Plasma applications in life science are currently emerging worldwide. Especially Plasma Medi-cine as a new independent field of scientific research leads to a growing interest in using low temperature atmospheric pressure plasma sources. Whereas todayâ?Ts commercially available plasma surgical technologies such as argon plasma coagulation (APC) or ablation are mainly based on lethal plasma effects on living systems, the newly emerging therapeutic applications will be based on selective, at least partially non-lethal, possibly stimulating plasma effects on living cells and tissue. Promising results could be obtained by different research groups worldwide revealing a huge potential for the application of low temperature atmospheric pressure plasma in fields such as tissue engineering, healing of chronic wounds, treatment of skin diseases, tumor treatment based on specific induction of apoptotic processes, inhibition of biofilm formation and direct action on biofilms or treatment of dental diseases. The development of suitable and reliable plasma sources for the different therapies requires an in-depth knowledge of their physics, chemistry and parameters. According to this, much basic research still needs to be conducted to minimize risk and to provide a scientific fundament for new plasma-based medical therapies. It is essential to perform a comprehensive assessment of physical and biological experiments to clarify minimum standards for plasma sources for appli-cations in life science and for comparison of different sources. This contribution intends to give an overview on a set of minimal investigations that have to be carried out to qualify plasma sources for potential applications in life science. It will discuss needs, prospects and approaches for the characterization of plasmas from the fundamental plasma physical and biological point of view. It is confined to the plasma sources developed and used by INP Greifswald and its network partners in various projects. After a general introduc-tion, selected specific plasma sources which are used for the investigation of various biological effects are presented. Regarding the manageability in everyday medical life, atmospheric pres-sure plasma jets (APPJ) and dielectric barrier discharges (DBD) are of special interest. A com-prehensive risk-benefit assessment was realized in the Campus PlasmaMed [1] using several in vitro and semi-in vivo models. [1] The research within the joint research project Campus PlasmaMed has been supported by the German Federal Ministry of Education and Research (grant no. 13N9779).

Because of last decadeâ?Ts intense research efforts on the application of low temperature plasmas in biology and medicine, today low temperature plasmas are poised to revolutionize healthcare. Researchers found ways whereby plasmas can be applied in direct contact with living tissues to deactivate pathogens; to stop bleeding with no damage to healthy tissue; to disinfect wounds and accelerate wound healing; and to induce apoptosis in some cancer cells, in vitro. These research activities constitute the basis of a new approach to healthcare and are the foundation of new field of research referred to as â?oPlasma Medicineâ? [1]. Although non-equilibrium plasmas generate various agents that could play biological roles (UV radiation, heat, charged particles), it was found that the oxygen-based and nitrogen-based chemically reactive species (ROS and RNS) are responsible for much of the impact on plasma- treated biological cells. Today, there is evidence that extra-cellular and intra-cellular ROS and RNS trigger signaling cascades within the cells that lead to certain outcomes. The focus of this paper is on low temperature plasma jets, generated in ambient air. Because the plasma plumes generated by jets are not confined between electrodes, their use in biological and medical applications became quite obvious. Therefore, the first part of this presentation will cover jet sources and their characteristics. This is followed by coverage of their wide field of applications in biology and medicine. This includes sterilization/decontamination, dental applications, destruction of pathogenic proteins, wound healing, and cancer studies. [1] M. Laroussi, â?oLow Temperature Plasmas for Medicine?â?, IEEE Trans. Plasma Sci., Vol. 37, No. 6, pp. 714-725, 2009.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Standard therapy consists of surgical resection to the extent that is safely feasible, followed by radiotherapy and chemotherapy. Despite recent advances in neurosurgery, radiotherapy and chemotherapy, current treatment regimens only have a marginal impact on patient survival. So far, the median survival is about 15 months. A crucial challenge is to develop and deliver effective drugs to cure this deadly disease. Non-thermal plasma displays features that are favorable in tumor biology. We could demonstrate that plasma is able to block cell proliferation, to induce S-G2-phase cell cycle arrest and mediate apoptosis. Not only high doses of non-thermal plasma, but also very short treatment times of about 10 seconds inhibit human glioma cell proliferation about 70 percent. Cell cycle arrest in S/G2- phase persists at least for 72h when cells were plasma treated once. Plasma effects could be mediated through the medium as well as by direct treatment of the cells without medium. Our data on glioma cells might open new applications in brain tumor biology using non-thermal plasma.

Nonequilibrium atmospheric pressure plasmas are used in various material processes. Recently, medical applications using nonequilibrium atmospheric pressure plasmas have appeared as interesting because cells are not vacuum-compatible and thermal damages on the cells are almost ignorable by tuning parameters of plasma properties and the experimental setup. Some groups in the world have applied the plasmas on the medical sterilization, wound healing, and cancer therapy. Mechanisms that cells are affected by plasmas are unclear, however, it is suggested that ultraviolet (UV) radiation, charged particles, and radicals such as reactive oxygen species (ROS) in and around plasmas might change biological properties on the cells. Understanding the interactions between plasmas and biological systems on the basis of such â?oparticle parametersâ? are important to apply plasmas to medicine. Epithelial ovarian carcinoma is one of the leading causes of death for women. One of the biggest issues is that it is difficult to find ovarian cancers at earlier stages and the five-year survival rate is pretty low. Thus, the innovative cancer therapeutic strategies are needed. In this study, we treated two independent ovarian cancer cell lines with nonequilibrium atmospheric pressure plasmas. We have previously succeeded to inactivate Penicillium digitatum spores using our developed high-density nonequilibrium atmospheric pressure plasmas. Here, we used the similar plasma unit equipped with a robot arm to control positions and exposure times accurately. The plasma unit is consisted of three regions: the gas diffusion region, the main discharge region, and the radical transportation region. While Ar gas was flowed, plasmas in the main discharge region were excited by applying 10 kV of a 60 Hz commercial power supply to two electrodes, and the radicals in the remote plasmas were produced through a plate and along with the gas flow exhausted from a slit. We tuned the parameters of the experimental setup so that the environments around the cells such as temperature, pH and volume of medium were not extremely disturbed by plasma treatments. Interestingly, most of plasma-treated ovarian cells were detached from the dish when they were continuously treated with plasmas for 10 minutes at the same spot on the dish without any movements of the plasma source, while non-treated cells stayed on the dish. Next, we scanned the plasma source all over the dish using a robot arm. In vitro cell proliferation assay showed that the plasma treatments significantly decreased the proliferation rates in the ovarian cell lines while normal cell lines were not damaged. Moreover, FACS analysis and Western blot using apoptotic markers showed the plasma-treated ovarian cells induced apoptosis. On the basis of these results, we propose the nonequilibrium atmospheric pressure plasma is a promising tool of anti-cancer therapy for the ovarian cancers.

Non thermal plasma (a ionized gas) is emerging as a novel tool for the treatment of living tissues for biological and medical purposes. Plasma considered as fourth state of matter and is obtained by a supply of energy. This excited gas contains free charges (electron, ions), free radicals, excited molecules, energetic photons (UV) and generates transient electric fields. In this study we described the comparative effect of coplanar DBD plasma on both cancer and normal cell line. Several previous studies have been demonstrated the efficacy of sterilization by plasma application and more recently, some studies have shown an antitumor effect of plasma in vitro on few cell lines. To this end we developed a friendly using coplaner and compact dielectric barrier discharge (DBD) plasma to access the anticancer effect of plasma using different type of cancer cells. We were evaluated non thermal coplanar DBD plasma as potential new anticancer treatment against different type of cancer and having less toxicity against normal cell lines. Keywords: DBD Plasma, Brain Cancer, Breast Cancer, Thyroid cancer and kidney cells.

Recently the potential application of non-equilibrium atmospheric pressure discharges in biology and medicine has grown significantly. Plasma interaction with living tissues and cells has not been understood to the extent of comprehensive biological and physical mechanisms, due to the complexity of both the plasma and the tissue. Many interpretations of the biological responses for disinfection by the plasma discharges have been reported, which has indicated that death is accompanied by lethal oxidation of membranes, electrostatic disruption of cell membranes, such as cell lysis, fragmentation, and biological responses as apoptosis. Penicillium digitatum, which is difficult to be inactivated and causes green mold of citrus fruits (postharvest disease), is categorized as fungi, and differs from other microorganisms such Escherichia coli and Bacillus subtilis. Fugal spores of Penicillium digitatum were disinfected by applying atmospheric pressure plasmas of Ar gas containing trace O₂. The results of this experiment indicated there was no contribution of ultraviolet radiation and no inactivation effects of NO, and O₃. Therefore we speculated that neutral oxygen species (O*) such as atoms and excited molecules were provided to inactivate. The free radical such as semiquinone (QH*) has been historically studied due to the identification of biological metabolic mechanisms, especially using the electron spin resonance (ESR) method. The ESR signal from Penicillium digitatum spores was observed at a g-value of ca. 2.004 with a line width of ca. 5G. To identify the signal, it was preliminarily assigned to a stable free radical, such as intracellular semiquinone. After germination of the spore, the signal was diminished with the change in the shape and color. It is noteworthy that the ESR signal was decreased when only oxygen plasma was discharged. The real time in situ ESR measurement signal decayed during O* irradiation. At the beginning of irradiation, a monotonic decrease with respect to time was evident and the regime then moved into exponential decrease. This behavior indicates that the rate-limiting step reflects a scavenging reaction with the supply of O*. These results provide evidence that O* plays a prominent role in the interaction of bacteria with plasmas. We have successfully obtained information regarding the reaction mechanism with free radicals and this real time in situ ESR method has proven to be a useful method to elucidate plasma-induced surface reactions on biological systems.

In agricultural fields and plant protection stations, pesticides are sprayed to protect crops from various insects and viruses. However, residual agricultural chemicals (e.g., thiabendazole, imazalil, and ortho-phenylphenol) also are harmful to the human body and the environment. Moreover, methyl bromide is an effective and widely used pesticide. In 2005, however, it was prohibited under the "Montreal Protocol on Substances that Deplete the Ozone Layer," because of its high ozone depletion potential. Non-equilibrium (low-temperature) atmospheric-pressure plasmas showed promise as a very effective system which causes minimal damage to crops, foods, seeds, humans, and the environment. Spore of Penicillium digitatum which causes green mold of citrus was used as a target sample. A high-density non-equilibrium atmospheric pressure plasma (NEAPP) applied for inactivating fungal spores of P. digitatum is introduced as an environmentally safe and rapid-inactivation method. The electron density of the NEAPP was approximately 1015 cm-3, which led to the generation of high-density reactive species. The contributions of ozone, UV radiation and ground-state atomic oxygen in the NEAPP on the inactivation of the spores are evaluated using absorption and emission spectroscopies. The absolute densities of ozone were measured by using ultraviolet absorption spectroscopy. The ozone density increased from 2 to 8 ppm with an increase in the distance from the plasma source, while the inactivation rate decreased. The inactivation rate of ozone was evaluated to be thousand times higher than that of an ozone generator using the integrated number density of ozone. In addition, it was clarified that the contribution of UV radiation to inactivation was not dominant for P. digitatum inactivation by NEAPP by filtering the active species using quartz plate. From these results, we can speculate that the inactivation efficiency of ROS will be larger than those of others. In order to investigate the effect of ground-state atomic oxygen as one of ROS, the inactivation of P. digitatum spores using an oxygen radical source that employs a high-density atmospheric-pressure O2/Ar plasma. The absolute O density was measured to be 1.4Ã-1014 and 1.5Ã-1015 cmâ?"3 using VUV absorption spectroscopy using a microdischarge hollow cathode lamp. The behaviors of the O densities as a function of O2/(Ar+O2) mixture flow rate ratio correspond to that of the inactivation rate. This result indicates that ground-state atomic oxygen is concluded to be the dominant species that causes inactivation. Plasma sources with radical densities guaranteed will be indispensable for evaluating the contributions of ROS to P. digitatum spores. The quantitative evaluation of individual species on the inactivation will help us to clarify the inactivation mechanism of microorganisms and to develop high-speed inactivation equipment using plasmas.

Atmospheric pressure non-equilibrium discharge plasmas and low-pressure discharge plasmas have been widely employed in biomedical applications such as sterilization of microorganisms and surface coating of biomaterials because such plasmas induce little thermal damage to biomaterials [1-2]. In this study, we investigated growth stimulation of radish sprouts using low pressure oxygen discharge in order to confirm stimulation ability. Experiments were performed using a capacitively coupled RF discharge reactor [3]. Pure O₂ or N₂ was supplied from the top of the reactor and the total pressure was 100 Pa. An RF voltage with a frequency of 13.56 MHz was applied to the powered electrode. The discharge power was 50 W. Optical emission spectra were obtained from the RF plasma with a spectrometer (Soma Optics S-2630) to obtain information on the radicals generated in the plasma. We employed seeds of radish sprouts (Raphanus sativus L.) in the plasma irradiation experiments. For each irradiation condition, 20 seeds were used. After plasma irradiation, the seeds were cultivated in a plant incubator at 22°C and 60% relative humidity in the dark with a pure water feed. Their weights and total lengths were measured after cultivation for 3, 5, and 7 days using an electric balance and an image analysis system, respectively. Seed husks were observed by scanning electron microscopy (SEM; Hitachi S-3400N). The chemical bonds on the surfaces of the seeds were investigated using a Fourier-transform infrared (FTIR) spectrometer (JASCO FT/IR-4200) in attenuated total reflectance (ATR) mode. Optical emission measurements show the atomic oxygen emission lines at 777.1 nm and 845 nm, and the line of O₂ at 762 nm. The average length of sprouts with O₂ plasma irradiation is 60% longer than that of sprouts without irradiation. In contrast, the average length of sprouts with N₂ plasma irradiation is nearly the same as that of the control. The surface morphology of seeds does not change even after O₂ plasma irradiation for 120 min. These results suggest that oxygen radicals play an important role in enhancing growth. [1] N. Hayashi, et al., Jpn. J. Appl. Phys. 50 (2011) 08JF04 (5 pages). [2] S. Kitazaki, et al., Proc. IEEE TENCON 2010 (2010) 1960. [3] S. Kitazaki, et al., Jpn. J. Appl. Phys. (in press). Work supported by JSPS.

The development of cold atmospheric plasma sources has paved the way for new and novel applications in health care. To proceed from the necessary basic research into safe and sustained utilization of the various technologies, a major interdisciplinary effort is needed, ranging from plasma physics and chemistry to microbiology, cell biology, medicine and lastly commercial considerations. In this talk the focus is on a review of current plasma technologies, plasma-cell interactions and, by comparison with topical medicine, on the expected benefits which "designer plasmas" may provide in current and future health care issues.

Basic researches on the characteristic of the bioplasma sources applicable to the living cell, especially to the human body will be investigated, along with the fundamental researches of mutual interactions between the bioplasma and organic-inorganic, liquid materials. We have investigated the nonthermal bioplasma sources and their interactions with microbial, fungi, yeast and living cells, especially to the blood immune cells. Also the diagnostic method for the hydroxyl radical density has been introduced from the bioplasma jet by the ultraviolet absorption spectroscopy. Also we report the electon temperature and density of bioplasma based on the collisional radiative model for application to the bacteria germicide caused by several reactive oxygen species in the atmospheric pressure. Herein, we have investigated the basic interactions of nonthermal dielectric-barrier discharge plasma with the Escherichia coli in morphologicl and biomolecular aspects under lethal dose. We have also investigated the influence of the nonthermal bioplasma on the cell proliferation and microglial cell death as well as the DNA damages. The pH of the blood plasma, the vitality of blood cells, and the cytokine production from white blood cells have been influenced by the bioplasma and their results will also be reported. We have also investigated the influence of the nonthermal bioplasma on the electron energy distribution in the valence band for the microbial and living cells. This work will contribute to the understanding of the exact biological pathways of plasma interaction with living organisms.

The successful and reliable sterilization of medical instruments is a basic requirement in each hospital. Conventional sterilization methods reach their limits since modern medical materials may be destroyed by autoclaving or by the contact with aggressive chemicals. Plasma sterilization, however, is able to meet these demands, because plasma can be made effective towards the germs but gentle towards the substrates. A successful implementation of a plasma sterilization method requires, however, a careful design of the plasma process and thorough understanding of the inactivation mechanisms. Plasma sterilization is a topic which started the earliest in the field of plasma medicine and which matured over the past two decades. But only recently, the first commercial plasma sterilization process entered the market. In this presentation, the concepts and pitfalls of plasma sterilization are highlighted and the recipes for a successful sterilization plasma equipment are discussed.

In the last twenty years new antibacterial agents approved by the U.S. FDA decreased whereas in parallel the resistance situation of multi-resistant bacteria increased. Thus, community and nosocomial acquired infections of resistant bacteria led to a decrease in the efficacy of standard therapy, prolonging treatment time and increasing healthcare costs. Therefore, the aim of this work was to demonstrate the applicability of cold atmospheric plasma for decolonisation of bacteria (MRSA, S. aureus and E. coli) using an ex vivo porcine skin model. Two different plasma devices were evaluated. After application of pure bacteria on the surface of the explants these were treated with cold atmospheric plasma treatment for up to 15 min. A decolonisation efficacy of 99.9% was achieved already after 6 min of plasma treatment. Longer plasma treatment times achieved a killing rate of 99.999% independently from the applied bacteria strains. Histological evaluations of untreated and treated skin areas upon cold atmospheric plasma treatment within 24 h showed no morphological changes as well as no significant degree of necrosis or apoptosis determined by the TUNEL-assay indicating that the porcine skin is still vital. This study demonstrates for the first time that cold atmospheric plasma is able to very efficiently kill bacteria applied to an intact skin surface using an ex vivo porcine skin model. The results emphasize the potential of cold atmospheric plasma as a new possible treatment option for decolonisation of human skin from bacteria in patients in the future without harming the surrounding tissue

Inactivation of microorganisms using a plasma processing method has attracted much attention recently, especially as a substitute for medical instrument sterilization methods. A plasma processing method possesses many advantages such as a low-temperature treatment and short processing time. We have reported that the rapid inactivation of spores of Penicillium digitatum using high-density non-equiribrium atmospheric-pressure plasma (NEAPP). The green mold of citrus is caused by spores of P. digitatum. We investigated the inactivation effects of ozone and UV radiation. These results showed that the inactivation rate of this plasma was thousand times larger than of that of an ozone generator using the integrated number density of ozone measured by UV absorption spectroscopy. In addition, the contribution of UV radiation toward inactivation was not dominant for P. digitatum inactivation by NEAPP. Some paper reports that the reactive oxygen species (ROS) such as O, OH, and so on is important factor for the inactivation due to oxidation-decomposition of cell membrane. In this study, we observed the spores of P. digitatum by using fluorescent microscopy in order to investigate the change such as oxidation after the plasma treatment. The spores inactivated by the NEAPP were compared with those by the UV treatment or moist heat treatment. Fluorescence image was observed by using an inverted microscope with charge-coupled device camera. The DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) which is carbocyanine was used as a vital fluorescence membrane dye. Octadecyl group of diI bind acyl group in the phospholipid of the cell membrane, so that the membrane is stained. The excitation wavelength of diI is 549 nm and fluorescent wavelength is 565 nm. NEAPP was used for the inactivation of P. digitatum. An alternative voltage (AC) of 6 kV was applied to two electrodes, and Ar gas of 3 slm was flowed through the gap between the electrodes. The distance between plasma and sample and exposure time were 25 mm and 5 min, respectively. The membrane of P. digitatum spore was successfully stained by DiI because it is difficult for fungi to be stained by fluorescent dye. The organelle such as cell nucleus, mitochondria, lysosome of some spores emits the fluorescent light by DiI in the case of the plasma treatment while the fluorescent of the cell organelle in the cases of the UV treatment and moist heat treatment was not observed. For the living cell, the DiI is not permeable because the membrane has selective permeability. On the other hand, the condition for UV and moist heat treatments were sufficient to kill the spores. Thus, these results indicate that the membrane was decomposed by ROS produced from the plasma and the DiI penetrate into the cell through the decomposed membrane. ROS is related to catalysis of peroxidation process of phospholipids, resulting in cellular death through DNA damage or lack of enzyme activity.

To successfully eliminate biofilm growing microorganisms is an important task/problem in both industrial and developing countries. Candida albicans is one of the main fungal species which is able to form biofilms on almost every surface, leading to skin and superficial mucosal infections. The worldwide increase of antifungal resistance against pharmaceuticals led to a decrease in the efficacy of standard therapies, to prolonging treatment times and therefore to increasing healthcare costs. In this study we demonstrate the applicability of cold atmospheric plasma for inactivating planktonic Candida albicans cells and Candida albicans biofilms. The plasma device used for these measurements is based on the Surface Micro-Discharge Technology. For igniting the plasma on a surface of 117 cm2 in area a voltage of 9 kVpp and a frequency of 1 kHz was used. The power consumption was approximately 0.2 W/cm2. For planktonic Candida albicans cells a killing efficacy of 99.9% of viable cells was achieved upon a plasma treatment time of 40 seconds. The reduction of viable Candida cells was increased further to up to 5 log steps when the plasma treatment time equaled 5 minutes. To achieve an optimum inactivation efficacy range of â?¥ 3log to 5log reduction of CFU different time points were selected for the treatment of the Candida albicans biofilm. Total eradication of the Candida albicans biofilm was achieved after a treatment time of 7 minutes (reduction of 5 log CFU). These results demonstrate that cold atmospheric plasma is able to inactivate extensive areas of biofilm growing Candida albicans very efficiently. Overall the antifungal plasma treatment of Candida albicans biofilms on inert surfaces seems to be a promising tool for industrial and clinical purposes where time saving is a critical point to achieve efficient disinfection of inanimate surfaces.

Ambient-gas plasma, or plasma created from air at ambient conditions, is emerging as a means of disinfecting surfaces. Possible applications of ambient-gas plasma disinfection include infection control in hospitals, emergency medicine and community settings. However, the parameters that determine the effectiveness of ambient-gas plasma disinfection, and the optimal conditions for its use, are not well-understood. We report results from a series of surface sterilization experiments to evaluate the practical suitability of ambient-gas plasma as a disinfectant and to determine the characteristics that are responsible for its antimicrobial effect. Treatment time is found to be a more significant parameter than discharge characteristics, at least within certain ranges, suggesting that the reaction between plasma species and biomolecular components of the microorganisms may be a more important process than the generation of reactive species in the plasma. In addition, we find that non-biological surfaces (e.g. metal, rubber, silicon) are considerably easier to disinfect than pig skin. However, plasma appears to perform at least comparably to the conventional antiseptic ethanol on pig skin, indicating that ambient-gas plasma treatment could be a promising strategy for both clinical and community infection control. Finally, we describe experiments using lipidomic and proteomic techniques that will better elucidate the mechanisms by which plasma reacts with biomolecules and kills microorganisms.

The maintenance of sterility and hygienic aspects in hospitals are crucial for the proper aid of patients. Therefore, atmospheric Surface Micro-Discharge (SMD) air plasma has been studied to evaluate its ability to inhibit bacterial growth on agar dishes and to sterilize surfaces inoculated with bacterial endospores. Geobacillus stearothermopilus ATCC 7953, Bacillus pumilus DSM 492, Bacillus atrophaeus ATCC 9732, Bacillus subtilis DSM 13019 have served as spore model and have been investigated according to EN/ISO 14937. The spore samples contained of the substrate (2cm2; initially ca. 2x106 vital spores) wrapped in air permeable Tyvek® sheet. The samples were treated for 1min, 3min, 5min plasma-on time plus 30s afterglow by SMD air plasma at closed conditions inside our plasma device. The experimental parameters for plasma ignition were mainly set to 1 kHz, 10 kVpp with sinusoidal waveform. The plasma power consumption was around 0.2 W/cm2. Although Geobac. stearothermophilus spores were more resistant than other spore types, they were completely inactivated within 5min. The inactivation efficacy was independent from the substrate material (stainless steel, PVC, PTFE, glass). At short treatment times, the afterglow could reduce the spore survivability by a factor of 4.5 compared to treatments without afterglow. Additional morphological observations of the same spore regions before and after plasma treatment by Scanning Electron Microscopy Imaging indicate that mainly the spore surrounding cell residues are affected and less the sporesâ?~ morphology itself. Furthermore, experiments with various gram negative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli DSM 1116, etc.), gram positive bacteria (lab. strain of Methicillin-resistant Staphylococcus aureus, Bacillus pumilus DSM 492, etc.) inoculated on agar dishes demonstrated that a 5- to 6-log reduction could be achieved within 30s of SMD plasma exposure; even in the case of the eukaryotic yeast Candida albicans ATCC 90028 there was a 4-log reduction. The dominant role of reactive oxygen species such as ozone and NO regarding the reduction of microorganism burden will be discussed. As conclusion, the atmospheric SMD air plasma can effectively reduce microorganism burden and therefore, might be appropriate for disinfection or even sterilization purposes.

Cold atmospheric plasma is a potential tool for biomedical applications, because it can produce many biologically active agents, e.g. reactive oxygen and nitrogen species, charged particles, UV photons. These agents affect on objects simultaneously. It is important to control the produced plasma components carefully depending on the purpose. For instance, there is a criterion in hand sanitation that the plasma should reduce bacterial density in the treated area with no negative effect on human cells. We have developed and tested a new atmospheric plasma device using Venturi effect. This effect gives a local low-pressure volume in the device. An air plasma discharge was ignited between two stainless-steel electrodes by a DC voltage of about 4 kV (current 3 mA constant) where the gas pressure can be controlled by changing gas flow speed. The plasma was dragged out to the ambient air with the gas flow. The gas pressure in the plasma production region was changed between 280 and 700 hPa and with all the pressures the plasma production was observed. This plasma flow was applied on Escherichia coli bacteria inoculated on agar plates (bacteria density about 0.5 x 10^6 cm^-2). By one-minute plasma treatment, a clear zone of inhibition was observed on plasma-treated agar plate. The size of inhibition zone became larger with increasing the gas pressure. The light emission was observed from the plasma production region by a spectrometer. Mainly the peaks of nitrogen 2nd positive system in UVA were observed. When the pressure was lower than 600 hPa, no peak at 777 nm corresponding atomic oxygen was observed. With the pressure higher than 600 hPa, the peak at 777 nm appeared. This result gives a possibility to control the reactive species components by changing the gas pressure and to tune the atmospheric plasma more precisely.

Based on recent advances in plasma technologies, it has been widely tried to use plasma treatments for injury to stop bleeding, better wound healing, and ablation of abnormal cells. Argon plasma coagulator (APC) is one of the representative successes, which is a commonly used in endoscopic surgeries. The effects of APC are more limited depth and able to apply for larger area than the other coagulation methods such as radiofrequency coagulators. These are the reason why APC is applied to coagulate superficial vascular lesions (sporadic angiodysplasia, gastric antral vascular ectasia and radiation proctitis). Whereas, it is not recommended to use in esophageal diseases due to the perforation risk (~3.6%), because unintentionally increased current has been pointed out as a cause of perforation through the arc-like plasma formation. In order to improve above concerns, we investigated blood coagulation and inflammatory reaction associated with procedures to stop bleeding. And then, we developed new devices eliminating heat and arc-like plasma formation but also accelerating coagulation. A new device equips fully coated electrodes with dielectric, and employs a dielectric barrier discharge with a peak-to-peak voltage of ~8 kV and frequency of ~60 kHz. Treatment with the device promptly coagulated bleeding blood from disrupted hemoral artery, except for apparent burned wounds. It was noteworthy that acute inflammatory reaction after plasma treatment was limited in superficial layer of dermis, while radiofrequency coagulator treatment extended into deep area of dermis. Moreover, coagulation by plasma treatment coated firmly bleeding injury, which appeared to play an important role on protecting from excessive inflammatory reaction, and triggering the favorable wound healing process. These results suggest the feasible to use our new device in medical procedures, and the possibility to dissect unknown pathophysiological mechanism that links between blood coagulation and inflammatory response in wound healing process.

Chronic wounds are thought to be caused in part by the persistence of aerobic microbes that deplete the local oxygen concentration and prevent oxygen dependent healing. Atmospheric pressure gas plasmas have been shown to be strong bactericidal agents and there is evidence that plasma treatment can kill bacteria in wounds and speed wound healing [1, 2]. However, there are variations in the outcome for different plasmas, bacteria, and supports. Plasma-generated reactive oxygen/nitrogen species (ROS/RNS) may be directly or indirectly responsible for bacterial elimination. To investigate which parameters are important in the context of chronic wounds, we adapted a five species multiphysics model of epithelial wound healing and used it to predict the efficacy of various plasma treatment protocols [3]. In the model, we assume that the only effect of plasma application to the wound is to reduce the bacterial load, and that this reduces the bacterial oxygen consumption in the wound. The model follows the spatial and temporal profiles within the wound of oxygen, chemo-attractants, capillary sprouts, blood vessels, fibroblasts and extracellular matrix material. We highlight the importance of prolonged effects of plasmas, and why an initial large reduction in the bacterial population may not be sufficient for improved healing. Although it is clear that current efforts to model wound healing in general and the effects of plasma in particular are in their early stage, the present results suggest several important directions for coupling plasma models with models of tissue biochemical responses. [1] G. Morfill et al., New J. Phys. 11 (2009) 115019 [2] G. Isbary, et al., Br J Dermatol. 163 (2010) 78 [3] J. A. Flegg et al., Bull. Math. Biol. 72 (2010) 1867

Cold plasmas at low/atmospheric pressure are widely utilized since almost 50 years to modify the surface of materials intended for biomedical applications, with the aim of driving the interactions of proteins, cells, and biological tissues with materials in lab wares, prostheses, biomedical devices and the like. In the last 20 years plasmas have been used also for sterilization and decontamination of surfaces of biomedical interest. More recently, in the new field of Plasma Medicine [1, 2], direct exposure of living tissues to atmospheric pressure air plasmas have started to be utilized for therapeutic uses, e.g. for sterilization and decontamination of wounds, for wound healing, for teeth bleaching and disinfection of teeth root canals, for blood coagulation, for cancer therapy, and for other uses. In some of these applications, cell activation has been measured, a phenomenon that may be properly used in certain applications, at reduced â?odoseâ? of plasma exposure (i.e., of oxygen and of nitrogen reactive species such as ozone and NOx gases, among others). In this talk we will show viability and cell growth data resulting from air DBD plasma exposure at different doses of different cell lines, Saos 2 and NHDF. At certain doses we have measured increased activity of the Saos 2 osteoblast cell lines. We believe that by properly tuning the dose of exposure of cells and biological tissues to air plasma it could be possible to stimulate positive effects on cell growth and tissue regeneration, that would turn useful in several branches of Medicine. References 1) Plasma Processes&Polymers 3(6/7), 2006; Special Issue on Plasma Processes for Biomedical Applications, organized by P. Favia. 2) Plasma Processes&Polymers 5(7), 2008; Special Issue on Plasma Medicine, organized by A. Frid-man and M. Laroussi. Acknowledgements S. Cosmai and D. Benedetti are gratefully acknowledged for their support in the lab.

Until the mid-1990s, indium was used for an alloy, solders and semiconductor materials. In 2001, a worker engaged as an operator of a wet surface grinder of indium tin oxide (ITO) targets died from bilateral pneumothorax due to interstitial pneumonia in Japan [1]. Following the first case of interstitial pneumonia consistent with occupational exposure to ITO was reported, 4 epidemiological surveys were studied among indium handling workers in Japan, and all studied showed that exposure to indium compounds caused pulmonary interstitial as well as emphysematous changes. Up to 2011, 8 cases of interstitial pneumonia in Japanese indium-exposed workers, 2 cases of pulmonary alveolar proteinosis (PAP) in US indium-exposed workers, 1 case of PAP in a Chinese indium-exposed worker [2]. We evaluated the toxicity of ITO and copper indium gallium diselenide (CIGS) particles when they were given into the lung of experimental animals. In the study of ITO, we evaluated the chronic pulmonary toxicity of ITO and In2O3, which is the main material of ITO, was also used in order to compare the toxicity of ITO, and it was instilled in equimolar amounts of indium to those of ITO [3]. Male Syrian golden hamsters were instilled ITO particles or In2O3 particles, twice a week, for 8 week. The hamsters were euthanized serially up to 78 week after the final instillation. From this study, the chronic pulmonary toxicity of ITO and In2O3 particles was confirmed. In the study of CIGS, the aim was to clarify the subacute pulmonary toxicity of CIGS solar cells in rats [4]. Male Wistar rats were given GIGS particles, intratracheally, twice a week, for 3 times. These rats were euthanized after 0, 1, 3 week from the final instillation serially. The present results clearly demonstrated that CIGS particles caused subacute pulmonary toxicity and absorption from lungs of CIGS particles was considerably slow. From these animal studies, it seems that the severity of lung lesions caused by ITO or In2O3 particles worsened with passage of time but not by CIGS particles, which developed from early period of observation. This difference of lung lesion appearance might depend on particle solubility in the body. Acknowledgements This study was funded in part by a Grant-in-Aid for Scientific Research (B) (19390164) and (23390164) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Incorporated Administrative Agency NEDO under METI in Japan. [1] T. Homma, et al. J. Occup. Health 45, 137 (2003). [2] K.Omae, et al. Int. Arch. Occup. Environ. Health 84,471(2011) [3] A.Tanaka, et al. J. Occup. Health 52, 14 (2010) [4] A. Tanaka, et al. Submitted.

Effect of plasma-exposure to bio-particles has been studied using B. subtilis, E. coli and bacteriophages. Non-Thermal Plasma has been used, and states of different biological components were monitored during the course of the exposure. Analysis of green fluorescent protein, GFP, introduced into E.coli cells proved that NTP causes a prominent protein damages without cutting peptide bonds. We have developed a biological assay which evaluates in vivo DNA damage of the bacteriophages. Different doses of the plasma were applied to wet state of λ phages. From the plasma-expossed λ phages, DNA was purified and subjected to in vitro DNA packaging reactions. The re-packaged phages consist of the DNA from discharged phages and brand-new coat proteins. Survival curves of the re-packaged phages showed extremely large D value (D=25 s) compared to the previous D value (D=3 s) from the discharged phages. The results indicate that DNA damage hardly contributed to the inactivation, and the damage in coat proteins is responsible for inactivation of the phages. We also report a single-molecule-based analysis of strand breakages on large DNA molecules induced by the plasma exposure. Single-molecule observation of DNA that involved molecular combing was used to measure the length of individual DNA molecules. The measured DNA length showed that plasma exposure caused a marked change in length of DNA molecules. The rate of plasma-induced strand breakage on large random-coiled DNA molecules was determined using a simple mathematical model. The measured rate shows good relation with the plasma exposure time, and could be used for safety evaluation of the plasma treated water.

This study uses stimulation by non-thermal (NT) Dielectric Barrier Discharge (DBD) plasma to investigate the role of electrochemical generated reactive oxygen (ROS) and reactive nitrogen species (RNS) on cells and their environment. It has been shown that these species have the potential to initiate cell signaling cascades, altering cellular and biological activity. The goal of this study is to determine exactly which part of the NT-plasma reaction generates the cellular response. Four main hypothesis were tested to determine whether the NT-plasma effect is: 1) physical (e.g electric currents or fields, UV radiation); 2) biochemical direct - a change occurs in the chemical composition of the media (separated method); 3) biochemical indirect - a growth factor is released into the media by the treated cells (transferred method) or 4) requires direct exposure by NT-plasma and the presence of media (direct method). In the direct method NT-plasma was directly applied to cells, in separated treatment the media alone was treated and applied to cells after the treatment, the transferred method is where media is transferred from a directly treated sample and applied to the test sample. These tests dissect out each aspect of NT-plasma treatment and its cellular interactions.

Cold atmospheric plasma (CAP) has already shown the ability to kill different microorganisms and to speed up wound healing. It can be applied in hospital to avoid nosocomial infection and also as a household product for domestic hygiene applications. Different CAP sources have been developed in recent years. Most of laboratory plasma sources use significant gas flow or high power amplifier for the plasma generations. Therefore they have very limited mobility. Our group recently developed a laboratory prototype of a portable CAP device. The device has a diameter of 3.8 cm and a length of 15 cm. The surface micro-discharge (SMD) electrode which has a sandwich structure is used for plasma production. The plasma area for applications is about 3 cm in diameter. The ambient air is used as the working gas therefore no additional gas flow is needed. The device is driven by integrated rechargeable batteries and can be charged up by a commercial 15 V dc power supply plugged into the mains. The total operation time of the device can be as long as one hour after the batteries is fully charged. In this contribution we present the characterizations for this portable air plasma device. The electrical property for the discharge is measured by using high voltage active probes. The dc voltage provided by the batteries is converted to high voltage pulses with peak-to-peak amplitude of 7 kV and with a repetition rate of 6.75 kHz. The high voltage pulses are applied to the SMD electrode and micro-discharge is then ignited. The current signal for the discharge (plasma production) consists of several narrow pulses per micro second. The current pulses are with typical frequency band in the range of 10 - 200 MHz. The power density for the discharge is about 0.5 W per square centimeter. The core plasma profile is modeled by using a finite element analysis and a particle-in-cell simulation. The kinetics of the electron adjacent to the SMD electrode is mainly studied. The spatial and the temporal evolutions of negative ions and electrons are considered for different conditions. The plasma chemistry generated by this portable air plasma device is diagnosed by using mass spectrometry and optical emission spectrometry. Bacterial experiments are carried out to address the bactericidal properties of this device.

Recent progress in plasma science and technology has enabled the development of a new generation of stable cold non-equilibrium plasmas operating at ambient atmospheric pressure. These plasmas are very efficient sources for energy transport through reactive neutral particles (radicals and metastables), charged particles (ions and electrons), UV radiation, and electro-magnetic fields. This includes the unique opportunity to deliver short-lived highly reactive species such as atomic oxygen and atomic nitrogen. Reactive oxygen and nitrogen species can initiate a wide range of reactions in biochemical systems, both therapeutic and toxic. The toxicological implications are not clear, e.g. potential risks through DNA damage, resulting in mutations and leading to late cancers. It is anticipated that interactions with biological systems will be governed through synergies between two or more species. Suitable optimized plasma sources are improbable through empirical investigations. Currently non-equilibrium plasmas initiate an undefined heterogeneous cascade of processes. Quantifying the power dissipation and energy transport mechanisms through the different interfaces from the plasma regime to ambient air, towards the liquid interface and associated impact on the biological system through a new regime of liquid chemistry initiated by the synergy of delivering multiple energy carrying species, is crucial. The major challenge to overcome the obstacles of quantifying energy transport and controlling power dissipation has been the severe lack of suitable plasma sources and diagnostic techniques. Diagnostics and simulations of this plasma regime are very challenging; the highly pronounced collision dominated plasma dynamics at very small dimensions requires extraordinary high resolution â?" simultaneously in space (microns) and time (picoseconds). Numerical simulations are equally challenging due to the inherent multi-scale character with very rapid electron collisions on the one extreme and the transport of chemically stable species characterizing completely different domains. This presentation will discuss our recent progress actively combining both advance optical diagnostics and multi-scale computer simulations. This combination allows us to overcome state-of-the-art limitations in quantifying and understanding energy transport mechanisms leading to new strategies for plasma control and manipulation towards superior concepts in plasma-medicine.

In the field of extinct or extant extraterrestrial life research on other planets and moons, the prevention of biological contamination is one of the most important requirements, and its detailed conditions are defined by the COSPAR planetary protection policy. Such a sterilization or decontamination procedure should be performed on the spaceprobes or rovers that land on the other environments. To avoid so-called back contamination, it is also necessary to sterilize them if the probes return to the earth. Currently, a dry heat microbial reduction method is the only applicable way to satisfy the demand, which could, however, damage the sophisticated components like integrated circuits. Therefore, as an alternative method, we investigate the application of low-temperature atmospheric pressure plasma on this subject. Recently, such a plasma exposure technique has been studied intensively from the viewpoint of bacterial sterilization, especially on a living tissue [e.g., 1]. We attempt to apply the related technique with respect to the planetary protection policy. To generate atmospheric pressure plasma in this study, surface micro-discharge technique was used where the temperature of the point of application is lower than 40 °C [2]. It generates the reactive species like NO, O₃, OH from the air component gas, and it is considered that the existence of those species plays an important role for the sterilization. Since the surfaces of spaceprobes and others are not flat, the treatment should be performed even on hidden positions. To study such effect, Geobacillus stearothermophilus spores or Escherichia coli (E. coli) placed on a stainless steal plate or an agar plate, respectively, were treated with the obstacles: a Teflon® cup for the spore treatment and stainless steal tubes for the E. coli treatment, respectively. In both cases, 10⁵ - 10⁶ reductions of colony forming units are observed after a certain plasma exposure time. The basic trend is the following: the higher the aspect ratio of the obstacle is, the longer the required treatment time is, which indicates that the diffusion of long lifetime species are important for the effective treatment of spores/bacteria on uneven surfaces. [1] M. Kong et al., New J. Phys. 11 (2009) 115012. [2] T. Shimizu et al., New J. Phys. 13 (2011) 023026.

Infected chronic wounds are both socioeconomic and medical problem. Cold atmospheric plasma (CAP) has already proven its efficacy in killing bacteria on agar plates but also in a first prospective randomized controlled trial in patients. As an add-on therapy CAPs proofed a highly significant decrease in bacterial load in 5 min plasma-treated wounds (34%, p<10-6, n=291, 36 patients) in comparison with wounds that received only standard wound care. This reduction is found in all kinds of germs, even multiresistant ones. Just as 2 min as well (40%, p<0.016, n=70, 14 patients). The treatment is very well tolerated and no side effects occurred until now (in total more than 2000 treatments in over 220 patients). The results of this study revealed the potential of atmospheric argon plasma treatment as a new approach to kill bacteria in terms of mutiresistancy. The observed bactericidal effect of plasma therapy relies on the synergy of reactive oxygen and nitrogen species, charged particles, electric fields, and UVR. The combination of these biologically active components makes plasma an efficient tool for fighting bacteria. With the same CAP device other dermatologic diseases were treated successfully, e.g. Hailey-Hailey disease. Otherwise CAPs failed in some diseases and revealed their limitations. New plasma devices using surrounding ambient air have not only greater bactericidal but also virucidal properties. These devices may herald a new era in public, personal, pet, and food hygiene, same as in decontamination. Investigations of human compatibility are promising.

Ambient gas plasma treatment is cytotoxic to cancer cells. Treatment of MCF-7 breast cancer cells by indirect dielectric barrier discharge device and subsequent flow cytometry shows that cytotoxicity correlates to an increase in intracellular reactive oxygen species. The effect of plasma treatment is different for normal, non-malignant cells. Cytotoxicity depends on total energy input as well as power, and the species created in plasma discharge. Plasma treatment of cells covered in media creates acidified nitrites in the media, which are known to have anti-cancer activity.

Recently, effects of electrical on plant or cell growth have been studied by many researchers. Growth stimulation characteristics of plants seeds are investigated by an atmospheric discharge irradiation into plasma seeds. The atmospheric pressure plasma gives influences both of the physical impact with energetic particles and chemical reactions on intracellular materials. The mechanism of the growth stimulation would be originated from extra reaction inside plant cells induced by active particles. In this experiment, redox reaction of thiol compound of cells of radish sprout are investigated using the atmospheric discharge torch plasma. Atmospheric pressure plasma torch is consisted of alumina ceramics tube and the steel mesh electrodes wound inside and outside of the tube, with the dimension of 130 mm in length and 3 mm in a diameter. When AC high voltage (8 kHz, 7 kV) is applied to the electrode gap, the barrier discharge plasma is produced inside the alumina ceramics tube. The plasma is blown outside with the gas flow in ceramics tube. The gas served in this experiment is pure oxygen and nitrogen. Radish sprouts seeds locate at 1 cm from the torch edge. The seeds are sown to the cultivation pot after the plasma irradiation. The length of the radish sprouts is recorded every 24 hours. The growth stimulation was estimated in the length of a stem and a root after the plasma irradiation. The stem length increases approximately 2.8 times at the cultivation time of 24 h. And the growth stimulation effect is found to be maintained for 40 h, after sowing seeds. The mechanism of the growth stimulation would be the redox reaction inside plant cells induced by oxygen radicals. The thiol quantity of radish sprouts, which is related to the redox reaction between cystein, cystine and the growth factors, would be modified by plasma irradiation. The oxygen plasma irradiation decreases the thiol quantity with the treatment period. This result indicates that the reduction type thiol is oxidized by oxygen radical. Decrease of the reduction type thiol in cells tends to activate transcription factors, and related growth-promoting factor activated by reduction type thiol are one of candidates of plants growth mechanism.

In recent years, atmospheric non-thermal plasma sources have attracted much attention in medical field because of its effectiveness for sterilization of medical devices and wound area. However, conventional plasma sources have some limitations in useable plasma gas species and so biological effect did not be confirmed with various gas plasmas. To generate various gas plasma we have developed damage-free multi-gas plasma jet source. It can generate stable plasma with various gas species and the plasma does not give thermal and electrical shock to the target. Mechanism of sterilization by plasma has been thought to be due to radicals or ultraviolet rays. And it is known that amount of radicals and emission intensity of ultraviolet are different according to plasma gas composition. In this research, biological effects of radicals and ultraviolet ray in various gas atmospheric plasmas were investigated. At first, argon, oxygen, nitrogen, air and oxygen mixed argon plasma were irradiated to E. Coli in agar medium. As a result, a clear difference was seen by plasma gas species in the bactericidal effect. Oxygen mixed argon plasma was most effective for sterilization of E. Coli. Secondly, ultraviolet spectrums from 200 nm to 400 nm in each plasma were measured. As a result, ultraviolet emission of nitrogen plasma show the highest intensity and it was over 10 times higher than oxygen mixed argon plasma. To investigate effect on human cell line, various gas plasmas irradiated to HeLa cells. As a result, HeLa cell was effectively killed by oxygen mixed argon plasma. The details of the plasma source and the results of these experiments will be presented.

Cold atmosphere pressure plasma (CAP) is one of the most promising prospective tools for the prevention of infectious diseases and nosocomial infections. It easily kills a wide range of microorganisms like bacteria, fungi, viruses and spores. A very crucial point is that CAPs are also able to inactivate bacteria resistant to antibiotics like multi resistant Staphylococcus aureus (MRSA) and verotoxin-producing Escherichia coli (EHEC), which can cause severe diseases. In addition to that, we were also able to demonstrate that the probability to build up resistance against CAP for both gram negative and gram positive bacteria is very low. The bactericidal mechanism by CAP is not clarified yet. However, it is believed that there are several processes which lead to inactivation of microorganisms. There is a common consensus that reactive species play a major role in this. Recent results show that reactive oxygen species cause peroxide emergence which finally leads to lipid peroxidation and other oxidative damages in the cell wall of the microorganisms. We also detected nitrites and nitrates in liquids after the plasma application and an uptake of NO into bacteria was observed. We did not see any breaks in dsDNA, neither in prokaryotic nor in eukaryotic epidermis cells. This finding leads to the assumption that the damage is mainly external and that the reactive species de-react with the cell wall and make it permeable for other species like nitrogen oxides. It is not clear how the reactive species behave as soon as they have entered the bacteria; however, in our parameter range, the reactive species are not able to break DNA double strand bonds.

Viruses are infectious nanoparticles composed of nucleic acids and proteins that depend on cells for energy. Viruses invade cells where they proliferate, resulting in disease. Sterilization, disinfection, and antisepsis are important for preventing diseases derived from pathogens such as viruses. Sterilization using gas plasma is promising. The gas plasma is generated by removing electrons and producing a highly excited mixture of charged nuclei and free electrons. The process is achieved by the application of sufficient energy, in the form of heat or an electromagnetic field, to gas. Gas plasma sterilization is a method potentially effective against all microorganisms including viruses. In this study, we investigated the effect of N2 gas plasma on influenza viruses and proteins. We used N2 gas plasma apparatus (BLP-TES, NGK Insulators, Ltd). As elevated temperature, UV emission, and oxidative stress production were detected during the N2 gas plasma generation, at least three major mechanisms of action (temperature, UV, and oxidative stress) are presumed in this system. By N2 gas plasma treatment, degradation of nucleoprotein and modification of lipids was induced in influenza virus. The degradation was also shown in treated bovine serum albumin (BSA), while the secondary structure was changed into increased α-helix and decreased β-turn. This effect was incompatible with elevated temperature, which induced decreased α-helix and increased β-sheet. In addition, although UV emission was seen, UV did not change secondary structure of proteins. Therefore, oxidative stress production such as radical generations may be important factor for secondary structure change, degradation, and modification of influenza virus components including proteins and lipids. Taken together, influenza virus components were effectively degraded or modified by N2 gas plasma. The new technology would contribute to infection prevention and contamination control such as sterilization of medical devices, safe drinking water and blood, production and preservation of products, and decontamination of surfaces.

Background: So far, investigations on cold atmospheric plasma have focused on hospital hygiene and treatment of wounds. Except for the investigation of the bacterial load of chronic ulcers there are no randomized controlled clinical trials on the use of cold plasmas in patients. To look into new potential indications for physical plasma and because some reports suggest plasma having antipruritic effects, we investigated the treatment of pruritus - the most frequent dermatological symptom that often represents a therapeutic challenge. Objectives: To assess the efficacy and safety of cold atmospheric argon plasma as add-on-therapy in pruritic dermatoses. Methods: 46 patients with various pruritic dermatoses were treated for 2 min daily with cold plasma in addition to standard treatment. All patients served as their own control which was treated with argon gas as placebo mode. The outcome measure was a long-term and short-term reduction in itching measured by means of a visual analogue score (VAS). Results: At baseline the VAS scores were comparable in both groups (plasma 4.57 [SD 2.38], argon 4.34 [SD 2.35]). There were no significant differences in VAS reduction between plasma and argon treatment: long-term VAS difference of 1.97 [SD 1.33] for plasma and 1.74 [SD 2.37] for argon (p=0.224, 95% CI: [-0.15; 0.60]), short-term VAS difference of 1.92 [SD 1.33] for plasma and 1.97 [SD 1.29] for argon (p=0.544, 95% CI: [-0.21; 0.11]). In both areas, patients experienced a significant reduction of pruritus at the end of therapy compared to baseline (plasma 1.97 [p<0.0001], placebo 1.74 [p<0.0001]). No relevant side effects occurred, and treatment was well-tolerated. Conclusions: Cold plasma application unfortunately did not lead to a higher pruritus reduction than placebo treatment. A significant reduction of pruritus compared to no effect was found at the end of therapy in both groups. Both treatment options had similar safety profiles.

Microplasma proved to be and efficient technology that replaced more conventional technologies for the surface treatment of polymers, NOx removal from exhaust gases, indoor air treatment or sterilization of bacteria. Our microplasma which is dielectric barrier discharge at atmospheric pressure non-thermal plasma was used for the surface treatment of L-lactic acid (C3H6O3) polymer film. The development and optimization of microplasma technologies depend on the clarification of microplasma physics. Emission spectroscopy is one of the methods for plasma analysis. Emission spectra were measured by an ICCD camera, a photomultiplier tube and a spectrometer to which was attached a fiber optic. Measurements were carried out at atmospheric pressure in N2, Ar and N2/Ar mixture. Discharge voltage was negative pulse, which has rise time of 100 ns, pulse width of 100 ns to1 us and frequency of 1 to 27 kHz. Emission spectrum showed high intensity peaks of N2 second positive band system peaks (N2 SPS), N2 first negative band system peaks (N2+ FNS), N2 first positive band system peaks (N2 FPS) for the discharge in N2 gas. OH peaks, Ar I peaks and N2 SPS peaks were observed in Ar and N2/Ar mixture. In surface treatment of polymer process, microplasma remote method could be effective. In this application the target surface was positioned at distance in order of millimeters from the electrodes. The process consisted in generating the active species between the electrodes and transporting them by way of the gas flow to the target. Thus the presence of active species outside the electrodes could be required. The spatial distribution of OH light emission corresponding to 308.9 nm peak and Ar I peak at 696.5 nm and N2 SPS peak at 337.1 nm outside the electrodes were measured. L-lactic acid (C3H6O3) polymer film, which has been developed for medical use, was treated by remote microplasma process in order to improve its hydrophilicity. The surface of polymer film was evaluated before and after the microplasma treatment using a contact angle meter and X-ray Photoelectron Spectroscopy (XPS) analysis. A modification of surface bonds was observed which explained the increase of hydrophilicity. The modification at of the polymer film surface versus discharge power, discharge time, gas composition and discharge voltage waveform was investigated. The effect of the microplasma on the surface of polymer film could be explained by the action of active species measured by emission spectroscopy technique.

Nonthermal atmospheric pressure plasmas have been employed for biomedical processing applications, because they provide high density radicals at a low gas temperature. Recently, nonthermal atmospheric pressure plasmas as well as low pressure plasmas have been employed for growth promotion of plant cells [1-3]. In this study, we have developed a scalable atmospheric dielectric barrier discharge (DBD) device for biomedical processing in a large area and have applied to growth promotion of bread yeast. The device consisted of 20 electrodes of a stainless rod of 1 mm in outer diameter and 60 mm in length covered with a ceramic tube of 2 mm in outer diameter. The electrodes were arranged parallel with each other at a distance of 0.2 mm. The dry yeast was set at 1 mm under the electrodes. The discharge voltage and frequency were 10 kV and 10kHz, respectively. The plasma treatment was carried out in the air. The treatment duration T_on was 50, 100 and 150 s. After the treatment, yeast was suspended in 1 ml yeast extract peptone dextrose (YPD) medium and agitated with a vortex mixer. 1 μl of sample was mixed with 199 μl YPD medium in a micro well plate and cultivated using a shaking incubator at 30^oC. To obtain growth curve of yeasts time evolution of 660 nm light absorbance of the samples was measured with a micro plate reader. For the control, the absorbance is almost constant until t = 1000 min. after the beginning of the cultivation, which corresponds to the lag phase, and then it exponentially increases with t, the exponential growth phase. For yeast with the plasma irradiation of 50 and 100 s, the absorbance increases from t = 0 min. At t = 1000 min., the maximum absorbance for T_on = 50 s is 3 times as high as that for the control. After t = 1000 min., the absorbance increases exponentially with t. For longer T_on of 100s and 150s, the yeast is inactivated and hence its growth is suppressed compared to the control. These results suggest that the DBD plasma irradiation reduces the lag phase of yeast cell division and there is an optimum duration of plasma irradiation for the growth promotion. [1] S. U. Kalghatgi, et al., Proc. IEEE Eng. Med. Biol. Soc. (2009) 6030. [2] N. Hayashi, et al., Jpn. J. Appl. Phys. 50 (2011) 08JF04 (5 pages). [3] S. Kitazaki et al., Jpn. J. Appl. Phys. (in press). Work supported by JSPS.

It is well known that for better and faster growth of plants water quality and composition is one of the crucial factors. Treatment of water with plasma results in change of its properties and chemical composition, which in turn may affect plant growth process and subsequently agriculture produce quality. Both non-thermal and thermal plasmas generated in air or in water produce a cocktail of reactive species, charges, electric fields, and ultraviolet radiation. Plasma treatment of water results in significant change of its properties like pH, oxidation-reduction potential (ORP), conductivity, and concentration of reactive oxygen and reactive nitrogen species (ROS and RNS). Here we report the results of an experimental study of the effect of water treated with different atmospheric plasmas on germination, growth rates, and sugar content of plants (via Brix measurement). In the study we have used 3 types of plasmas: spark discharge ignited in water, gliding arc discharge, and dielectric barrier discharge. It is shown that effects of these plasmas on chemical composition of various types of water are qualitatively different. Non-thermal DBD plasma results in lower (acidic) pH, and production of significant amount of oxidizing species (e.g. H2O2 and O2-). Gliding arc discharge also causes significant acidification of water, but accompanied by production of reactive nitrogen species (NO, NO2 and NO3). Spark discharge treatment results in neutral or higher (basic) pH depending on initial water composition, and production of RNS. The effects of plasma treated water on plant germination and growth will be presented and discussed.

Metal nanoparticles have recently presented a number of promising methods including detection and diagnostic platforms not only in biology and medicine and also in chemical and physical sensors. Di-electrical properties of metal nanoparticles e.g. gold, silver have importance to sense the changes in micro- and/or microenviroment and these changes are dependet on their size, charge and dimension. Localized surface plasmon resonance (LSPR) spectroscopy of metal nanoparticles is an alternative technique for detection of infectious agents such as virus, bacteria, fungi and protozoa. The requirement of fast, flexible, easy-to-use diagnostic tools is an increasing challenge in healthcare systems. To overcome these problems, we aimed to develop a new platform in order to detect some dreadful diseases caused by viruses e.g. HIV. In our study, we first modified polymeric support material with chemical oxidizing reagents, polymeric substrates and chemical etching processes and then performed layer-by-layer surface chemistry including 11-Mercaptoundecanoic acid (MUA), N-(3-Dimethylaminopropyl)-N�-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), respectively. To capture HIV virus particles in samples, viral surface recognizing elements such as gp120 antibody were bound with the help of avidin-biotin interactions. As a preliminary result, polymeric substrate concentration indicated a parallel increase in wavelength shift and 107-fold dilution of HIV viruses caused wavelength change from 554±0.7nm to 560±1.8nm.

Recently, there have been considerable interests in biological applications such as sterilization in the medical field and pesticide spraying in the agricultural field. In an extirpation of pest before harvesting and storage, the environmental impact and human exposure should however take into account. Plasma sterilization has been paid attention as an alternative technique to pesticides sterilization. In the our previous study, we found that bactericidal rate for Penicillium digitatum (PD) spores depended on the partial pressure of O₂ gas in O₂/Ar atmospheric pressure plasma irradiation. For the evaluation of bactericidal rate, it demands culture for several days after plasma treatment. Thus, it requires quick evaluation methods. For this purpose, terahertz time domain spectroscopy (THz-TDS) is suitable because this technique enables to quick observation through detection of vibrational modes staying THz region arisen from macromolecules in the PD. In this study, we have developed in situ evaluation method by using the THz-TDS, and succeeded in sensing of PD response after plasma irradiation. Also this study aims to clarify the mechanisms of interaction between the plasma and macromolecules in the microorganisms. Sample was PD spores prepared on cover glass by dropping, suspension, and drying. The cover-glass samples were treated for 10 min by the non-equilibrium atmospheric pressure plasmas generated by applying AC voltage (9kV, 60Hz) with Ar gas flow of 3 slm, and distanced 10 mm between the plasma and the sample. In the THz-TDS, we measured transmission spectra and calculated differences after plasma irradiation referenced by background spectrum taken before the irradiation. As in differencing spectrum of PD spore between before and after plasma irradiation, intensity of spectral features at 0.5-0.9 THz region was decreased with correlated with plasma irradiation periods. The features were known to be intermolecular vibrational modes of molecular cluster. So it was possibly considered that biological molecules of PD spores are destroyed by the plasma treatment. In addition, the intensity of the spectral depended on plasma exposure time. Therefore, we successfully demonstrated evaluation of PD using terahertz time-domain spectroscopy.

Low-temperature gas discharges generated at atmospheric pressure, commonly known as cold atmospheric plasmas (CAPs), are known to effectively inactivate many different microorganisms. Much of the evidence has been established under laboratory conditions with the microorganisms, often in planktonic growth phase, deposited on a variety of flat and often non-porous surfaces (e.g. glass slides, polymeric or metallic surfaces, and membrane filters). Such surfaces are ideally for gas plasmas that are essentially a surface processing technique. To develop the cold atmospheric plasmas into a robust decontamination technology, key features of practical contamination challenges must be used to challenge the capability and limit of the CAP technology. In particular, most contaminated surfaces are highly porous and bacteria are known to be capable of migrating from the surface into surface cavities and crevices thus escaping the action of the plasma. In addition, microorganisms associated with infection are in a sessile growth form and with the physical protection of a biofilm, an extracellular polysaccharide matrix. Logically the question of plasma penetration depth arises. When the obvious remedy of elevated plasma doses as a compensation for limited plasma penetration is considered, the related question of plasma toxicity also arises. In this presentation, we present results of plasma inactivation of microorganisms, both in planktonic and sessile growth states that are deposited on plant tissues, animal tissues and skins. Discussions of the level of plasma penetration and its mechanisms are presented, and implications to practical applications of the CAP technology are suggested.

The therapeutic use of plasmas for wound healing and the biocidal use of plasmas for sterilization ultimately requires control of the flux of radicals, ions and photons onto the biological surface, and the electric fields induced in those materials. In this talk, we discuss results from computational investigations of the interaction of atmospheric pressure plasmas (APP) with tissues, bacteria and wounds. The plasma modeling platform used in these studies is a 2-dimensional hydrodynamics simulation including radiation transport, surface chemistry, liquid phases and kinetic transport of ions. Molecular dynamics (MD) simulations are used to predict the sputter yield of ions and radicals with cellular surfaces such as lipid layers. We first address the delivery of plasma activated species to these biological surfaces by direct and remote plasma sources. In direct plasma sources, emphasis is on the synergistic interaction of the plasma with the biological surface â?" that is the properties of the biological surface (e.g., wound) affect the properties of the plasma. In remote plasmas, emphasis is on the ability to deliver plasma activated species over long distances, as in endoscopic surgery, and into small spaces, as in surface sterilization. We then address the interaction of plasma produced species with cellular surfaces and biological fluids. Using the energy resolved ions fluxes from the gas phase models and sputter yields from the MD simulations, we evaluate the likely interaction of APPs with cellular surfaces. We find that typical dielectric barrier discharges sustained in air over the period of a few seconds can significantly erode cell membranes.

Atmospheric microplasma has been intensively studied for various application fields, since this technology has features shown here: (1) Generated around only 1 kV under atmospheric pressure, (2) Discharge gap of only 10 to 100 um, (3) Dielectric barrier discharge [1]. Low discharge voltage atmospheric plasma process gives us various applications such as indoor air control including sterilization, odor removal, surface treatment, and would be suitable for plasma-life science field such as medical application (â?oPlasma medicineâ?). In this article, the basic study for plasma-life science will be presented. One life science application of microplasma is â?osterilizationâ?. The sterilization process was carried out with active species generated between the electrodes [2]. The active species were observed by emission spectrometry. The spectra showed the existence of active species, and the microplasma had typical characteristics of nonthermal plasma. Sterilization of E. coli was confirmed after microplasma treatment with Ar gas. The bacteria shape was changed after the microplasma process. Surface treatment by long life active species of materials used for the medical field such as glass, polymer film and others could be also possible [3]. One idea may arise; if we were trying to sterilize the bacteria on the surface or if these materials were human or animal skin. Yes, that would be an application of â?oPlasma medicineâ?. How would it work for human beings or animals? Let us try to find out what is the â?oKey playerâ? for this process. It should be noted that the plasma process must be the safe, when the target is the soft matter, especially for human being. Surface potential and temperature change after microplasma exposure will be also presented to confirm the safeties of this process. References [1] K. Shimizu, et al., â?oEmission Spectroscopy of Pulsed Power Microplasma for Atmospheric Pollution Controlâ?, IEEE Trans. on IAS, Vol. 46, No. 3, pp.1125-1131, 2010. [2] K. Shimizu, et al., â?oStudy of Sterilization and Disinfection in Room Air by Using Atmospheric Microplasmaâ?, Pharmaceutica Analytica Acta, Special issue title: PK/PD: Antifungal and Antibacterial, No. 2, Vol. 11, 2153-2435-S1-001, 2011(in press). [3] K. Shimizu, et al., â?oSurface Treatment of Polymer Film by Atmospheric Pulsed Microplasma : Study on Gas Humidity Effect for Improving the Hydrophilic Propertyâ?, Jpn. J. Appl. Phys. Vol. 50, No. 8, 2011.

Production of ROS (reactive oxygen species) and RNS (reactive nitrogen species) is involved in the regulation of multiple cellular-level processes, including signal transduction, activation of phagocytes, and cell proliferation and apoptosis. Also, ROS/RNS have been shown in some cases to have therapeutic effects in various diseases (e.g. cancer and chronic wounds). It is well established that atmospheric pressure plasmas generate a wide variety of ROS/RNS in air near room temperature, including O2*, O3, OH, NO, HNO2, and H2O2. Therefore, the plasma-generated ROS/RNS have significant potential to realize a radical therapeutic approach. A challenge, however, is to control the plasma chemistry in the gas phase in such a way that the therapeutic potential can be realized. Also, aqueous-phase chemical reactions are thought to play crucial roles because cells and tissues to be treated are mostly immersed in or contact with water or other solutions. We have developed various means to modulate the plasma-generated ROS/RNS in gas and aqueous phase using surface micro-discharges in air. Also, a zero-dimensional plasma-neutral chemistry numerical model, including 50 species and over 600 reactions, has been developed to support the experimental observation and give further insight into the fundamental mechanisms of the highly non-linear plasma chemistry. Recent measurements demonstrate that gas phase air plasma chemistry can show surprising complexities. In the presentation, we will report the effect of the modulation of plasma chemistry through gas/aqueous-phase UV absorption measurement and discuss the correlation with antibacterial efficacy.

Non-thermal atmospheric pressure dielectric barrier discharge applied to the surface of a liquid creates a chemically and physically metastable substance.We investigated the properties and lifetime of the substance and we show that they depend on the treatment conditions such as gas atmosphere and liquid medium used, treatment dose, and other parameters. When deionized water is used, the metastable substance becomes a strong oxidizer. We show that direct exposure of deionized water to neutral and charged species produced in plasma creates a strong oxidizer and acidic substance in this water which, for the lack of a better term, we termed plasma acid. Plasma acid can remain stable for relatively long time and its oxidizing power may be linked to the significant lowering of its pH. We report experiments that demonstrate plasma acidâ?Ts metastability. We also show that observed pH of as low as 2.0 cannot be completely accounted for by the production of nitric acid; and that the conjugate base derived from superoxide is at least partly responsible for both, lowering of the pH and increase in the oxidizing power of the solution.

Energetic particle bombardment from plasma and ions are of great interest for biological materials. However, one can realize that these interactions are quite difficult to investigate because of the complexity of the structure and composition of living organism such as cells and tissues. Various secondary particle emissions from biomaterials are quite useful for characterization techniques. In particular, information on the molecular structural and chemical state is invaluable for biological material analysis, because most biological molecules have similar chemical components, i.e. H, C and O. Secondary molecular ions emitted from biological samples are used to visualize molecular distribution, and molecular imaging is a promising technique for fields such as biomarkers, metabolite characterization, histology, oncology and drug development.
We have developed a novel molecular imaging technique employing swift heavy ions and cluster ions and successfully obtained molecular images of cells and tissues. The molecular distribution of small peptides (up to 1 kDa) was clearly imaged with a lateral resolution of around 5um [1], opening a new opportunity in organic and biological material analysis. This technique was used to obtain secondary ion images of animal cells (3T3) with phospholipid ions [2]. Molecular images of tissues, such as muscles, cerebrums and cerebellums could be observed and compared with images obtained with other techniques, namely fluorescence microscopy and matrix-assisted laser desorption ionization (MALDI). The new technique successfully enables molecular imaging at pressures above 2000 Pa, which is sufficient for water vapor pressure [2], and was named â?owet-SIMSâ? because it provides new opportunities in biological analysis of cells or tissue samples under wet conditions.
Recent progress in this technique will be presented and discussed in view of its possible application in the analysis of biological materials.

[1] Y. Nakata, Y. Honda, S. Ninomiya, T. Seki, T. Aoki and J. Matsuo, J. Mass Spectrom. 44, 128 (2009)
[2] J. Matsuo, S. Ninomiya, H. Yamada, K. Ichiki, Y. Wakamatsu, M. Hada, T. Seki, T. Aoki, Surf. Interface Anal. 42, 1612 (2010)

*This work is partially supported by the New Energy and Industrial Support Organization (NEDO), the (Japanese) Ministry of Economy Trade and Industry (METI) and the Core Research for Evolutional Science and Technology (CREST) program of the Japan Science and Technology Agency (JST).

For the diversified plasma applications to regenerative medicine and medical treatment, we carried out the experiments about the direct irradiation to the tissues and cells using an atmospheric pressures plasma. Especially, we sought to clarify the relationship between nitric oxide (NO) and growth factors in cells. The plasma source using helium (He) gas has a coaxial structure having a tungsten wire for the plasma generation installed inside a glass capillary, and a grounded tubular electrode wrapped on the outside of the capillary. According to the dependency of cell numbers of mouse fibroblasts (NIH 3T3) cells against the plasma irradiation, when only He gas was flowed, the growth of cells was inhibited as the floatation of cells caused by gas agitation was promoted. In contrast, there was no floatation of cells and healthy growth was observed when plasma was generated. On the other hands, in an experiment testing the effects of plasma irradiation on small and middle animals (rat and mini-pig) that were artificially given burn wounds, no evidence of electric shock injuries was found in the irradiated area. In fact, the observed evidence of healing and improvements of the burn wounds. From the results of NO concentration change in the medium using a NO sensor, no large change in the NO concentration is observed for the medium without NIH 3T3 cells. In contrast, for the medium containing NIH 3T3 cells, the NO concentration reaches a maximum value and it subsequently decreases gradually. Moreover, according to the confocal laser microscopy images of the calcium ion distribution, fluorescence from Ca²⁺ ions was observed from plasma flow only. NO is produced by calcium-binding protein (calmodulin), which combines with Ca²⁺ ions released from a cell with NO synthase (NOS) surface.

During the last century and the beginning of the current one enormous progress in the field surgery has been made and led to the development of minimally invasive surgery. Surgery has evolved from laparotomy to simple endoscopy for exploration of the abdominal and pelvic cavities and currently the use of advanced 3-dimentional articulations. Gynecologic surgeons were always at the forefront of the novel techniques. Energy sources have evolved as well. Dr William Bovie introduced electosurgery in the 1920s. Electosurgery can produce monopolar and bi-poloar frequencies for tissue dissection and homeostasis. This led to development of vessel sealing devices aimed at reducing tissue destruction and operating time. Further innovation came with the introduction of laser technologies. Which revolutionized the surgical field of medicine. The combination of cutting and haemostatic properties improved effectiveness and safety during surgical procedures. Argon plasma handheld device produces low flow neutral argon gas with a mixture of high-energy argon atoms, ions and electrons that emerge from the tip of the hand-piece in a precise jet stream, and has a safety profile superior to laser technology. We have better understanding of pathophiolsogy of certain cancers or enigmatic disease like endometriosis. Endometriosis is the presence of endometrial glands in ectopic locations. Endometriosis effects 15% of all reproductive aged women, however it is found in 70% of women with chronic pelvic pain and 50% of women with infertility. Endometriosis can be commonly found on the ovary, pelvic peritoneum, uterosacral ligaments, fallopian tubes, appendix, bowel, cervix, vagina, and pelvic lymph nodes. It is rarely located in urinary tract, diaphragm, umbilicus, surgical scars, arm, legs, etc. Treatment requires precision and accuracy and may involve any organ in the body. Gynecologic cancer account for three of the top ten new cases of malignancy and malignancy related death in the United States. Endometriosis is an independent risk factor for epithelial ovarian cancer. Risk of malignant transformation in ovarian endometriosis is approx 1% to 2.5%. We review properties of Argo plasma and held device that make it ideal for the treatment of these conditions Neutral argon plasma can be utilized as a multi-functional device that has vaporization, coagulation, and superficial cutting capacities with minimal thermal spread and acceptable outcomes. The use of neutral argon plasma appears to be efficacious and safe for the complete treatment of endometriotic implants

Plasma applications in medical science and biological treatments have shown remarkable progress with increasing interests worldwide recently. Considering that biological molecules (biomolecules) such as DNA, amino acids and proteins, which are the building blocks of cells and organs in a hierarchy manner, are basically composed of organic chemical compounds, it is significantly essential to perform fundamental studies in molecular and/or atomic levels for understanding plasma interactions with soft-materials (organic materials) during exposure with plasmas, which contain ions, radicals, electrons and photons. In the plasma interactions with soft-materials, it is of great importance to know that bond-dissociation energies of soft materials are typically less than 10 eV and fundamental processes involved in plasma exposure may contribute to modification and/or degradation of the soft-materials in complex synergetic manners via ions, radicals, electrons and photons. So far, the authors have carried out investigations on plasma interactions with polymers on the basis of surface analysis using x-ray photoelectron spectroscopy (XPS) for development of low-damage processing of soft-materials, in terms of physical interactions via ion kinetic energy and chemical interactions via radicals and photos from plasmas. The studies have been carried out using low-damage plasma generation and control technologies via employing low-inductance antenna (LIA) modules [1-3] to sustain inductively coupled plasmas (ICPs), which can provide one of the solutions to realize high-density plasma production with low sheath-edge potential. Ion energy distributions measured with a mass-separated ion energy analyzer showed significantly suppressed ion energy at the sheath edge as low as 6 eV [3], which is essential for studies of plasma interactions with soft-materials having bond dissociation energies as low as or less than 10 eV. Based on the studies carried out so far, the preset works in this paper extend the investigations further on plasma interactions with amino acids, which are the building blocks of proteins, as convenient model systems in studies of interaction with plasmas. Experimental results obtained from plasma exposures to amino acids will be presented in terms of physical and chemical effects via variations of chemical bonding states via XPS analysis. References [1] Y. Setsuhara, T. Shoji, A. Ebe, S. Baba, N. Yamamoto, K. Takahashi, K. Ono and S. Miyake, Surf. Coatings. Tehcnol. 174-175 (2003) 33. [2] Y. Setsuhara, K. Takenaka, A. Ebe, K. Nishisaka, Plasma Process. Polym. 4 (2007) S628. [3] Y. Setsuhara, K. Cho, K. Takenaka, M. Shiratani, M. Sekine, M. Hori, Surf. Coatings Technol. 202 (2008) 5225.

Objectives: To elucidate the effects of low-temperature plasma (LTP) treatment on a mature matrix-rich biofilm. Methods: In this series of in vitro experiments we used a portable LTP torch operated at an airflow rate of 1.10 L/s. A matrix-rich Streptococcus mutans biofilm was formed on saliva-coated hydroxiapatite, in batch cultures with media containing 1% sucrose, at 37 °C and 5% CO2 for 5 days. At the end of the experimental period, the mature biofilm received a one-time LTP treatment (30 sec). The samples were placed on a table at distance of 3 cm from the nozzle outlet and then exposed to the plasma. Control samples were treated using airflow only. After treatment the biofilms were analyzed for bacterial viability, culturing the treated biofilm samples; biomass and polysaccharide composition by colorimetric assays; polysaccharide structure by glycosyl linkage analysis; and biofilm morphology by environmental scanning electron microscopy (ESEM). Results: When a single LTP treatment was applied on the mature biofilm, although as expected there was no difference in the biomass and insoluble polysaccharides â?"we processed the biofilms immediately after treatmentâ?" there was a remarkably reduced viability of S. mutans. ESEM results showed the single LTP treatment dramatically affected the biofilm morphological characteristics, damaging the matrix. The bacteria were most likely exposed to the surroundings. Differences in the structure of the insoluble polysaccharides can be seen in the glycosyl linkage analysis, indicating that more 6-linked polysaccharides were present in the insoluble polysaccharides from biofilms treated with LTP. This composition reflects a polysaccharide that is easily disrupted. Conclusion: Treating a mature matrix-rich biofilm with LTP leads to the disruption of the extracellular polysaccharide matrix, apparently exposing bacteria within the biofilm to the LTPâ?Ts antibiotic effect.

Biological molecules such as aromatic peptides can undergo sublimation due to their volatile nature and this makes them attractive monomers for the vapor deposition process. Aromatic dipeptides, diphenylalanine and dityrosine, used in this study have an ability to form nanotubes and nanofibrils both in organic-aqueous solutions and during vapor deposition process, due to hydrogen bonding and Ï?-stacking. In particular, plasma enhanced chemical vapor deposition (PECVD) has been used as the technique for the deposition of peptide nanotubes on a variety of substrates. Dipeptides were sublimed into a reactive plasma species using a home-built reactor and allowed to deposit onto substrates, downstream from the plasma zone. Plasma deposition allows control over the growth of homogenous and dense array of nanotubular structures. In this study, we have investigated the impact of the conditions used in the plasma deposition process on the morphology and the characteristics of the deposited nanotubes. Several parameters such as pressure in the plasma chamber, time of deposition, flow rate of the ionizing species and the RF plasma power source conditions have been varied. We have used a variety of techniques such as SEM, TEM, contact angle measurements, nanoindentation and AFM to quantify the effects of the deposition conditions on the peptide nanostructures.

Objectives: This study evaluated the effect of low-temperature plasma (LTP) on hydrophilicity of polymethyl methacrylate (PMMA) resin and Candida albicans adhesion. Methods: PMMA resin discs were polished to a surface roughness (Ra) value of 0.30 ± 0.05 um. The contact angle obtained on the PMMA resin discs subjected or not to argon-LTP treatment was measured by the captive bubble method. The argon plasma was applied during 1 minute at a constant flow rate of 5.0 l/min. The same treatment conditions were conducted with only argon gas flow without plasma activation as a negative control. The contact angles were also measured after 15, 30, 60 and 120 minutes of treatment to evaluate the effect maintenance over time. The surface chemistry of PMMA samples submitted to LTP treatment was also evaluated by X-ray photoelectron spectroscopy (XPS). For microorganism adhesion, salivary pellicle was formed on additional samples after the same treatment conditions described previously. Initially, Candida albicans (ATCC 14053) was cultured for 18 hours (5%CO2, 37°C). The cells were suspended in Yeast Mold (YM) broth, incubated for 5 hours (5%CO2, 37°C), centrifuged in adsorption buffer solution, and standardized to 1 x 107 cells/mL. The discs were positioned in cell culture plates and 2ml of the microorganism suspension was added and remained under stirring for up to 2 hours at 37°C. The samples were stained with gram crystal violet and images were obtained by a digital camera. Results: The discs submitted to LTP treatment before formation of salivary pellicle exhibited a more hydrophilic surface (P<0.05) and this effect was maintained up to 120 minutes following treatment. XPS analysis revealed different surface chemistry between the LTP-treated and non-treated samples. Images revealed patterns of C. albicans adhesion on LTP-treated and non-treated discs. Conclusion: The LTP treatment influences the surface chemistry of PMMA resin and improves the material hydrophilicity, which may influence the microorganism adhesion.

This study reports on non-stoichiometric hydroxyapatite (HA) coatings doped with silica ions, which possess both a higher resorption rate in comparison to stoichiometric HA, and an improved bioactivity. Plates of Ti, Ti6Al4V and 316 L SS were used as substrates. The target consisted of a powder of Si-containing HA (Si-HA) Ca10(PO4)5.5(SiO4)0.5(OH)1.5 was used. Thin nanostructured CaP-based coatings of Si-containing non-stoichiometric HA were deposited by rf-magnetron sputtering at the power level in the range 30-290 W, negative substrate bias up to 100 V, a pressure of 0.1 Pa for 30-180 min [1, 2]. The thin coatings were characterized by EDX, ESEM, XRD, IR spectroscopy, and pull off test. Biological trials on Si-HA films were done in vitro with culture of prenatal stromal cells of human lung (PSCHL) of FL-42 line (obtained from Bank of Stem Cells Ltd, Tomsk). We used carcinogen-free cell populations of different shape and size with limited life period, maintaining a stable karyotype during passages. The cells were free from foreign viral (AIDS, hepatitis, herpes etc.) and fungous agents. After defrosting, the cell viability as tested with trypan blue 0.4% according to ISO 10993-5 was 91-93 %. The discs tested (surface area 1.77 cm2) were placed into wells of 24-well plates (Orange Scientific, Belgium); and cell suspension in the concentration of 30000 viable karyocytes in 1 ml of osteogenic culture medium was added. Cell culture on plastics was used as growth control. The as-deposited Si-containing CaP-based coatings are dense, pore-free and their composition resembled that of the precursor target composition. A low rf-power density (0.1-0.5 WÃ-cm-2) resulted in amorphous or low crystalline CaP-coating structure, an increase in rf-power level (>0.5 WÃ-cm-2) induced the coating crystallization. The negative substrate bias allowed to vary the Ca/P ratio in the range of 1.53 to 4 [2]. The adhesion strength of the coatings was higher than 40 MPa, i.e. it met the requirements of ISO 13779. The prenatal stromal cells of human lung cells had diverse morphological forms in the case of short-term (4 days) contact with coatings tested. For example, 85-90 % of cells had a round or ellipsoid shape. Fibroblast-like cells were observed in the areas of significant microroughness. Cells also spread into the valleys of artificial surface. Such cells in the surface valleys showed a high expression of alkaline phosphatase (ALP). ALP is considered as general marker of maturation and osteogenic differentiation of stromal stem cells [3]. Thus, the Si-containing rf-magnetron sputter deposited coatings are prospective to be used in stomatology or craniofacial medicine, where the initial material surface porosity should be preserved. REFERENCES 1. Pichugin, V.F., et al. Chem. Mater. Sci., 2011. 5: p. 863-869. 2. Surmenev, R.A., et al. Surf. Coat. Technol., 2011. 205: p. 3600-3606. 3. J.N.M. Heershe, J.A. Kanis (Eds.), 1990, Amsterdam: Elsevier Science Publishers.

One of the promising plasma processes for life sciences is a creation of nano-bio conjugates applied to drug delivery systems, gas sensors, field-effect transistors, and so on. In particular, the synthesis of various kinds of nanoparticles using gas-liquid phases plasmas [1-3] is advantageous in that toxic stabilizers and reducing agents are unnecessary and the process is continuous during the plasma irradiation. In addition, the nanoparticles conjugated with carbon nanotubes (CNTs) and biomolecules such as DNA are very fascinating materials in electronic, magnetic, optical, and biological applications. In this paper, the gold nanoparticles (AuNPs) conjugated with the CNTs and/or DNA are synthesized using a novel plasma technique combined with introduction of ionic liquids or aqueous solution. First, we successfully generate the gas-liquid interfacial discharge plasma (GLIDP) using an ionic liquid, in which the large sheath electric field is formed on the ionic liquid and the plasma ion irradiation to the ionic liquid with high energy is realized [4]. Second, it is found that the high energy ion irradiation to the ionic liquid is effective for the synthesis of the AuNPs [5]. Furthermore, the controlled ion irradiation to the ionic liquid including a carboxyl group can realize the density-controlled synthesis of the AuNPs on the CNTs by dissociation of the ionic liquid and the controlled functionalization of the CNTs by the dissociated carboxyl group. Third, the size- and morphology-controlled AuNPs covered with DNA are synthesized using the GLIDP with aqueous solution [6], where DNA prevents the AuNPs from further clustering, resulting in the small-sized AuNPs. The synthesized AuNPs conjugated with DNA can be encapsulated into the CNTs using the DC electric field [7]. The CNTs work as vectors to deliver DNA into living cells because the CNTs have the unique ability to easily penetrate cell membranes with low cytotoxicity [8]. [1] S. A. Meiss, M. Rohnke, L. Kienle, et al., ChemPhysChem 8 (2007) 50. [2] J. Hieda, N. Saito, and O. Takai, J. Vac. Sci. Technol. A 26 (2008) 854. [3] T. Kaneko, Q. Chen, T. Harada, and R. Hatakeyama, Plasma Sources Sci. Technol. 20 (2011) 034014. [4] T. Kaneko, K. Baba, and R. Hatakeyama, J. Appl. Phys. 105 (2009) 103306. [5] T. Kaneko, K. Baba, T. Harada, and R. Hatakeyama, Plasma Proc. Polym. 6 (2009) 713. [6] Q. Chen, T. Kaneko, and R. Hatakeyama, J. Appl. Phys. 108 (2010) 103301. [7] Q. Chen, T. Kaneko, and R. Hatakeyama, Curr. Appl. Phys. (2011) in press. [8] D. Pantarotto, R. Singh, D. McCarthy, et al., Angew, Chem. Int. Ed. 43 (2004) 5242.

In this study, we aim to develop a plasma processing technology to modify the surface properties of micro- and nano-structured materials for bio-and medical-applications. We also intend to reveal the physical and chemical roles of plasma on surface modification of nano-structured materials and to develop a plasma technology to functionalize the surface of magnetic nanoparticles (MNPs), carbon nanotubes (CNTs) or polymer substrate itself for medical applications such as drug delivery system, magnetic resonance imaging, bio-chip sensors, and so on. For the application of MNPs as a drug delivery system, we carried out amino group surface functionalization of graphite-encapsulated iron compound nanoparticles using low-pressure Ar plasma pre-treatment and ammonia plasma post-treatment followed by oxidized dextran immobilization. We have found that the Ar plasma pre-treatment represents an important step to increase the amino group addition on the MNP surface. After Ar and ammonia plasma treatments, the dispersion property of nanoparticles in dextran solution was significantly improved. The successful dextran immobilization was confirmed by X-ray photoelectron spectroscopy (XPS) and high resolution-transmission electron microscopy (HR-TEM) analyses followed by amino group derivatization using 4-(trifluoromethyl)-benzaldehyde (TFBA). As an evidence for the covalent bonding between nanoparticles and dextran, the area percentage of deconvoluted C = N peak at ~389.6 eV increased from 0% to 10.53±1.30% with increasing the dextran concentration. The result is consistent with the evidenced decreasing of the free amino group percentage from 68.09±5.10% to 14.73±5.89% on the nanoparticles surfaces after dextran immobilization. Similar plasma processing has been carried out to introduce the amino group onto the surface of dotted CNT arrays. Results of fluorescence microscope measurement indicated that the amino group was selectively introduced over the dotted CNT surface. The present result indicates the possibility to immobilize additional biomolecules onto the CNT arrays, which would be demanded for the application as bio-chip sensor. In the talk, we will also present recent experimental results on the plasma processing of biomolecules, such as amino acids, peptides, and microorganisms, discussing the interaction between plasma and biomolecules by the results of several methods such as XPS, high-performance liquid chromatography (HPLC) and quadrupole mass spectrometer (QMS).

One of the major challenges for the development of analytical devices for biological analysis relies on the ability to design advanced surfaces with controlled interaction with the biological entities. Surface functionalization techniques provide those bio-interfaces: appropriate surface physico-chemical properties are able to control the conformation and activity of the immobilized biomolecules. The subsequent technological step is the combination of different bio-functions in micro- and nano-patterns on the surfaces. For instance, structuring the surface in adhesive and non adhesive zone in order to preferentially guide the cell growth is one of the most promising tools for the development of cell chips and for tissue engineering.The requirement of further integration scales and the study of the special behaviour of the biomolecules interacting with nanostructured materials have been the two main motivations for the development of submicron patterning techniques. For instance a strong increase of magnitude of sensitivity in biosensing devices together with lower detection limits have been demonstrated. We show some examples of micro- and nano-functional surfaces provided by plasma processes and self assembled monolayers in combination with Electron Beam Lithography and Colloidal lithography, and their application as platforms for molecular detection and stem cell cultures. Adsorption of IgG on micro- and nano-patterned surfaces show that the protein adsorbs on the pillars, which results in a higher detection sensitivity in an immunoreaction with anti-IgG. Enzyme-Linked Immunosorbent Assay and SPR imaging experiments were set-up showing that nano-patterned surface constrains the immobilization of the antibodies in a biological reactive configuration, thus significantly improving the device performances as compared to more conventional non-patterned or disordered patterned surfaces. We show with different methods (SPR, QCM, ELISA) that the detection sensitivity improvement increases as the size of the patterns decreases. Finally, different protein micropatterns were used for stem cell culturing, showing that stem cell maintenance and differentiation can be controlled by the nature and topography of the protein spots.

In several recent communications from these laboratories, we have described observations that thin organic layers which are rich in primary amine (C-NH2) groups are very efficient surfaces for the adhesion of mammalian cells, even for controlling the differentiation of stem cells. We prepare such deposits by plasma polymerisation at low pressure (thin films designated â?oL-PPE:Nâ?, for â?oLow-pressure Plasma Polymerised Ethylene containing Nitrogenâ?)(1), at atmospheric (â?oHighâ?) pressure (â?oH-PPE:Nâ?)(2), or by vacuum-ultraviolet photo-polymerisation (â?oUV-PE:Nâ?)(3). More recently, we have also investigated a commercially available material, Parylene diX AM(4). In the present communication we shall, first, briefly describe those fabrication techniques; next, and more important, we discuss the comparative results of physico-chemical characterisations of those various organic deposits, which deliberately contain varying concentrations of N, [N], and amine groups, [NH2]. These investigations include solubility measurements in aqueous cell-culture media, and studies of structural properties by XPS (with and without chemical derivatisation with TFBA), FTIR, SEM, among others. Finally, we present certain selected cell-response results that pertain to applications in vascular and orthopaedic medicine; we discuss the influence of surface properties on the observed behaviours of various cell lines, with particular emphasis on possible electrostatic attractive forces due to positively charged C-NH3+ groups and negatively charged proteins and cells, respectively.(5) (1) F.-E. Truica-Marasescu, M.R. Wertheimer, Plasma Process. Polym. 5, 44 (2008). (2) P.-L. Girard-Lauriault et al., Plasma Process. Polym. 5, 631 (2008) (3) F. Truica-Marasescu, M.R. Wertheimer, Macromol. Chem. Phys. 209, 1043; Erratum: 209, 2061 (2008) (4) J. Lahann, D. Klee, H. Höcker, Macromol. Rapid Comm. 19, 441 (1998) (5) A. St-Georges-Robillard et al., Proc. 14th IEEE Int. Symp. on Electrets (ISE14), Montpellier, France, Aug. 2011

Plasma treatment of materials is used since more then 50 years to develop thin films for different kinds of applications. At least since the sixties of the last century these films are used in the fields of medicine and pharmacy. Due to the fact that polymers are applied to design low-weight devices and to realize different geometries very easily, the films are mainly deposited onto polymeric substrates. It is a characteristic property of plasma treatment that chemical functionalities are created revealing a long term availability if they are part of a thin polymeric film. Thus thin layers with good adhesion, a defined amount of chemical functionalities and stability to sterilization processes are generated. This fits to the needs for medical application. In principle plasma processing offers different approaches for the treatment as well as for the deposition of thin films with a variable amount of functionalities available for reaction with bio-molecules. In this contribution advantages and disadvantages of the different deposition strategies will be discussed. The interaction of materials with biological systems can be divided in three subsystems. First, the interaction with bio-molecules, where the binding of molecules with specific activities on one hand and the minimizing of unspecific protein adsorption on the other hand can be influenced by plasma treated surfaces of medical devices. Second, the interaction between bacteria and surfaces can be modulated via deposition of thin films with bacteriostatic or bacteriocidic properties on devices. Third, the interaction of surfaces with mammalian cells can also be influenced to enhance the cell growth and cell proliferation for the development of test kits or implants. This contribution will focus on an application of surface treatment to scavenge endotoxine moieties during blood apheresis. Beside the preparation of the mentioned devices also the analytical tools necessary for film development and control of its properties are shortly stressed as well. A correlation between physico-chemical properties of the applied plasma polymerized films and the biological requirements will be tried. References 1. C. Oehr et al, Plasma Processes and Polymers Wiley-VCH 2005 p 23-37, p 39-49, p309-317 2. V. Sciarratta et al, Plasma Process. Polym. 2006,3, 532-539 3. J. Barz et al, Plasma Process. Polym. 2006,3, 540-552 4. M. Haupt, J. Barz, C. Oehr, Plasma Process. Polym. 2008,5, 33-43 5. Patents: a) PCT/SE2005/001088, b) PCT/EP2010/007110

Surface properties together with topographical features have a dramatic impact on the initial interactions of cells with materias. One of the problem that is generally observed on scaffolds is a difficult ingrowth of cells inside the structure, resulting in poor compatibility of such materials for in vivo applications. Surface modification of porous 3D materials by means of plasma deposition, grafting or etching processes is an exciting field of research due to its impact on several fields of applications including biomedicine, tissue engineering and membranes. The versatility and ability of plasma processes to modify only the outermost layer of a material can render them competitive with respect to wet chemistry approaches in the field of biomedical materials; surface modification plasma processes, in fact, possibly coupled with chemical immobilization strategies of biomolecules, can confer unique surface properties to materials in terms of wettability, similarities with the Extra cellular Matrix (ECM) biological environment, and compatibility with cells and tissues. In this talk we will show results obtained by plasma processing 3D interconnected porous polymer scaffolds for Tissue Engineering. In particular, it will be shown how it is possible to enhance cell adhesion, growth and colonization in porous scaffolds where gradient of surface compositions are induced from the external (e.g., hydrophobic, slightly cell-repulsive) to the internal (e.g., hydrophilic, cell-adhesive) side of the scaffolds. In certain processes hydroxyapatite-based nanocomposite coatings were plasma deposited for orthopedical applications. Polycaprolactone (PCL) 3D scaffolds were modified with several RF (13.56 MHz) deposition and treatment plasma processes. Materials were characterized by means of water contact angle, XPS, SEM, and FT-IR techniques. Cell-growth experiments were run with cell-lines to check the efficiency of several treatments to enhance/accelerate cell in-growth inside scaffolds. Acknowledgements: S. Cosmai and D. Benedetti are gratefully acknowledged for their support in the lab. The national project PRIN 2008 - 20089CWS4C and the project LIPP â?oLaboratorio Industriale Pugliese dei Plasmiâ? of Regione Puglia are gratefully acknowledged for financial support.

Polymeric materials have been attracted many R&D attentions to be used in biomedical applications such as blood-contacting devices. Attempts were made to evaluate the changes in heamocompatibility properties of poly ethylene terephthalate surface by grafting acrylic acid (AAc) and immobilizing heparin on PET surface through two step plasma treatments for the first time. PET surface was modified by using a new method namely â?otwo-step plasma treatmentsâ? (TSPT). In the first step, the films were pre-treated with low temperature glow discharge oxygen plasma and immersed into the aqueous monomer solution of AAc. The second step was carried out by plasma polymerizing of pre-adsorbed reactive monomer on the surfaces of dried pre-treated films. Finally, heparin immobilization was performed in the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide. All films were characterized by attenuated total reflection Furrier transformer infrared (ATR-FTIR) spectroscopy and scanning electron microscopy (SEM). Roughness of the hydrogel and heparinized layers in wet state were measured by atomic force microscopy (AFM). The surface hydrophilicity and blood compatibility of the studied films were evaluated on the basis of water contact angle and platelet adhesion measurements (LDH Test). The AFM results showed that the heparin formed a uniform layer onto the PET with comparatively high level of anti-thrombin-binding capacity. The results of measuring water contact angle show that surfaces became very hydrophilic after the subsequent immobilization of heparin via the carbodi-imide chemistry. In vitro studies by LDH method showed that platelet adhesion onto modified surfaces with heparin was drastically reduced in comparison with un-modified PET. The modified PET surface demonstrated significant improvement on anticoagulation activities of human blood in comparison with un-treated PET. Based upon LDH data the amount of platelet adhering on the heparin-immobilized films decreased significantly in comparison with AAc-grafted-PET and un-treated PET. These results prove that the hydrophilicity and hemocompatibility of PET films could be effectively improved by immobilizing the heparin biomacromolecule on the surface using two step plasma treatments.