Glassy carbon (GC) electrodes are widely used in electroanalytical applications especially in bioelectrochemistry. Their use starts with an efficient surface cleaning and activation protocol, mostly based on surface polishing steps. We studied the use of an oxygen plasma exposure of GC electrodes to replace common polishing procedures. The cyclic voltammetry (CV) responses of ferrocyanide and ferrocene-dimethanol were used to compare brand new, surface-polished and plasma-treated GC electrodes. Plasma treatment induces CV responses with improved features, close to theoretical values, as compared to other methods. The plasma effects were quasi-stable over a week when electrodes were stored in water, this being explained by increased surface energy and hydrophilicity. Furthermore, when electroreduction of diazonium was performed on GC electrodes, the surface blockade could be removed by the plasma. Thus, a short oxygen plasma treatment is prone to replace polishing protocols, that display person-dependent efficiency, in most of the experiments with GC electrodes.

This study aims at sensing in situ reactive oxygen and nitrogen species (RONS) and specifically superoxide anion (O2•-) in aqueous buffer solutions exposed to cold atmospheric plasmas (CAPs). CAPs were generated by ionizing He gas shielded with variable N2/O2 mixtures. Thanks to ultramicroelectrodes protected against the high electric fields transported by the ionization waves of CAPs, the production of superoxide and several RONS was electrochemically directly detected in liquids during their plasma exposure. Complementarily, optical emissive spectroscopy (OES) was used to study the plasma phase composition and its correlation with the chemistry in the exposed liquid. The specific production of O2•-, a biologically reactive redox species, was analyzed by cyclic voltammetry (CV), in both alkaline (pH 11), where the species is fairly stable, and physiological (pH 7.4) conditions, where it is unstable. To understand its generation with respect to the plasma chemistry, we varied the shielding gas composition of CAPs to directly impact on the RONS composition at the plasma-liquid interface. We observed that the production and accumulation of RONS in liquids, including O2•-, depends on the plasma composition, with N2-based shieldings providing the highest superoxide concentrations (few 10s of micromolar at most) and of its derivatives (hundreds of micromolar). In situ spectroscopic and electrochemical analyses provide a high resolution kinetic and quantitative understanding of the interactions between CAPs and physiological solutions for biomedical applications.

The reactivity of platinized ultramicroelectrodes (Pt-black UMEs) towards superoxide anion O-2(.-), an unstable Reactive Oxygen Species (ROS), and its relatives, H2O2 and O-2, was studied. Voltammetric studies in PBS demonstrate that Pt-black UMEs provide: i) a well-resolved reversible redox signature for O-2(.-) detected in both alkaline and physiological buffers (pH 12 and 7.4); ii) irreversible oxidation and reduction waves for H2O2 at pH 7.4. The oxygen reduction reaction (ORR) at Pt-black surfaces solely yields H2O2 (2 electrons/2 H+) at physiological pH. Consequently, Pt-black UMEs allow to sense different ROS including superoxide anion for future biomedical or physico-chemical investigations.

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

Cold Atmospheric Plasma (CAP) is a novel promising tool developed in several biomedical applications such as cutaneous wound healing or skin cancer. Nevertheless, in vitro studies are lacking regarding to CAP effects on cellular actors involved in healthy skin healing and regarding to the mechanism of action. In this study, we investigated the effect of a 3 minutes exposure to CAP-Helium on human dermal fibroblasts and Adipose-derived Stromal Cells (ASC) obtained from the same tissue sample. We observed that CAP treatment did not induce cell death but lead to proliferation arrest with an increase in p53/p21 and DNA damages. Interestingly we showed that CAP treated dermal fibroblasts and ASC developed a senescence phenotype with p16 expression, characteristic morphological changes, Senescence-Associated β-galactosidase expression and the secretion of pro-inflammatory cytokines defined as the Senescence-Associated Secretory Phenotype (SASP). Moreover this senescence phenotype is associated with a glycolytic switch and an increase in mitochondria content. Despite this senescence phenotype, cells kept in vitro functional properties like differentiation potential and immunomodulatory effects. To conclude, we demonstrated that two main skin cellular actors are resistant to cell death but develop a senescence phenotype while maintaining some functional characteristics after 3 minutes of CAP-Helium treatment in vitro .

Cold Atmospheric Plasmas (CAPs) are now currently used for numerous applications concerning the surface treatment of solid materials at the macro-, micro- and nano- scales. Surface modifications produced lead to new functionnalities induced by the interactions of the materials with the different energy components (electrons, ions, photons, reactive species) of these partially ionised gases, produced at atmospheric pressure and temperature closed to the room one. Another large area of applications is related to the treatment of liquids in which it is possible to increase the chemical reactivity, thus allowing nanoparticles synthetisis or bacterial decontamination. In this presentation we will first discuss on the physical and chemical properties of these specific plasmas. Their principles of manufacturing will be summarized and particular attentions will be indicated on the environment around the plasma-surface interaction zone. A non-exhaustive state of the art will provide a better understanding of the physical mechanisms associated with their propagation up to the material to be treated. A large biological applications field will be then shown, illustrated by the analyses of modifications produced on steels, polymers but also on physiological liquids and living cells. Methods of the interactions characterizations are numerous and we will present some known techniques but with an innovative approach as for instance the IR-ATR spectroscopy for tracking of modifications of polymers surface. The insitu electrochemistry1 has been also developed during the plasma exposure of liquids and results obtained will be given, showing that it is possible to measure very low current in the nA range and in the presence of high electric fields used for the plasma formation. Bio-chemically Reactive Oxygen and Nitrogen Species (RONS) production (H2O2, NO2-) can thus be followed and quantified, as well as the detection of short-living species.

The understanding of plasma-liquid interactions is of major importance, not only in physical chemistry, chemical engineering and polymer science, but in biomedicine as well as to better control the biological processes induced on/in biological samples by Cold Atmospheric Plasmas (CAPs). Moreover, plasma-air interactions have to be particularly considered since these CAPs propagate in the ambient air. Herein, we developed a helium-based CAP setup equipped with a shielding-gas device, which allows the control of plasma-air interactions. Thanks to this device, we obtained specific diffuse CAPs, with the ability to propagate along several centimetres in the ambient air at atmospheric pressure. Optical Emission Spectroscopy (OES) measurements were performed on these CAPs during their interaction with a liquid medium (phosphate-buffered saline PBS 10 mM, pH 7.4) giving valuable information about the induced chemistry as a function of the shielding gas composition (variable O2/(O2 + N2) ratio). Several excited species were detected including N2+(First Negative System, FNS), N2(Second Positive System, SPS) and HO˙ radical. The ratios between nitrogen/oxygen excited species strongly depend on the O2/(O2 + N2) ratio. The liquid chemistry developed after CAP treatment was investigated by combining electrochemical and UV-visible absorption spectroscopy methods. We detected and quantified stable oxygen and nitrogen species (H2O2, NO2-, NO3-) along with Reactive Nitrogen Species (RNS) such as the peroxynitrite anion ONOO-. It appears that the RNS/ROS (Reactive Oxygen Species) ratio in the treated liquid depends also on the shielding gas composition. Eventually, the composition of the surrounding environment of CAPs seems to be crucial for the induced plasma chemistry and consequently, for the liquid chemistry. All these results demonstrate clearly that for physical, chemical and biomedical applications, which are usually achieved in ambient air environments, it is necessary to realize an effective control of plasma-air interactions.

Cold atmospheric pressure plasmas (CAPPs) are known to have bactericidal effects but the mechanism of their interaction with microorganisms remains poorly understood. In this study the bacteria Escherichia coli were used as a model and were exposed to CAPPs. Different gas compositions, helium with or without adjunctions of nitrogen or oxygen, were used. Our results indicated that CAPP induced bacterial death at decontamination levels depend on the duration, post-treatment storage and the gas mixture composition used for the treatment. The plasma containing O2 in the feeding gas was the most aggressive and showed faster bactericidal effects. Structural modifications of treated bacteria were observed, especially significant was membrane leakage and morphological changes. Oxidative stress caused by plasma treatment led to significant damage of E. coli. Biochemical analyses of bacterial macromolecules indicated massive intracellular protein oxidation. However, reactive oxygen and nitrogen species (RONS) are not the only actors involved in E. coli’s death, electrical field and charged particles could play a significant role especially for He-O2 CAPP.

Atmospheric pressure guided ionization waves (GIWs) that are driven by ns/μs-pulsed high voltages, are promising tools in the biomedical field allowing for the effective production of reactive species and metastables without thermal damages of the specimens that are exposed. In most cases, plasma is produced in noble gases using dielectric barrier discharge (DBD) devices of more-or-less sophisticated geometries. In this study, a compact low-cost DBD reactor of very simple geometry is presented. It is fed with pure helium and driven by positive μs-pulsed high voltage (amplitude: 4.5–8 kV, pulse width: 1–10 μs) of audio frequencies (5–20 kHz), while it operates consistently for long time periods in a wide range of conditions. The produced plasma exhibits propagation lengths up to 4 cm and rich chemical reactivity is established outside the reactor, depending on the device’s experimental parameters. Besides, the dielectric tube’s temperature during plasma operation is an important factor, which is linked to the plasma characteristics. This temperature and its variations are thoroughly investigated herein, along with GIWs electrical features versus the electrical parameters of the pulsed power supply. Accordingly, it is demonstrated that not all of the operational windows are adequate for thermal-free operation and suitable operating conditions of this system are proposed for diverse applications, such as biomedical (low gas temperature is a prerequisite) and surface treatments of solid materials (low temperatures are not required).

Compelling evidence suggests that Cold Atmospheric Pressure Plasma (CAPP) has potential as a new cancer therapy. However, knowledge about cellular signaling events and toxicity subsequent to plasma treatment is still poorly documented. The aim of this study was to focus on the interaction between 3 different types of plasma (He, He-O 2 , He-N 2 ) and human epithelial cell lines to gain better insight into plasma-cell interaction. We provide evidence that reactive oxygen and nitrogen species (RONS) are inducing cell death by apoptosis and that the proteasome, a major intracellular proteolytic system which is important for tumor cell growth and survival, is a target of (He or He-N 2 ) CAPP. However, RONS are not the only actors involved in cell death; electric field and charged particles could play a significant role especially for He-O 2 CAPP. By differential label-free quantitative proteomic analysis we found that CAPP triggers antioxidant and cellular defense but is also affecting extracellular matrix in keratinocytes. Moreover, we found that malignant cells are more resistant to CAPP treatment than normal cells. Taken together, our findings provide insight into potential mechanisms of CAPP-induced proteasome inactivation and the cellular consequences of these events.

Cold Atmospheric Plasmas (CAPs) are increasingly used for biomedical applications, their various reactive components must be then better determined. We demonstrate that peroxynitrite (ONOO−) is effectively a major reactive species generated by CAPs.

Growth of gas hydrates as fast-growing polycrystalline crusts at interfaces between water and guest phases is well documented, but the mechanisms of hydrate growth on solid substrates are much less known. We report here on cyclopentane (CP) hydrate spreading on glass (fused silica) under CP. As seen for methane hydrate by Beltran and Servio (Cryst. Growth Des. 2010, 10, 4339-4347), CP hydrate grows on glass as a "halo" radiating from the contact line of a primary drop. Complementary optical microscopies at micron resolution here allow identification of the mechanisms of halo growth and melting. We conclude that forms of water on the substrate control halo spreading, namely, a precursor film near the contact line and a breath figure (dew) condensed from the CP (halo spreading at <= 2 mu m s(-1) at T approximate to 0 degrees C or subcooling similar to 7 degrees C), and "leap-frogging" (at similar to 10 mu m s(-1)) over secondary drops left behind by melting a previous halo. Halo thickening, about 5 nm s(-1), is attributed to water condensation, either incorporation of water dissolved in CP (like ablimation) or settling of water "fog" from the CP. Halos spread slower on untreated, compared to hydrophilic, glass, an effect attributed to the quantity of water present on the substrate; a similar trend is noted when the CP phase is not pre-equilibrated with water prior to the experiment. No hydrate halo was detected on hydrophobized (silane-treated) glass, where the breath figure is absent.

INTRODUCTION:Cold plasma is a partially ionized gas generated by an electric field at atmospheric pressure that was initially used in medicine for decontamination and sterilization of inert surfaces. There is currently growing interest in using cold plasma for more direct medical applications, mainly due to the possibility of tuning it to obtain selective biological effects in absence of toxicity for surrounding normal tissues,. While the therapeutic potential of cold plasma in chronic wound, blood coagulation, and cancer treatment is beginning to be documented, information on plasma/cell interaction is so far limited and controversial.METHODS AND RESULTS:Using normal primary human fibroblast cultures isolated from oral tissue, we sought to decipher the effects on cell behavior of a proprietary cold plasma device generating guided ionization waves carried by helium. In this model, cold plasma treatment induces a predominantly necrotic cell death. Interestingly, death is not triggered by a direct interaction of the cold plasma with cells, but rather via a transient modification in the microenvironment. We show that modification of the microenvironment redox status suppresses treatment toxicity and protects cells from death. Moreover, necrosis is not accidental and seems to be an active response to an environmental cue, as its execution can be inhibited to rescue cells.CONCLUSION:These observations will need to be taken into account when studying in vitro plasma/cell interaction and may have implications for the design and future evaluation of the efficacy and safety of this new treatment strategy.

He/N-2 cold atmospheric plasma treatment of E. coli suspension leads to a fast and efficient inactivation process. Significant generation of ammonium is reported. The formation of NH4+ species in saline solution treated by cold atmospheric plasma is proposed for the first time as the main process responsible for the fast bacterial inactivation in pH-buffered solutions, at ambient temperature and physiological pH.

This paper is devoted to the study of gas flow fields related to helium atmospheric pressure guided streamer (plasma bullet) propagation in the air. For very weak up to moderate helium flows, the modification induced to the gas flow field by the plasma ignition is demonstrated; it is shown that the turbulent flow region is expanded and two conditions must be fulfilled regarding the working gas profile in the air for streamer propagation, i.e., laminar flow and high concentration in this laminar flow region.

Cover: Non-thermal plasma elicits various reactive oxygen species which play different roles in dysfunction of proteins. Taking the model protein horseradish peroxidase as an example, this work demonstrates how to quantitatively assess the plasma induced oxidative damage of protein through a spectroscopic approach. Further details can be found in the article by Qing Huang et. al. on page 731.

Today, it is necessary to better understand the role of reactive species in plasmas on biological objects in order to use plasma processes for biomedical applications. Even when well-known plasma set-ups producing one main reactive species in a controlled environment are used, it is still difficult to understand reactive species interaction processes with biological systems. In this study, nanoimaging with the most sensitive mass spectrometry technology, and the use of isotopic 15N2 or/and 18O2 containing-Ar gas mixtures to produce the plasma is proposed in order to obtain isotopic and elemental data on microwave late afterglow treated Escherichia coli bacteria. By this way, new data dealing with reactive species binding processes on plasma treated bacteria by using NanoSIMS50 mass spectrometer are obtained.

In this paper, a dielectric-barrier discharge (DBD) of Ar:N 2 mixtures at atmospheric pressure is studied in relation to plasma jet production. The discharge takes place in a capillary dielectric tube, and it is sustained by positive high-voltage pulses. It is herein claimed that, apart from the traditional DBD (which is developed perpendicularly to the gas flow and can produce afterglows), plasma jet propagates simultaneously parallel to the gas flow due to ionization waves guided by the dielectric tube. A quite simple electrical model is applied to simulate the proposed DBD and plasma jet coexistence. The concept of capacitive behavior of both barrier discharge and jet is taken into account. This model in combination with UV-visible optical emission spectroscopy and conventional photography supports the aforementioned statements. The dependence of the jet domination against the afterglow on the gas mixture is demonstrated, and the N 2 additive at 2.5 %-3% in Ar is found to be the most efficient for jet production. The model runs with the measured interelectrode voltage as input and yields the circuit total current as output. Matching between the measured current and the model output is attempted by adjusting two free parameters. This tuning of the free parameters provides reliable values for the electrical components of the model. Thus, a simple mean for realistic representation of both DBD and jet is achieved. This tool could be applied to the design of plasma jet reactors.

Liposomes (LIPs) consist of phospholipid bilayers and are currently being used as carriers for drug delivery and targeting [1]. Hydrophilic active substances may be loaded in LIPs and for characterization of such formulations a standard method is to disrupt LIP membranes with detergent (as Triton X-100) and measure released active concentration. However, the presence of the detergent may interfere with the analytical procedure used. We have performed a preliminary study in order to understand if plasma jet could be useful for LIP disruption. A jet device similar to the one presented in [2] has been used. The system is fed with high purity helium at atmospheric pressure and driven by sinusoidal high voltage (10 kHz, 11 kV peakto-peak). As hydrophilic substance we used a highly fluorescent compound, calcein, at a concentration of 100 mM at which its fluorescence intensity (FI) is quenched, permitting easy determination of its leakage from vesicles (since FI is de-quenched due to dilution, when the encapsulated molecules are released in the aqueous dispersion media) [3]. MultiLamellar Vesicle (MLV) and Small Unilamelar (SUV) liposomes, encapsulating calcein (100mM) and consisting of egg lecithin (PC) or saturated lipid DSPC (which forms more rigid bilayers) were prepared by thin film hydration and probe sonication (for SUV) [1]. Vesicle hydrodynamic mean diameter and size distribution were determined by dynamic light scattering (Malvern, Nanosizer). The liposomes were subjected to cold plasma at different lipid concentrations and for different time periods (as seen in Table 1).

Wood or cellulosic materials protection against fungi is possible by binding covalent molecules as quaternary ammonium compounds (QAC) on the hydroxyl reactive groups present in cellulose. This work firstly consists in the synthesis of a specific quaternary ammonium molecule containing a long aliphatic chain and an epoxide reactive group: Glycidyldimethyldodecylammonium chloride (GDDAC). Fungicidal properties of this molecule have been proven. Secondly, afterglows issued from atmospheric pressure Dielectric Barrier Discharges (DBDs), can assist QAC grafting on cellulose. The efficiency of the reaction is improved by adding a few percentage of oxygen in nitrogen DBD. Antifungal efficiency of GDDAC grafting on wood sawdust is finally shown demonstrating the great interest of such process.