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The main bactericidal components of cold atmospheric plasma (CAP) are thought to be reactive oxygen and nitrogen species (RONS) and UV-radiation, both of which have the capacity to cause DNA damage and mutations. Here, the mutagenic effects of CAP on Escherichia coli were assessed in comparison to X- and UV-irradiation. DNA damage and mutagenesis were screened for using a diffusion-based DNA fragmentation assay and modified Ames test, respectively. Mutant colonies obtained from the latter were quantitated and sequenced. CAP was found to elicit a similar mutation spectrum to X-irradiation, which did not resemble that for UV implying that CAP-produced RONS are more likely the mutagenic component of CAP. CAP treatment was also shown to promote resistance to the antibiotic ciprofloxacin. Our data suggest that CAP treatment has mutagenic effects that may have important phenotypic consequences.

Refereed/Peer-reviewed This study compares how a helium plasma jet activates peracetic acid (PAA) from tetraacetylethylenediamine (TAED) and acetic acid (AA). Hydrogen peroxide (H2O2) generated from the plasma jets reacts with TAED resulting in the formation of PAA which further dissociates into AA. The by-product AA can also react with H2O2 to form PAA, which might also be useful for antimicrobial applications when coupled with plasma. Equivalent concentrations of TAED and AA solutions are used to compare the formation of PAA after activation with a helium plasma jet. Our results showed that the concentrations of both H2O2 and PAA in plasma-activated TAED (PAT) are higher than plasma-activated AA (PAAA), and that PAT is more efficient in reducing the growth of Pseudomonas aeruginosa and Staphylococcus aureus; the pathogens commonly found in wounds. The results are attributed to the presence of more acetyl donor groups in TAED, resulting in the formation of higher concentrations of PAA and H2O2.

Deposition chemistry from plasma is highly dependent on both the chemistry of the ions arriving at surfaces and the ion energy. Typically, when measuring the energy distribution of ions arriving at surfaces from plasma, it is assumed that the distributions are the same for all ionic species. Using ethyl acetate as a representative organic precursor molecule, we have measured the ion chemistry and ion energy as a function of pressure and power. We show that at low pressure (

Antibiotics are commonly used as the first line of defense in the treatment of infectious diseases. However, the rise of antimicrobial resistance (AMR) is rendering many antibiotics less effective. Consequently, effective non-antibiotic antimicrobial strategies are urgently needed to combat AMR. This paper presents a strategy utilizing cold plasma for the "on-demand"activation of acetyl donor molecules. The process generates an aqueous-based antimicrobial formulation comprising a rich mixture of highly oxidizing molecules: peracetic acid, hydrogen peroxide, and other reactive oxygen and nitrogen species. The synergistic potent oxidative action between these molecules is shown to be highly effective at eradicating common wound pathogenic bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) and at inactivating a virus (SARS-CoV-2). Refereed/Peer-reviewed

Current methods used to treat non-muscle invasive bladder cancer are inadequate due to a high recurrence rate after surgery and occurrence of adverse events such as interstitial pneumonia following intravesical instillation therapy. Low-temperature plasma is a new form of physical therapy that provides a rich source of reactive oxygen species (ROS). Oxidative solutions, created by pre-treatment of aqueous media with plasma before application to target cells, lead to the destruction of cancer cells through oxidative stress pathways. This study focuses on the effects of plasma activated media (PAM) in bladder cancer cells. PAM treatment increases up regulation of p21 and down regulation of Cyclin D and CdK4 leading to cell cycle arrest, and concomitantly depolarises the mitochondrial membrane leading to increased mitochondrial ROS production. Cell cycle arrest and increased mitochondrial ROS production induce apoptosis in bladder cancer cells in vitro and in a bladder cancer tumour in vivo via the pro-apoptotic caspase 3/cytochrome c pathway. These observations highlight the potential of plasma activated solutions as a new adjuvant therapy in the clinical treatment of bladder cancer.

Improved retention of desirable chemical structures during plasma polymerization requires rational tailoring of plasma-phase conditions. Using ethyl trimethylacetate, we studied the effects of pressure and power on the contribution of intact molecular ions to deposition and retention of ester groups. The abundance of protonated molecular ions in plasmas varies with pressure and power, but the functionality of plasma polymers, assessed by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry, is not correlated. Together with high ion flux, the ion energy distribution was found to be a key parameter and needs to be tailored to enable the soft landing of ions on the surface after traversing the sheath. The compromise between the abundance of ions and their energy distribution is optimal near the transition between the α and γ plasma phases. Refereed/Peer-reviewed

A conical assembly of six plasma jets arranged in a rectangular pattern for biomedical applications is presented. The conical configuration increases the separation distance between individual tubes within the assembly that reduces interference between individual plasma jets and enables the jets to converge at the output, facilitating more uniform treatment as opposed to plasma jet arrays operated in parallel. Electrical and optical diagnostics of the plasma discharges and measurements of H2O2 and [Formula: see text] production in de-ionized water are used to characterize the potential suitability of the device for biomedical applications. Particularly, it was found that the efficiency in H2O2 (an important bacterial disinfection agent) production by the conical assembly of six plasma jets was more than nine-fold higher compared to its single plasma jet counterpart and that this could be achieved at a biocompatible temperature of below 300 K. Therefore, the device may find use in biomedical applications, particularly where larger area treatments are required such as for certain wounds and cancer tumors that can span areas of tens of cm2.

To investigate the potential role of the hydroxyl radical (•OH) in cold atmospheric plasma (CAP) jet treatment, two fluorescence-based methodologies are utilised to measure DNA strand breaks. The first comprises a model system of a double-stranded DNA oligomer, where the respective strand ends are tagged with fluorophore and quencher molecules; and the second, a cell culture system reporting DNA strand breaks using the γ-H2AX assay. During the various CAP jet treatments, optical emission spectroscopy is used to detect the •OH in the gas phase and electron spin resonance is used to detect the •OH in solution. The CAP jet production of the •OH is shown to correlate to CAP jet induced DNA damage both with the DNA model and in biological cells. Results indicate that the CAP jet induces a higher degree of DNA damage when the CAP plume is in contact with the target solution. The potential of a ‘plasma screen’ based upon a hydrogel film, as a method to remove the DNA-damaging •OH species from reaching skin cells, is shown to significantly reduce DNA damage whilst facilitating the delivery of hydrogen peroxide. These findings could aid in the development of CAP jet-based applications where DNA damage is the objective (e.g. in cancer treatment) and others where it is to be avoided, e.g. in open-wound treatment and dermatology.

Plasma polymerization of (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) yields thin films containing stable nitroxide radicals that have properties analogous to that of nitric oxide (NO) without short lifetimes. This property gives TEMPO films a wide variety of potential applications. Typically, control of the final film chemistry is difficult and the plasma discharge conditions must be tailored to in order to maximize the retention of these nitroxide groups during the polymerization and deposition process. In this study, plasma diagnostics and surface analysis of the deposited films were carried out to determine the optimal plasma conditions for the retention of nitroxide groups. These techniques included energy-resolved mass spectrometry, heated planar probe ion current measurements, deposition rate measurements, and x-ray photoelectron spectroscopy (XPS). Results show that operating the plasma with a combination of low input powers and high pressures produces a collisional discharge in which fragmentation of the TEMPO molecule is suppressed, leading to good retention of nitroxide groups. Ion energy distribution functions and quartz crystal microbalance measurements support the soft landing theory of ion deposition on the substrate within this γ-mode, in which the flux of low energy, soft landed ions form the primary contribution to film growth. XPS analysis of deposited polymers shows 75.7% retention of N - O groups in the polymer films deposited in a 25 Pa 5 W discharge. © 2020 Author(s).

The mechanisms of plasma in medicine are broadly attributed to plasma-derived reactive oxygen and nitrogen species (RONS). In order to exert any intracellular effects, these plasma-derived RONS must first traverse a major barrier in the cell membrane. The cell membrane lipid composition, and thereby the magnitude of this barrier, is highly variable between cells depending on type and state (e.g. it is widely accepted that healthy and cancerous cells have different membrane lipid compositions). In this study, we investigate how plasma-derived RONS interactions with lipid membrane components can potentially be exploited in the future for treatment of diseases. We couple phospholipid vesicle experiments, used as simple cell models, with molecular dynamics (MD) simulations of the lipid membrane to provide new insights into how the interplay between phospholipids and cholesterol may influence the response of healthy and diseased cell membranes to plasma-derived RONS. We focus on the (i) lipid tail saturation degree, (ii) lipid head group type, and (iii) membrane cholesterol fraction. Using encapsulated molecular probes, we study the influence of the above membrane components on the ingress of RONS into the vesicles, and subsequent DNA damage. Our results indicate that all of the above membrane components can enhance or suppress RONS uptake, depending on their relative concentration within the membrane. Further, we show that higher RONS uptake into the vesicles does not always correlate with increased DNA damage, which is attributed to ROS reactivity and lifetime. The MD simulations indicate the multifactorial chemical and physical processes at play, including (i) lipid oxidation, (ii) lipid packing, and (iii) lipid rafts formation. The methods and findings presented here provide a platform of knowledge that could be leveraged in the development of therapies relying on the action of plasma, in which the cell membrane and oxidative stress response in cells is targeted. Refereed/Peer-reviewed

Electrically generated cold atmospheric plasma is being intensively researched for novel applications in biology and medicine. Significant attention is being given to reactive oxygen and nitrogen species (RONS), initially generated upon plasma-air interactions, and subsequently delivered to biological systems. Effects of plasma exposure are observed to millimeter depths within tissue. However, the exact nature of the initial plasma-tissue interactions remains unknown, including RONS speciation and delivery depth, or how plasma-derived RONS intervene in biological processes. Herein, we focus on current research using tissue and cell models to learn more about the plasma delivery of RONS into biological environments. We argue that this research is vital in underpinning the knowledge required to realize the full potential of plasma in biology and medicine. Physical effects of plasma can be seen to depths of several hundred micrometers within tissue.Plasma-derived RONS are likely to be delivered millimeters into tissues.Speciation reveals that RONS delivered by plasma into tissue fluid and tissue are predominately stable secondary RONS - for example, H 2 O 2 , NO 2 - , and NO 3 - .The plasma generation of RONS within a hydrated target is influenced by the target matrix that can enhance or reduce the RONS concentrations and act as a reservoir of RONS.It is likely that the concentration of these plasma-derived RONS exceeds hundreds of micromoles, even at depths of several millimeters within tissue.Oxygen concentration at the time of plasma treatment significantly influences RONS generation within a hydrated proteinaceous target. Refereed/Peer-reviewed

This study investigates how addition of a 2nd ground electrode in an argon plasma jet influences the production of hydrogen peroxide (H2O2) in deionised water (DIW). Briefly, plasma is ignited by purging argon gas through a quartz tube at 1 l min−1 and applying a sinusoidal voltage of 7 kV (peak–peak) at 23.5 kHz to a high voltage stainless steel needle electrode sealed inside the quartz tube surrounded by one or two copper ring(s) that served as the ground electrode(s) situated downstream of the high voltage electrode. The mechanisms of H2O2 production are investigated through the electrical and optical plasma properties and chemical analysis of the treated DIW. We discover that the addition of a 2nd ground electrode results in higher accumulation of charges on the inner wall surface of the quartz tube of the plasma jet assembly resulting in an increase in the discharge current and dissipated power. This further leads to an increase in the electron temperature that more than doubles the H2O2 production through dissociative recombination of water vapour molecules, whilst still maintaining a biological tissue tolerable gas temperature. The double ground electrode plasma jet is shown to be highly effective at reducing the growth of common wound pathogens (Pseudomonas aeruginosa and Staphylococcus aureus) in both planktonic and biofilm states whilst inducing a low level of cytotoxicity in HaCaT keratinocyte skin-like cells under certain conditions. The information provided in this study is useful in understanding the complex physicochemical processes that influence H2O2 production in plasma jets, which is needed to optimise the development of plasma sources for clinical applications.

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887-1896, 2018.

We investigated a new approach to control protein and cell attachment inside 96-well polystyrene plates. The wells were first coated with bovine serum albumin (BSA) to inhibit cell and protein attachment. The BSA-coated wells were then treated with a helium microplasma jet for increasing times that resulted in gradual removal of BSA from the surface. It was found that the amount of enzyme and cell attachment could be controlled in the wells where BSA was only partially removed by the microplasma jet. In addition to the surface coverage of BSA, the new surface chemistry induced by the microplasma jet treatment also had an important role in the control of enzyme and cell attachment. In summary, microplasma jet treatment of BSA-coated polystyrene wells is a simple and effective method for controlling enzyme and cell attachment. This might find use for high-throughput screening of new cell culture platforms where control over the level protein, enzyme or cell adherence is needed in order to maintain a specific cell function. Refereed/Peer-reviewed

Here, a reaction-based indicator displacement hydrogel assay (RIA) was developed for the detection of hydrogen peroxide (H2O2) via the oxidative release of the optical reporter Alizarin Red S (ARS). In the presence of H2O2, the RIA system displayed potent biofilm inhibition for Methicillin-resistant Staphylococcus aureus (MRSA), as shown through an in vitro assay quantifying antimicrobial efficacy. This work demonstrated the potential of H2O2-responsive hydrogels containing a covalently bound diol-based drug for controlled drug release.

The ability of plasma-generated hydrogen peroxide (H 2O 2) to traverse bacterial biofilms and the subsequent fate of the generated H 2O 2 has been investigated. An in vitro model, comprising a nanoporous membrane impregnated with artificial wound fluid and biofilms of varying maturity was treated with a helium-driven, cold atmospheric pressure plasma (CAP) jet. The concentration of H 2O 2 generated below the biofilms was quantified. The results showed that the plasma-generated H 2O 2 interacted significantly with the biofilm, thus exhibiting a reduction in concentration across the underlying nanoporous membrane. Biofilm maturity exhibited a significant effect on the penetration depth of H 2O 2, suggesting that well established, multilayer biofilms are likely to offer a shielding effect with respect to cells located in the lower layers of the biofilm, thus rendering them less susceptible to plasma disinfection. This may prove clinically significant in the plasma treatment of chronic, deep tissue infections such as diabetic and venous leg ulcers. Our results are discussed in the context of plasma-biofilm interactions, with respect to the fate of the longer lived reactive species generated by CAP, such as H 2O 2

We explore how to configure an argon atmospheric-pressure plasma jet for enhancing its production of hydrogen peroxide (H2O2) in deionised water (DIW). The plasma jet consists of a quartz tube of 1.5 mm inner diameter and 3 mm outer diameter, with an upstream internal needle electrode (within the tube) and a downstream external cylindrical electrode (surrounding the tube). The plasma is operated by purging argon through the glass tube and applying a sinusoidal AC voltage to the internal needle electrode at 10 kV (peak–peak) with a frequency of 23.5 kHz. We study how the following operational parameters influence the production rate of H2O2 in water: tube length, inter-electrode separation distance, distance of the ground electrode from the tube orifice, distance between tube orifice and the DIW, argon flow rate and treatment time. By examining the electrical and optical properties of the plasma jet, we determine how the above operational parameters influence the major plasma processes that promote H2O2 generation through electron-induced dissociation reactions and UV photolysis within the plasma core and in the plasma afterglow; but with a caveat being that these processes are highly dependent on the water vapour content from the argon gas supply and ambient environment. We then demonstrate how the synergistic action between H2O2 and other plasma generated molecules at a plasma induced low pH in the DIW is highly effective at decontaminating common wound pathogens Gram-positive Staphylococus aureus and Gram-negative Pseudomonas aeruginosa. The information presented in this study is relevant in the design of medical plasma devices where production of plasma reactive species such as H2O2 at physiologically useful concentrations is needed to help realise the full clinical potential of the technology.

Functionalising surfaces using polymeric thin films is an industrially important field. One technique for achieving nanoscale, controlled surface functionalization is plasma deposition. Plasma deposition has advantages over other surface engineering processes, including that it is solvent free, substrate and geometry independent, and the surface properties of the film can be designed by judicious choice of precursor and plasma conditions. Despite the utility of this method, the mechanisms of plasma polymer growth are generally unknown, and are usually described by chemical (i.e., radical) pathways. In this review, we aim to show that plasma physics drives the chemistry of the plasma phase, and surface-plasma interactions. For example, we show that ionic species can react in the plasma to form larger ions, and also arrive at surfaces with energies greater than 1000 kJ∙mol–1 (>10 eV) and thus facilitate surface reactions that have not been taken into account previously. Thus, improving thin film deposition processes requires an understanding of both physical and chemical processes in plasma.[Figure not available: see fulltext.] Refereed/Peer-reviewed

Skin has a remarkable capacity for regeneration; however, with an ever aging population, there is a growing burden to the healthcare system from chronic wounds. Novel therapies are required to address the problems associated with nonhealing chronic wounds. Novel wound dressings that can encourage increased reepithelialization could help to reduce the burden of chronic wounds. A suite of chemically defined surfaces have been produced using plasma polymerization, and the ability of these surfaces to support the growth of primary human skin cells has been assessed. Additionally, the ability of these surfaces to modulate cell migration and morphology has also been investigated. Keratinocytes and endothelial cells were extremely sensitive to surface chemistry showing increased viability and migration with an increased number of carboxylic acid functional groups. Fibroblasts proved to be more tolerant to changes in surface chemistry; however, these cells migrated fastest over amine-functionalized surfaces. The novel combination of comprehensive chemical characterization coupled with the focus on cell migration provides a unique insight into how a material's physicochemical properties affect cell migration. Refereed/Peer-reviewed

We employed UV-vis spectroscopy to monitor real-time changes in the oxygen tension and concentration of reactive oxygen and nitrogen species (RONS) in deionized (DI) water during treatments with helium (He) and argon (Ar) gas plasma jets. He and Ar gas jets are both shown to de-oxygenate DI water with He being more efficient than Ar, whilst the plasma jets deliver and regulate the concentrations of hydrogen peroxide (H 2 O 2 ), nitrite (NO 2 - ) and nitrate (NO 3 - ) in DI water. The H 2 O 2 and NO 3 - production efficiency varied between He and Ar plasma jets, but was similar for NO 2 - . Whilst DI water fully equilibrated with ambient air prior to treatment (de-oxygenated by both plasma jets) when DI water was first de-oxygenated by an inert gas jet treatment, both plasma jets were found to be capable of oxygenating DI water. These insights were then used to show how different combinations of plasma jet and inert gas jet treatments can be used to modulate O 2 tension and RONS chemistry. Finally, potential further improvements to improve control in the use of plasma jets in regulating O 2 and RONS are discussed. © 2018 The Japan Society of Applied Physics.

We investigate the ability to disrupt and limit growth biofilms of Pseudomonas aeruginosa using application of cold atmospheric pressure (CAP) plasma. The effect of the bio-film's exposure to a helium (CAP) jet was assessed at varying time points during biofilm maturation. Results showed that the amount of time during biofilm growth that CAP pressure was applied has a crucial role on the ability of biofilms to mature and recover after CAP exposure. Intervention during the early stages of biofilm formation (0-8 h) results in a 4-5-log reduction in viable bacterial cells (measured at 24 h of incubation) relative to untreated biofilms. However, CAP treatment of biofilm at 12 h and above only results in a 2-log reduction in viable cells. This has potentially important implications for future clinical application of CAP to treat infected wounds.

Plasma polymerization was used to coat a melt electrospun polycaprolactone scaffold to improve cell attachment and organization. Plasma polymerization was performed using an amine containing monomer, allylamine, which then allowed for the subsequent immobilization of biomolecules, i.e., heparin and fibroblast growth factor-2. The stability of the plasma-polymerized amine-coating was demonstrated by X-ray photoelectron spectroscopy analysis and imaging time-of-flight secondary ion mass spectrometry revealed that a uniform plasma amine-coating was deposited throughout the scaffold. Based upon comparison with controls, it was evident that the combination scaffold aided cell ingress and the formation of distinct fibroblast and keratinocyte layers.

While plasma polymerization is used extensively to fabricate functionalized surfaces, the processes leading to plasma polymer growth are not yet completely understood. Thus, reproducing processes in different reactors has remained problematic, which hinders industrial uptake and research progress. Here we examine the crucial role pressure plays in the physical and chemical processes in the plasma phase, in interactions at surfaces in contact with the plasma phase, and how this affects the chemistry of the resulting plasma polymer films using ethanol as the gas precursor. Visual inspection of the plasma reveals a change from intense homogeneous plasma at low pressure to lower intensity bulk plasma at high pressure, but with increased intensity near the walls of the chamber. It is demonstrated that this occurs at the transition from a collision-less to a collisional plasma sheath, which in turn increases ion and energy flux to surfaces at constant RF power. Surface analysis of the resulting plasma polymer films show that increasing the pressure results in increased incorporation of oxygen and lower cross-linking, parameters which are critical to film performance. These results and insights help to explain the considerable differences in plasma polymer properties observed by different research groups using nominally similar processes. Refereed/Peer-reviewed

Historically, there have been two opposing views regarding deposition mechanisms in plasma polymerisation, radical growth and direct ion deposition, with neither being able to fully explain the chemistry of the resultant coating. Deposition rate and film chemistry are dependent on the chemistry of the plasma phase and thus the activation mecha nisms of species in the plasma are critical to understanding the relative contributions of various chemical and physical routes to plasma polymer formation. In this study, we investigate the roles that hydrogen plays in activating and deactivating reactive plasma species. Ethyl trimethylacetate (ETMA) is used as a representative organic precursor, and additional hydrogen is added to the plasma in the form of water and deuterium oxide. Optical emission spectroscopy confirms that atomic hydrogen is abundant in the plasma. Comparison of the plasma phase mass spectra of ETMA/H 2 O and ETMA/D 2 O reveals that (1) proton transfer from hydronium is a common route to charging precursors in plasma, and (2) hydrogen abstraction (activation) and recombination (deactivation) processes are much more dynamic in the plasma than previously thought. Consideration of the roles of hydrogen in plasma chemistry may then provide a more comprehensive view of deposition processes and bridge the divide between the two disparate schools of thought. Refereed/Peer-reviewed

Plasma-activated water ( PAW) is receiving much attention in biomedical applications because of its reported potent bactericidal properties. Reactive oxygen and nitrogen species (RONS) that are generated in water upon plasma exposure are thought to be the key components in PAW that destroy bacterial and cancer cells. In addition to developing applications for PAW, it is also necessary to better understand the RONS chemistry in PAW in order to tailor PAW to achieve a specific biological response. With this in mind, we previously developed a UV-vis spectroscopy method using an automated curve fitting routine to quantify the changes in H2O2, NO2 %, NO3 % (the major long-lived RONS in PAW), and O-2 concentrations. A major advantage of UV-vis is that it can take multiple measurements during plasma activation. We used the UV-vis procedure to accurately quantify the changes in the concentrations of these RONS and O-2 in PAW. However, we have not yet provided an in-depth commentary of how we perform the curve fitting procedure or its implications. Therefore, in this study, we provide greater detail of how we use the curve fitting routine to derive the RONS and O-2 concentrations in PAW. PAW was generated by treatment with a helium plasma jet. In addition, we employ UV-vis to study how the plasma jet exposure time and treatment distance affect the RONS chemistry and amount of O-2 dissolved in PAW. We show that the plasma jet exposure time principally affects the total RONS concentration, but not the relative ratios of RONS, whereas the treatment distance affects both the total RONS concentration and the relative RONS concentrations. (C) 2018 The Japan Society of Applied Physics Refereed/Peer-reviewed

Amine containing plasma polymer films are of interest due to their ability to bind biomolecules either covalently or electrostatically. One issue with generating such plasma polymers is the need to generate sufficient amine density on the surface to enable binding, while simultaneously maintaining the chemical, physical stability of the surface in aqueous media. Here we investigate the relationship between plasma parameters, film stability for two commonly used monomers, allylamine AA, ethylenediamine EDA. Plasma polymer films from AA, EDA were produced at radio frequency RF powers between 2 and 20 W at a constant monomer flowrate. Deposition rate, ion flux, ion energy, plasma phase mass spectrometry were used to investigate the plasma-surface interactions. Film stability was assessed by comparing X-ray photoelectron spectroscopy XPS, atomic force microscopy AFM measurements before, after washing in phosphate buffered saline PBS. The results show that films generated from EDA plasmas are generally unstable in aqueous media, while films generated from AA plasmas exhibit higher stability, particularly those deposited at high RF power. The chemical, physical stability of the films is then related to the mechanisms of deposition, the energy density provided to the surface during film growth.

The influence of the electrical and optical properties of a spatially varying He microplasma on the gradient surface modification of an adsorbed protein layer inside a microfluidic chip was investigated. Microplasma was generated inside a native polydimethylsiloxane (PDMS) microchannel. Excited and metastable states of He and N2 (from air) potentially played a major role in the removal/modification of protein during the discharge period. In-between discharge events, N2(A) metastables can also induce bond breaking and surface modification. The purge time of the feed gas was shown to be important in the use of small scale microplasma sources for surface processing or protein modification. Refereed/Peer-reviewed

Despite growing interest in the application of atmospheric plasma jets as medical treatment strategies, there has been comparatively little research on the potential genotoxic and cytotoxic effects of plasma jet treatment. In this study, we have employed the cytokinesis block micronucleus cytome (CBMN-Cyt) assay with WIL2-NS B lymphoblastoid cells to test the potential genotoxicity, as well as the cytotoxicity, of toxic species generated in cell culture media by an argon (Ar) plasma jet. Elevated levels of cell death (necrosis) and occurrence of chromosomal damage (micronuclei MN, nculeoplasmic bridge NPBs and nuclear bus, Nbuds) were observed when cells were exposed to plasma jet-treated media. These results provide a first insight into how we might measure the genotoxic and cytotoxic effect of plasma jet treatments (both indirect and direct) in dividing human cells.

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887-1896, 2018.

The quantification of primary and secondary amines in plasma polymerized allylamine and plasma copolymers of allylamine/octadiene films was carried out using chemical derivatization followed by X-ray photoelectron spectroscopy. Data was analyzed using a combination of the peak-fit-analysis method (PFA) and the quantitative-elemental-analysis method (QEA). The latter method overestimates the secondary amine contribution, since it does not account for the additional reaction of TFAA with OH groups that may be present as a result of surface aging effects. In the current work the PFA method has been extended to enable the separation of –OH groups and secondary amines, thus allowing for a more precise determination of the secondary amine groups even after surface aging.

Plasma processing is widely used to provide novel surface modifications to materials for a variety of applications. Typically, the systems used to carry out these modifications are poorly characterised. Here we describe the basics of a global model for a capacitively coupled asymmetric parallel plate radiofrequency plasma system routinely used to produce plasma polymers. An analytical global model was developed for argon, for which cross-sections are known, at a constant pressure of 1 Pa, and includes an electrical model and a power balance. The main parameters of interest were ion flux and self-bias voltages. The argon modelling results were then compared to experimental results for a range of operating gases (argon, oxygen, amines, acids, alcohols, ethers, siloxanes) including both saturated and unsaturated compounds with molecular weights ranging from 40 to 162 g · mol−1, for different inter-electrode separations and from 2 to 50 W using an Impedans OctIV probe. Importantly, it is shown that the RF power transfer efficiency is dependent on the gas. The results show that the argon model results can be used to predict the plasma parameters for other gases when the RF power transfer efficiency is taken into account.

Cold atmospheric pressure plasma jets (plasma) operated in ambient air provide a rich source of reactive oxygen and nitrogen species (RONS), which are known to influence biological processes important in disease. In the plasma treatment of diseased tissue such as subcutaneous cancer tumors, plasma RONS need to first traverse an interface between the plasma-skin surface and second be transported to millimeter depths in order to reach deep-seated diseased cells. However, the mechanisms in the plasma generation of RONS within soft tissues are not understood. In this study, we track the plasma jet delivery of RONS into a tissue model target and we delineate two processes: Through target delivery of RONS generated (primarily) in the plasma jet and in situ RONS generation by UV photolysis within the target. We demonstrate that UV photolysis promotes the rapid generation of RONS in the tissue model target's surface after which the RONS are transported to millimeter depths via a slower molecular process. Our results imply that the flux of UV photons from plasma jets is important for delivering RONS through seemingly impenetrable barriers such as skin. The findings have implications not only in treatments of living tissues but also in the functionalization of soft hydrated biomaterials such as hydrogels and extracellular matrix derived tissue scaffolds. Refereed/Peer-reviewed

This paper reports a method for producing protein arrays on antifouling PEO-like coatings using a “non-contact” atmospheric pressure helium microplasma array. ToF-SIMS characterization of PEO-like coatings after short plasma treatment revealed the antifouling plasma polymer was largely modified/removed within the microplasma-exposed regions. Incubation of microplasma-patterned PEO-like coatings with a fluorescently labeled streptavidin resulted in selective and uniform protein adsorption within the modified regions. A protein sensing application was demonstrated where a target red fluorescent protein (RFP) was only captured on the anti-RFP functionalized regions. This indicates the adsorbed antibody protein on the microplasma-treated PEO-like regions retained its bioactivity.

Growth factors (GFs) play an important role in biological processes such as cell proliferation, differentiation and angiogenesis. GFs are known to bind to glycosaminoglycans (GAGs) in the extracellular matrix, aiding projection from degradation and pooling the GFs for quick response to biological stimuli in vivo. GFs are typically expensive and have a relatively short half-life in culture media, requiring regular replenishment. Here the cooperative binding of GF to a plasma polymerised surface decorated with heparin, and the subsequent culture of primary human dermal fibroblasts (HDFs) is investigated. A simple one-step technique suitable for coating a wide range of different substrates was utilised. Substrates such as culture-ware, scaffolds, bandages and devices for implantation could be coated. The modified surface was compared to standard culture techniques of addition of GF to the media. Results demonstrate that surface bound heparin and FGF-2 have a greater effect on cell proliferation especially at reduced serum concentrations. With performance equivalent to supplementing the media achieved at as little as 1% total FGF-2 added. The protective cooperative effect of FGF-2-GAG bound to modified surface at the interface could lead to reduced costs by reduction of FGF-2 required. Furthermore, for applications such as chronic non-healing wounds, bandages can be produced modified by plasma and decorated with GAGs that could utilise and protect important GFs. This would effectively re-introduce important biomolecules which are protected by GAG binding into a harsh environment. Refereed/Peer-reviewed

An oxygen plasma treatment was employed to modify the surface of carbon electrodes used in capacitive deionization (CDI). X-ray photoelectron spectroscopy analysis of samples showed that oxygen plasma is mainly attaching oxygenated groups on the PTFE binder used in these electrodes. By functionalizing the binder it can increase the hydrophilicity of the electrode surface and increase the available specific surface area. 2.5 min of plasma treatment resulted in the largest improvement of CDI performance of electrodes. Thermodynamic study of CDI performance showed that the modified electrodes followed Langmuir and Freundlich isotherms resulting from the increased interaction between the enhanced electrodes and water. The kinetic study showed that the CDI process followed a pseudo-first order adsorption kinetics. The calculated adsorption rate constants suggested that plasma modification can accelerate ion adsorption of electrodes. Refereed/Peer-reviewed

This multi-centre investigation explores the variability that results from using the power/flow rate ratio (W/F) to describe plasma treatment and plasma polymerization processes. Results from fourteen reactors of different design and spread across ten laboratories, showed that the chemistry of the treated and deposited polymer/plasma polymer films is highly variable between reactor systems, and that there was no clear pattern linking these variations to other properties of the reactor systems (e.g. pressure, volume, electrode configuration). Although W/FM provides a useful rule-of-thumb for process optimization within a single system, it does not provide sufficient information to enable the same plasma polymer to be produced on a different system.

We investigate an approach for the patterning of reactive oxygen and nitrogen species (RONS) onto polystyrene using atmospheric-pressure microplasma arrays. The spectrally integrated and time-resolved optical emission from the array is characterized with respect to the applied voltage, applied-voltage frequency and pressure; and the array is used to achieve spatially resolved modification of polystyrene at three pressures: 500, 760, and 1000 Torr. As determined by time-of-flight secondary ion mass spectrometry (ToF-SIMS), regions over which surface modification occurs are clearly restricted to areas that are exposed to individual microplasma cavities. Analysis of the negative-ion ToF-SIMS mass spectra from the center of the modified microspots shows that the level of oxidation is dependent on the operating pressure, and closely correlated with the spatial distribution of the optical emission. The functional groups that are generated by the microplasma array on the polystyrene surface are shown to readily participate in an oxidative reaction in phosphate buffered saline solution (pH 7.4). Patterns of oxidized and chemically reactive functionalities could potentially be applied to the future development of biomaterial surfaces, where spatial control over biomolecule or cell function is needed. Refereed/Peer-reviewed

Culture surfaces that substantially reduce the degree of cell manipulation in the delivery of cell sheets to patients are described. These surfaces support the attachment, culture, and delivery of multipotent adult progenitor cells (MAPC). It was essential that the processes of attachment/detachment to the surface did not affect cell phenotype nor the function of the cultured cells. Both acid-based and aminebased surface coatings were generated from acrylic acid, propanoic acid, diaminopropane, and heptylamine precursors, respectively. While both functional groups supported cell attachment/detachment, amine coated surfaces gave optimal performance. X-ray photoelectron spectroscopy (XPS) showed that at a primary amine to carbon surface ratio of between 0.01 and 0.02, greater than 90% of attached cells were effectively transferred to a model wound bed. A dependence on primary amine concentration has not previously been reported. After 48 h of culture on the optimized amine surface, PCR, functional, and viability assays showed that MAPC retained their stem cell phenotype, full metabolic activity, and biological function. Consequently, in a proof of concept experiment, it was shown that this amine surface when coated onto a surgical dressing provides an effective and simple technology for the delivery of MAPC to murine dorsal excisional wounds, with MAPC delivery verified histologically. By optimizing for cell delivery using a combination of in vitro and in vivo techniques, we developed an effective surface for the delivery of MAPC in a clinically relevant format. Refereed/Peer-reviewed

There is a growing literature database that demonstrates the therapeutic potential of cold atmospheric plasma (herein referred to as plasma). Given the breadth of proposed applications (e.g. from teeth whitening to cancer therapy) and vast gamut of plasma devices being researched, it is timely to consider plasma interactions with specific components of the cell in more detail. Plasma can produce highly reactive oxygen and nitrogen species (RONS) such as the hydroxyl radical (OH•), peroxynitrite (ONOO-) and superoxide () that would readily modify essential biomolecules such as DNA. These modifications could in principle drive a wide range of biological processes. Against this possibility, the reported therapeutic action of plasmas are not underpinned by a particularly deep knowledge of the potential plasma-tissue, -cell or -biomolecule interactions. In this study, we aim to partly address this issue by developing simple models to study plasma interactions with DNA, in the form of DNA-strand breaks. This is carried out using synthetic models of tissue fluid, tissue and cells. We argue that this approach makes experimentation simpler, more cost-effective and faster than compared to working with real biological materials and cells. Herein, a helium plasma jet source was utilised for these experiments. We show that the plasma jet readily induced DNA-strand breaks in the tissue fluid model and in the cell model, surprisingly without any significant poration or rupture of the phospholipid membrane. In the plasma jet treatment of the tissue model, DNA-strand breaks were detected in the tissue mass after pro-longed treatment (on the time-scale of minutes) with no DNA-strand breaks being detected in the tissue fluid model underneath the tissue model. These data are discussed in the context of the therapeutic potential of plasma. Refereed/Peer-reviewed

With ambient mass spectrometry, we followed the transport of neutral gas species and ionic species through a 3.2 mm thick agarose tissue model target during He non-thermal atmospheric pressure plasma (NT-APP) jet treatment. We found that the neutral gas species are unable to efficiently penetrate the agarose target. But both positively and negatively charged ionic species readily penetrate through the agarose target, following an initial time-lag period of several minutes. Interestingly, we also found that the ionic species are easily hydrated. The trends in the He NT-APP jet transport of ionic species observed in this study correlate well with the He NT-APP jet transport of reactive oxygen and nitrogen species (RONS) through agarose tissue model targets that was investigated in previous studies. Therefore, mass spectrometry might prove to be a useful tool in the future for analyzing the dosages of NT-APP-generated RONS in real biological tissues. Refereed/Peer-reviewed