the liquid increases. There is a higher rate of formation of Cys-SOH through the intermediate product Cys-SOH 2 , which regenerates O 2aq and OH aq from O 3aq (equation (28)). OH aq converts Cys-SOH to Cys-SO 2 H (equation (29)). When the fluence of OH to the liquid decreases (with increasing O 2 inlet fraction), Cys-SOH is not converted to Cys-SO 2 H as readily. However, OH fluences of at least 6 × 10 11 cm −2 are sufficient to promote formation of the second oxidation state with densities on the order of 10 14 cm −3 .
Though there is a decrease in Cys-SO 2 H production from the lowest inlet O 2 fraction to the highest, there is an increase in final Cys-SO 3 (25)). As the densities of other aqueous RONS (O aq , OH aq ) decrease with increasing inlet O 2 , formation of Cys-SSO 3 H becomes more viable due to increasing densities of O 2aq . Reduction in RONS concentrations also reduces competition between reactions resulting in thiol radical sites (equations (15)- (17)) and reactions of the native cysteine molecule to form cystine (equation (18)). The final density of Cys-SSO 3 H is 5.2 × 10 14 cm −3 for f O2 = 0.2% and 1.8 × 10 15 cm −3 for f O2 = 3%.
As Cys-SNO formation is dependent upon NO aq (reaction (36) in table 3), production of Cys-SNO is expected to be highest when the NO aq production is highest (f O2 = 0.8%, when the NO fluence is also highest). The final Cys-SNO density is 1.9 × 10 13 cm −3 when f O2 = 0.8% and 1.4 × 10 13 cm −3 when f O2 = 0.2% (base case conditions). Cys-SNO production suffers at higher O 2 inlet fractions, though it is not as greatly affected by increasing O 2 flow rate as other oxidation products. This lack of sensitivity results from the fluence of NO not being as greatly affected by changing O 2 inlet fraction when the N 2 inlet fraction is held constant. As the rate coefficient for the formation of Cys-SNO at a thiol radical site is 2-3 orders of magnitude smaller than the rate coefficient of other oxidation products with the thiol radical site (reaction (36) in table 3), increased Cys-SNO production likely requires NO fluences to be orders of magnitude higher. However, NO fluences on the order of a few times 10 17 are sufficient to produce Cys-SNO with densities on the order of 10 13 cm −3 .
Cystine production is reduced as inlet O 2 increases (2.1 × 10 17 cm −3 f O2 = 0.1% to 9 × 10 16 cm −3 at f O2 = 3%). At f O2 = 0.2%, when O aq and OH aq densities are highest (and O fluence is highest), thiol radical production occurs most readily by reaction of cysteine with O aq and OH aq (equations (15) and (16)). When densities of thiol radical sites are high, the fast conversion of two thiol radical sites to cystine occurs more quickly than other ROS can react to form oxidation products (equation (18)). When fluences of ROS are reduced, the production of cystine decreases. Less RONS are available to participate in H abstraction, resulting in a lower density of thiol radical sites. Competition between RONS reactions that form oxidation products and the near immediate reaction of two thiol radical sites to form cystine is reduced due to the lower density of thiol radical sites. More thiol radical sites can be oxidized. The production of Cys-SO 3 H is increased due to the increase in O 2aq discussed above, though depletion of the native cysteine molecule is reduced at higher O 2 inlet fractions. Total fluences of H-abstracting RONS on the order of 10 18 cm −2 are sufficient to promote Habstraction that results in at least 84% depletion of the native cysteine molecule.
Control of hydrogen abstraction from cysteine is critical if oxidation products are preferred over the formation of cystine. If full conversion of the cysteine to oxidation products is desired, lower fluences of plasma-produced RONS for longer treatment times may be preferable. If nitrosylated products are also desired, the gas-phase production of NO should be maximized while also controlling the rate of thiol radical site formation.

Nitrogen inlet fraction
N 2 inlet fraction was varied (f N2 , from 0%-5%) while holding the O 2 inlet fraction (f O2 = 0.2%) and the total inlet flow rate (1010 sccm) constant to investigate the consequences of RNS production on cysteine oxidation product formation. Of special interest here is the formation of the nitrosylated product, Cys-SNO. Maximizing NO additions to organic molecules may be desirable for applications in wound healing and cancer treatment. For example, the presence of NO during wound treatment lowers risk of infection and wound complication by promoting increased cell motility, providing anti-bacterial effects, and influencing gene regulation [59]. Final fluences of select RONS that contribute to the formation of cysteine oxidation products as a function of inlet N 2 fraction are shown in figure 11(a). The final densities of cysteine oxidation products as a function of f N2 are shown in figure 11(b).
Increasing the N 2 flow rate while holding the O 2 flow rate constant increases the density of NO x species produced in the gas phase and decreases the fluence of ROS (O, OH, HO 2 ) onto the liquid. O is consumed by NO x formation while formation of HO 2 and OH require O, O 2 , and H 2 O dissociation products such as H. Under low power conditions, O 2 and N 2 will be preferentially dissociated over H 2 O. The maximum density of gas-phase NO produced is about 5 × 10 14 cm −3 for f N2 = 1% or higher. Increasing the N 2 fraction beyond 1% does not result in significant increases in the NO density when the O 2 inlet fraction is held constant at f O2 = 0.2%. The fluence of NO to the liquid is also not terribly sensitive to f N2 (around 3 × 10 18 cm −2 for all f N2 ). At this flow rate, production of NO is limited by power and the production of O atoms and not the availability of N 2 . The production of the Cys-SNO product is also not significantly increased for N 2 inlet fractions higher than 1%. The maximum Cys-SNO density after five minutes of plasma treatment is 1.5-1.6 × 10 13 cm −3 .
Varying N 2 inlet fraction while holding O 2 inlet fraction constant results in more depletion of the native cysteine molecule than when O 2 inlet fraction is varied. Varying N 2 inlet fraction results in near complete depletion of the native cysteine molecule for all N 2 inlet fractions, whereas only 84% of the cysteine molecule was depleted for large values of f O2 . When N 2 inlet fraction is varied with the O 2 inlet fraction being low, the production of O 3 by three-body processes does not deplete O. The fluence of O 3 stays low (around 10 15 cm −2 ) while the fluences of O, OH, and HO 2 remain high enough (5-6 × 10 18 cm −2 ) that hydrogen abstraction from the thiol group (equations (16)-(18)) dominates. The high density of thiol radical sites promotes rapid formation of cystine, resulting in a near constant density of cystine (1 × 10 17 cm −3 ) regardless of the N 2 inlet fraction. Since the fluences of ROS remain high as the inlet N 2 increases, and formation of oxidation products Cys-SO 2 H, Cys-SO 3 H, and Cys-SSO 3 H depend only on ROS, not RNS, the final densities of these products are not sensitive to varying N 2 inlet fraction at constant O 2 . The variation in these species is 6-7 × 10 13 cm −3 Cys-SO 2 H, 2-3 × 10 16 cm −3 Cys-SO 3 H, and 5 × 10 14 cm −2 Cys-SSO 3 H over the range of f N2 when there is 0.2% O 2 in the inlet.
Increasing f N2 beyond 0.2% does not result in a significant change in Cys-SNO production, as the fluence of NO (3 × 10 18 cm −2 ) is limited by the O 2 inlet fraction. As discussed in the previous section, NO fluences on the order of a few times 10 17 cm −2 result in Cys-SNO densities on the order of 10 13 cm −3 . Higher production of Cys-SNO requires higher NO fluence. Higher NO fluence could potentially be achieved by adjusting the N 2 inlet fraction and the O 2 inlet fraction together.

Concluding remarks
A reaction mechanism for the CAP treatment of cysteine in water was developed and applied to the investigation of cysteine oxidation and production of nitrosylated products using a global plasma-and liquid-chemistry model. The reaction mechanism was validated against experimental data for COST-jets sustained in He/H 2 O and He/O 2 treating cysteine in water. Both gas mixtures result in depletion of the native cysteine molecule while promoting full oxidation to Cys-SO 3 H. As the presence of NO and its incorporation into oxidation products is often desirable for wound treatment applications, experiments that treated cysteine solution with a COSTjet sustained in He/N 2 /O 2 mixtures were performed. Small amounts of nitrosylated Cys-SNO are formed with He/H 2 O and He/O 2 due to mixing of the effluent of the COST-jet with the ambient air. Experimental results showed that the nitrosylated Cys-SNO product is produced in much higher quantities after treatment using the He/N 2 /O 2 mixture due to the controlled and larger production of NO x .
The validated reaction mechanism and model were used to investigate reaction pathways and mechanisms contributing to the oxidation of cysteine by the COST-jet sustained in He/N 2 /O 2 mixtures. Results indicate that some selectivity over some oxidation products can be achieved through changing plasma operating parameters such as gap distance and inlet gas flow rate of O 2 and N 2 . The oxidation of the cysteine molecule is sensitive to the gas-phase production of ROS responsible for the initiating hydrogen abstraction step. If the flux of ROS to the liquid is too high, hydrogen abstraction occurs too quickly and formation of cystine dominates over formation of oxidation products. If the Cys-SNO product is desired, the inlet fraction of N 2 should be increased though tuning other operating parameters (i.e. rf power, total flow rate) may be more effective in selecting for the nitrosylated product.
Only reactions at the cysteine thiol (-SH) site were considered in this work, as the thiol site is the most reactive site on the cysteine molecule. However, hydrogen abstraction can occur at the amino site, along the alkane backbone, or at the carboxyl site as well. Future work should consider whether contributions from these sites also play important roles in the oxidation of the cysteine molecule, as these groups are common in living organisms. Oxidation at sites other than the thiol site on other molecules will contribute to, for example, bacterial inactivation due to oxidative stress.
Cell media solutions usually contain an array of organic and inorganic molecules (i.e. vitamins, proteins) that also likely consume plasma-produced RONS, and with which cysteine derived radical species may react. The fundamental processes discussed in this investigation of RONS reacting with cysteine would be components of that larger, more complex reaction mechanism. We expect that initiating reactions of RONS with other organic molecules will be similar to those discussed here for cysteine. The greatest uncertainty is then reactions of those activated molecules with other organic molecules, leading to structures analogous to cystine or cross-linked-like molecules.

Data availability statement
The data that support the findings of this study are either contained within this paper or available from the corresponding author upon reasonable request.