The efficacy of cold plasma in a pre-clinical model of various cancer types (lung, bladder, breast, head, neck, brain and skin) was demonstrated. Both in-vitro and in-vivo studies revealed that cold plasmas selectively kill cancer cells. It was shown that: (a) cold plasma application selectively eradicates cancer cells in vitro without damaging normal cells. (b) Significantly reduced tumor size in vivo. Cold plasma treatment led to tumor ablation with neighbouring tumors unaffected. These experiments were performed on more than 10 mice with the same outcome. It was found that tumors of about 5mm in diameter were ablated after 2 min of single time plasma treatment. The two best known cold plasma effects, plasma-induced apoptosis and the decrease of cell migration velocity can have important implications in cancer treatment by localizing the affected area of the tissue and by decreasing metastasic development. In addition, cold plasma treatment has affected the cell cycle of cancer cells. In particular, cold plasma induces a 2-fold increase in cells at the G2/M-checkpoint in both papilloma and carcinoma cells at ~24 hours after treatment, while normal epithelial cells (WTK) did not show significant differences. It was shown that reactive oxygen species metabolism and oxidative stress responsive genes are deregulated. We investigated the production of reactive oxygen species (ROS) with cold plasma treatment as a potential mechanism for the tumor ablation observed. Simulations of the cold plasma interaction with tumor performed showed reasonable agreement with experimental evidence.

Thermal plasmas and lasers have been used in medicine to cut and ablate tissues and for coagulation. Non-equilibrium atmospheric pressure plasma (NEAPP; non-thermal plasma) is a recently developed, non-thermal technique with possible biomedical applications. Although NEAPP reportedly generates reactive oxygen/nitrogen species, electrons, positive ions, and ultraviolet radiation, little research has been done into the use of this technique for conventional free radical biology. Recently, Prof. Masaru Hori&’s team (Plasma Nanotechnology Research Center, Nagoya University) developed a NEAPP device with high electron density. Here Electron spin resonance revealed hydroxyl radicals as a major product. To obtain evidence of NEAPP-induced oxidative modifications in biomolecules and standardize the measurement of these modifications, we evaluated lipid peroxidation and DNA modifications in various in vitro and ex vivo experiments. Conjugated dienes increased after exposure to linoleic and a-linolenic acids. An increase in 2-thiobarbituric acid-reactive substances was also observed after exposure to phosphatidylcholine, liposomes or liver homogenate. Direct exposure to rat liver in medium produced immunohistochemical evidence of 4-hydroxy-2-nonenal- and acrolein-modified proteins. Exposure to plasmid DNA induced dose-dependent single/double strand breaks and increased the amounts of 8-hydroxy-2&’-deoxyguanosine and cyclobutane pyrimidine dimers. These results indicate that oxidative biomolecular damage by NEAPP is dose-dependent and thus can be controlled in a site-specific manner. Simultaneous oxidative and UV-specific DNA damage may be useful in cancer treatment. Other recent advancements in the related studies of non-thermal plasma in Nagoya University Graduate School of Medicine will also be introduced. References: Toyokuni S. Adv Drug Deliv Rev 65: 2098, 2013; Akatsuka et al. PLoS One 7: e43403, 2012; Toyokuni S. Cancer Sci 100, 9, 2009; Toyokuni. Pathol Int 49: 91, 1999; Toyokuni S et al. FEBS Lett 358: 1, 1995

Plasma-cells/tissues interaction system is a complex system that contains multiple inputs from plasma. Plasma consists of electrons, ions, radicals, UV/VUV, an electric field, and so on. Cells possess signaling circuits to process the information from multiple inputs. Thus, understanding processes from plasma inputs to physiological outputs is essential to develop plasma devices and materials generated by plasma-treatments. Intracellular molecular mechanisms of cancer cell death in plasma-activated solutions are particularly important for clinical applications of plasma-activated solutions to anti-cancer chemotherapy.
We have previously reported that plasma-activated medium (PAM) selectively killed glioblastoma brain tumor cells and induced apoptosis [1]. PAM also showed anti-tumor effects on anti-cancer drug resistant ovarian cancer cells in vitro and in vivo [2]. We further investigated the intracellular molecular mechanisms of apoptosis of PAM-treated glioblastoma. The survival and proliferation signaling network is particularly important for development and maintenance of cancer cells, and signaling pathways such as a PI3K/AKT pathway and a RAS/MAPK pathway in the survival and proliferation signaling network are constitutively activated in various cancer cells to continuously promote cell growth and inhibit apoptosis. We found that activities of AKT, ERK (a MAPK), and mTOR molecules in the signaling pathways were down-regulated in PAM-treated glioblastoma brain tumor cells [3].
In this study, we have further investigated several signaling pathways in and around the survival and proliferation signaling network as well as ROS/RNS and DNA damage signaling. We propose plasma-activated solutions as promising cancer chemotherapy based on our understanding mode of actions.
[1] H. Tanaka, M. Mizuno, K. Ishikawa, K. Nakamura, H. Kajiyama, H. Kano, F. Kikkawa, and M. Hori, "Plasma-Activated Medium Selectively Kills Glioblastoma Brain Tumor Cells by Down-Regulating a Survival Signaling Molecule, AKT Kinase," Plasma Medicine, vol. 1, pp. 265-277, 2011.
[2] F. Utsumi, H. Kajiyama, K. Nakamura, H. Tanaka, M. Mizuno, K. Ishikawa, H. Kondo, H. Kano, M. Hori, and F. Kikkawa, "Effect of Indirect Nonequilibrium Atmospheric Pressure Plasma on Anti-Proliferative Activity against Chronic Chemo-Resistant Ovarian Cancer Cells In Vitro and In Vivo," Plos One, vol. 8, p. e81576, 2013.
[3] M. M. Tanaka H., Ishikawa K., Nakamura K., Utsumi F., Kajiyama H., Kano H., Maruyama S., Kikkawa F., and Hori M., "Cell survival and proliferation signaling pathways are downregulated by#12288;plasma-activated medium in glioblastoma brain#12288;tumor cells," Plasma Medicine, in press.

Cancer therapy by indirect plasma-activated medium (PAM) exposure has been reported some effects on anti-proliferative activity against cancer cells such as carcinoma and glioma. [1-3] Although the atmospheric pressure plasma (APP) consists of various active particles such as charged species, neutral species, radicals, energetic photons, etc. In this study, we focused on clarification of generation of reactive species such as radicals in the PAM. We have detected elecron-spin-resonance (ESR) signals originating generation of radicals in the PAM. From analysis of kinetic behavior of the ESR signals, we will discuss about anti-proliferation effects on the basis of experimental results for chemical modication on the PAM. Preliminarily we will discuss about generation mechanism of active species in the cell culture medium by exposing reactive species such as O atoms. On the basis of experimental evidence of the generated chemical species, we propose the effect of free radicals generated by the plasma on the biological response under exposure of the plasma activated medium.
Acknowledgments: This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas, “Plasma Medical Innovation” (No. 24108001) from the Ministry of Education, Culture, Sports, Science and Technology of Japan
References: [1] F. Utsumi et al., PLoS one 4 (2013) e81576; [2] H. Tanaka et al., Plasma Medicine 3 (2013) 265; [3] S. Iseki et al. Appl. Phys. Lett. 100 (2012) 113702.

As one of the cold atmospheric plasma sources, radio-frequency atmospheric pressure glow discharges (RF APGDs) have attracted considerable attention for the past decades because of their interesting physics and their widespread applications, especially those in the biomedical and biotechnological fields, for example, for the genome mutation of organisms, sterilization of heat-sensitive medical instruments, therapeutic effects with plasma acting on living tissues, etc. [1-3]. For promoting such novel applications of plasmas, it is essential to study the fundamental processes in a RF APGD plasma system, including the energy and particle balance mechanisms, gas flowing effects, interactions between the partially ionized gas and the surrounding air, and the action mechanisms of the plasma jet on the biomaterials and cells, etc. In recent years, we have conducted both numerical and experimental studies on the characteristics of the RF APGD plasmas and tried to employ the plasma jet for the genome mutation breeding of more than 40 kinds of microorganisms as well as plants. In this paper, the recent progresses concerning the fundamental research on the physical features and biological effects of the RF APGD plasmas, their mutation breeding applications for different types of organisms, as well as development of the ARTP (atmospheric and room temperature plasma) mutation system, will be reviewed. The obtained results show that the RF APGD plasma jet is a powerful and environmental friendly mutagenesis tool for organisms with a high mutation rate, diverse mutation and high genetic stability. Keywords: radio-frequency glow discharge; atmospheric pressure plasma; fundamental process; biological effects; mutation Acknowledgement: This work has been supported by the Tsinghua University Initiative Scientific Research Program (No. 2011Z01019), National Natural Science Foundation of China (No. 10972119), and JST CREST of Japan. References [1] Kong MG, Kroesen G, Morfill G, Nosenko T, Shimizu T, van Dijk J, Zimmermann JL (2009) Plasma medicine: an introductory review. New J Phys 11:115012 [2] von Woedtke Th, Reuter S, Masur K, Weltmann K-D (2013) Plasmas for medicine. Phys Rep 530 (4):291-320 [3] Li H-P, Wang Z-B, Ge N, Le P-S, Wu H, Lu Y, Wang L-Y, Zhang C, Bao C-Y, Xing X-H (2012) Studies on the physical characteristics of the radio-frequency atmospheric-pressure glow discharge plasmas for the genome mutation of Methylosinus trichosporium. IEEE Trans Plasma Sci 40 (11):2853-2860

[Background and Aim]
Nonalcoholic steatohepatitis (NASH) has become a significant problem with increase of obesity and type II diabetes mellitus. Although oxidative stress has been implicated in the pathogenesis of NASH, anti-oxidative treatments have thus far exhibited only limited success. We previously showed that electron discharge at the extremely low energy level exhibited anti-oxidative efficacies against H⁺, glutathione disulphide and Fe³⁺ in vitro. [1] Here, we examined the reductive potency of the electron discharge at the low energy levels on the rat model of type II diabetes-induced NASH. [2]
[Methods]
We designed a non-invasive device using dielectric barrier discharge system, which is capable to supply the plenary discharge electron onto the skin surface at nano ampere level/cm². [3]
We have prepared the type II diabetic NASH model rats induced by administration of streptozotocin and a high fat diet. The efficacy of the discharged electron on the NASH rats was examined with an electron treatment at 5mu;A for four weeks.
[Results]
The rats at 8 weeks old exhibited elevation of blood glucose, serum alanine aminotransferase (ALT), and hepatic peroxide product, malondialdehyde (MDA). When the rats were treated with 5mu;A, values of ALT and MDA were significantly decreased (p<0.05). In addition, gradual but steady decreasing tendency of glucose levels was observed comparing to the untreated group, although the values did not reach statistical significant (p=0.074). The serum values of MDA, ALT, and glucose were correlated significantly. Progression of fibrosis as measured by serum hyaluronic acid and histological examination was not affected by the treatment in this model. The present studies suggest that possible application of the discharged electron treatment at a micro ampere level should be examined by performing clinical studies on the diseases in which oxidative stress is implied as a pathogenesis factor.
[Conclusions]
Anti-oxidative electron treatment attenuated the pathogenically elevated liver inflammation and oxidative stress, together with presumably impaired glucose metabolism in NASH rat model.
[References]
[1] Dozen M., Enosawa S., Tada T., Hirasawa K., Inhibition of hepatic ischemic reperfusion injury using saline exposed to electron discharge in a rat model. Cell Med. 5, 83-87 (2013)
[2] Enosawa, S., Dozen, M., Tada, Y., Hirasawa, K. Electron therapy attenuated elevated alanine aminotransferase and oxidative stress values in type 2 diabetes-induced non-alcoholic steatohepatitis of rats. Cell Med. 6, 63-73 (2013)
[3] T. Ishida, K. Hirasawa, M. Dozen, Y. Tada. in Conference Proceedings of 2011 International Symposium on Electrical Insulating Materials (Kyoto, Japan, 2011).

Biological application employing a non-equilibrium plasma processing has been attracted much attention. Foodborne diseases encompass a wide spectrum of illnesses and are a growing public health problem worldwide. They are the result of ingesting contaminated foodstuffs, and range from diseases caused by a multitude of microorganisms to those caused by chemical hazards. Non-thermal plasmas could be an effective method for killing pathogens and reduce pathogens on the surface of fruits, vegetables, fishes, and meats. Reactive oxygen species (ROS) induces Redox reactions in metabolism or cell membrane, denaturalization of DNA or protein, and some biological responses, depending on the dose or flux of factors produced from plasmas.
The albumins are a family of globular proteins. The albumin family consists of all proteins that are water-soluble, are moderately soluble in concentrated salt solutions, and experience heat denaturation. Albumins are commonly found in blood plasma, and are unique from other blood proteins in that they are not glycosylated. A number of blood transport proteins are evolutionarily related, including serum albumin, alpha-fetoprotein, vitamin D binding protein and afamin.
In this study, for elucidating mechanism on the plasma interaction of living tissues with reactive oxygen species, we investigated mass-spectral changes of protein treated by non-equilibrium atmospheric pressure plasma.
Non-equilibrium atmospheric plasma was generated by applying alternative current of 8 kV to two electrodes. Flow rate of Ar gas was 4.26 slm. 3% Bovine Serum Albumin(BSA) solution was prepared from phosphate buffered saline and Bovine Serum Albumin (Wako, 014-15092). Then, the sample was spotted on a 40-mm dish and exposed by the plasma. Matrix used for matrix-assisted laser desorption/Ionization time-of-flight mass spectroscopy (MALDI) were made a mixture of 4 ul of sinapic acid and acetoni-trile (MeCN) and 0.1% trifluoroacetic acid (TFA).
The MALDI spectra were clearly observed at m/z = 33257 and 66506. The peak at 33257 is assigned to be bivalent ion of BSA and that at 66506 is assigned to be univalent ion of BSA 1). The signal intensities decreased by 5 minutes plasma treatment.
Reference
1) M. Adamczyk et al.,Bioconjugate Chem. 5 (1994) 631.

Various stimuli or stresses cause responses of microorganisms such as activation, functional depression, and cell death, depending on the dose or flux of factors produced from plasmas. Reactive oxygen species (ROS) induces Redox reactions in metabolism or cell membrane, denaturalization of DNA or protein, and some biological responses.
Optical microscopy techniques provide information on the distribution and dynamics of biomolecules at the cellular level. Fluorescence microscopy has contributed greatly to our understanding of these processes. However, it relies on the use of fluorescent labels or dyes. These labels may be toxic or destructive to cells, and are subject to photobleaching. It is, therefore, difficult to use them for studying long-term biological dynamics within living cells. Non-invasive label-free imaging techniques are desirable because they would allow long time observations without photobleaching in living cells or tissue where labeling is not always possible.
Raman microscopy is a label-free imaging technique that offers contrast based on vibrational frequencies that are characteristic of chemical bonds. However, spontaneous Raman scattering has very weak signals, so a long integration time is required to achieve a good signal to noise ratio.
A coherent anti-Stokes Raman scattering microspectroscopy(CARS), which is non-linear Raman scattering spectroscopy with high sensitivity, is attractive method to monitor the change in molecular structure of microorganisms. CARS also permits nondestructive molecular signal without any labeling. In this study, we developed system of multiplex CARS and analyzed molecular structures of the microorganisms.
Two lasers were used to generate a coherent anti-Stokes frequency beam, which can be enhanced by resonance. Picosecond laser beams with 1064 nm and supercontinuum (450 to 2000nm) were employed as the pump and the Stokes beams, respectively. The pump and Stokes laser beams were collinearly overlapped and tightly focused into a sample using an objective lens on the microscope. The emitted CARS signal is measured with a spectrometer and CCD.
CARS spectrum of a budding yeast cell (Saccharomyces cerevisiae W303a) was observed. Vibrational spectra were obtained in the fingerprint region (between 1800 cm^-1 and 800 cm^-1), which exhibits many skeletal vibrations that are highly sensitive to molecular structure. The CARS signals at 1655 cm^-1 and 1440 cm^-1 were assigned to the superposition of the C=C stretch of lipid chains and the amide I mode of proteins and CH bend mode, respectively. The band at 1602 cm^-1 originates from mitochondria and its intensity sharply reflects the metabolic activity of mitochondria. The Vibrational spectra structures of a budding yeast cell were successfully observed with multiplex CARS.

Recently, nonequilibrium atmospheric-pressure plasma jet (APPJ) technology has emerged as a novel tool in medicine. We previously reported that APPJ irradiation changes the permeability of the cell membrane without killing the cell and discussed the relevant factors [1]. The ability to control the permeabilization would make possible minimally invasive transfection and drug delivery, which greatly enhances medical, biological, and pharmaceutical research and development. However, it is not clarified how the stresses associated with APPJ application affect biochemical pathways in living cells. Therefore, we have visualized basic intracellular signaling molecules after APPJ irradiation and investigated plasma-induced cell response.
We generate atmospheric pressure plasma using low frequency (LF) (frequency: 10 kHz, voltage: V_p-p kV) with He gas flow, which is irradiated to the bound cells with various fluorescent probes. After the plasma irradiation, we observed the fluorescence that indicates intracellular or extracellular signaling molecules concentration through a confocal laser scanning microscope. As a result, it is observed that intracellular ions concentration increase after APPJ irradiation, which means APPJ irradiation excites cellular activation.
[1] S. Sasaki, M. Kanzaki, and T. Kaneko, Appl. Phys. Express 7 (2014) 026202.

We studied the pulmonary toxicity of indium-tin oxide (ITO), indium oxide (In₂O₃) and indium hydroxide (In(OH)₃), which are used for the raw materials of flat panel displays, on laboratory animals. One hundred and eight male Wistar rats were given 10 mg/kg as indium of ITO, In₂O₃ or In(OH)₃ particles, intratracheally, twice a week, for a total of 5 times, during an 2 week. Control rats were given vehicle only, comprising distilled water. During 3 weeks, these rats were euthanized serially and the toxicological effects were determined. Body weight gain was significantly suppressed in the In(OH)₃ -treated rats compared with the control group, but not in the ITO- or In₂O₃-treated rats. Relative lung weight among all the indium-treated groups was significantly increased compared to that in the control group throughout the observation period. Furthermore, that in the In(OH)₃ group was significantly higher than that in the ITO or In₂O₃ groups. The content of indium in the lung was constant during the observation period in the all indium-treated groups. Serum indium levels in the In(OH)₃ -treated rats were extremely higher, from 100 to 300 times, than that in the ITO- or In₂O₃-treated rats at each time point. Histopathologically, foci of slight to severe pulmonary inflammatory response with diffuse alveolar or bronchiolar cell hyperplasia, expansion of the alveolar spaces and exudation to alveolar spaces were present in all the indium-treated groups throughout the observation period. Interstitial fibrotic proliferation was seen in the In(OH)₃ -treated rats only. The severity of these lesions in the In(OH)₃ -treated rats was more severe in comparison with the ITO- and In₂O₃-treated rats.
The present results clearly demonstrated that ITO and In₂O₃ or In(OH)₃ particles caused pulmonary toxicity when repeated intratracheal instillations were given to rats. Furthermore, lung toxicity of In(OH)₃ was extremely strong comparison with ITO and In₂O₃. Accordingly, a great deal of attention should be paid to the toxicity of In(OH)₃ particles in addition to ITO and In₂O₃ particles.

Treatments based on non-thermal atmospheric pressure plasmas have been widely employed for researches of biomedical and agricultural applications [1], because plasmas produce a cocktail of reactive species that can react with cells and may change cell activity. Plasmas can offer novel treatments of many diseases, improvement crop productivity, and enhancement biofuel productivity. Here we have demonstrated that atmospheric air pressure dielectric barrier pressure (DBD) irradiation to plant seeds has influence on concentrations of chlorophyll and carotenoids in Raphanus sativus L. Experiments were carried out with a scalable dielectric barrier discharge device. 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 discharge voltage and current were supplies 9.2 kV and 0.2 A. As samples for supernatant extract analysis, we employed Raphanus sativus L. plants after 7 days cultivation. Chlorophyll and carotenoids were extracted from 0.5 gram of fresh leaves mixed with acetone and homogenized in a mortar. Mixtures were filtered and washed several times with acetone. Absorbance of supernatant was measured with a spectrophotometer and pigment concentration was calculated using the formula of Holm and Wetsttein [2]. The concentration of Chlorophyll are 0.76 mg/g without plasma irradiation, 0.92 mg/g for 3 minutes plasma irradiation, and 0.59 mg/g for 10 minutes of plasma irradiation. The concentration of carotenoids are 0.14 mg/g without plasma irradiation, 0.19 mg/g for 3 minutes plasma irradiation, and 0.10 mg/g for 10 minutes of plasma irradiation. The concentration variation of these two kinds of pigment suggests that plasma irradiation to plant seeds influences cell activity.
This work was partly supported by JSPS KAKENHI grant numbers 24340143 and 24108009
[1] S. Kitazaki, K. Koga, M. Shiratani, and N. Hayashi, MRS Proceedings 2012, vol.1469, DOI: 10.1557/opl.2012.966
[2] M. Markovic, et al., Arch. Biol. Sci., 64, 531-538, 2012

We investigated the inactivation mechanisms of Penicillium digitatum spores using an atmospheric-pressure plasma or a flux-defined oxygen radical source, which can supply only neutral oxygen species. [1-6]
However, many kinds of microorganisms are living in liquid phase. So, it is very important for investigating the effects of the neutral oxygen radical produced in the gas phase on activation and inactivation of cells in liquid.
In this study, we have investigated dose-dependences of neutral radicals on the inactivation of E. coli in liquid and the activation as well as the inactivation of budding yeast cells in liquid using the atmospheric-pressure plasma.
In the inactivation process of E. coli in liquid, the neutral oxygen radicals were supplied from the gas phase using the oxygen radical source. The distance between the radical exit and the suspension surface was varied. The effects of pH value of the suspension on the inactivation were also investigated. From the results, we have found that O(³P_j) in the gas phase contributes to the inactivation of E. coli cells in liquid and that the pH condition is independent of the inactivation rates of E. coli cells treated with neutral oxygen radicals unlike treated with plasma.
Moreover, we investigated the effects of oxygen radicals on promotion and repression of proliferation as well as inactivation of budding yeast cells, which is widely used as a model eukaryote.
The results indicate that cell growth is promoted ~ 20% at O(³P_j) dose from 6×10¹⁶ to 2×10¹⁷ cm^-3 using the budding yeast cells (Saccharomyces cerevisiae W303a) suspended with phosphate buffered saline of 3ml under the O(³P_j) flux of 2.3×10¹⁷ cm^-2s^-1 while it is repressed ~ 10% from O(³P_j) dose of 3×10¹⁷ to 1×10¹⁸ cm^-3 and the cells are inactivated over 1×10¹⁸ cm^-3.
These results suggest that cell growth from promotion to repression can be controlled by the dose of oxygen radicals.
References
[1] M. Ito,T. Ohta, and M. Hori, Journal of the Korean Physical Society, 60, 937 (2012).
[2] S. Iseki, T. Ohta, A. Aomatsu, M. Ito, H. Kano, Y. Higashijima, M. Hori, Appl. Phys. Lett., 96, 153704 (2010).
[3] S. Iseki, H. Hashizume, F. Jia, K. Takeda, K. Ishikawa, T. Ohta, M. Ito, and M. Hori, Applied Physics Express,.4, 116201 (2011).
[4] H. Hashizume, T. Ohta, T. Mori, S. Iseki, M. Hori, M. Ito, Japanese Journal of Applied Physics 52, 056202 (2013).
[5] H. Hashizume, T. Ohta, J. Fengdong, K. Takeda, K. Ishikawa, M. Hori, M. Ito, Applied Physics Letters, 103, 153708 (2013) .
[6] H. Hashizume, T. Ohta, K. Takeda, K. Ishikawa, M. Hori, M. Ito, Japanese Journal of Applied Physics 53, 010209 (2014).

This study aims to elucidate the interaction between plasma and cell membrane by treating artificial lipid bilayer of cell membrane [1] with dielectric barrier discharge (DBD). The artificial planar lipid bilayers formed at solid-liquid interfaces are called supported lipid bilayers (SLBs). The SLBs before and after DBD irradiation were observed with fluorescence and atomic force microscopy (AFM).
DOPC (dioleoylphosphatidylcholine) and Rb-DOPE (rhodamine B-dioleoylphosphatidylethanolamine) were chosen as a material of lipid bilayer. SLB was formed by the vesicle fusion method [1] and treated with the DBD apparatus [2,3]. The DBD apparatus is composed of the parallel plate electrodes, each of which is covered with a quartz plate. Since SLB is stable in a liquid solution, the lower quartz plate of the apparatus has a dent, in which SLB on substrate was mounted. DBD was generated in Ar gas between the electrodes by applying low frequency (~ 15 kHz) high voltage (15 kV) for 120 s. The gap distance was 1.5 mm. The thickness of the buffer solution above the SiO₂/Si substrate was ~0.47 mm, and the solution was carefully treated so as not to expose the SiO₂/Si surface to the air.
After the DBD irradiation, micropores on the SLB were observed with fluorescence microscopy. The generation of pores was not significant until the irradiation time of 30 s, but the area fraction of the pores increased with time for irradiation times longer than 60 s. We investigate the surface morphology of the SLB on the submicron scale using AFM. After the DBD irradiation, small pits appeared on the SLB surface. We recognized 41 pits in 1 mu;m² of the AFM image, and their lateral and vertical average sizes were 16.4±4.4 nm in diameter and 0.81±0.19 nm in depth, respectively. [3]
We tested heat and chemical treatments without DBD irradiation. When SLB was heated up to 70^#9675;C, some white spots whose fluorescence intensity was higher than the other area were seen on the SLB. The white spots were also observed when H₂O₂ solution was added to SLB, indicating different phenomena from those by DBD irradiation. We then used equilibrium chemicals (HNO₃ and H₂O₂) for the stimulation of SLB. Dark defect regions appeared after the addition of HNO₃ at the concentration of 37 mM. The estimated pH value of the solution in this case is 1.4. Using pH paper, we confirmed that the DBD irradiation of the buffer solution in the same conditions as that for SLB did not change the pH. This result indicates that transient active species play critical roles during the poration in the SLB.
References
[1] Ryugo Tero, Materials, 5 (2012) 2658-2680.
[2] Yoshiyuki Suda, Akinori Oda, Ryo Kato, Ryuma Yamashita, Hideto Tanoue, Hirofumi Takikawa, Ryugo Tero, Japanese Journal of Applied Physics, submitted.
[3] Ryugo Tero, Yoshiyuki Suda, Ryo Kato, Hideto Tanoue, Hirofumi Takikawa, Applied Physics Express, in press.

The development of a safe and damage-free gene transfection technique is needed for several advanced medical technologies such as gene therapy, regenerative medicine, etc. Although the viral vector method is widely used for the gene transfection procedure, this method has risks of pathogenicity expression or neoplastic transformation. As a risk free method, a technique that uses discharge plasma irradiation was invented by Satoh, who is one of the authors, in 2002. However, this method has two problems. One is the trade-off relationship between transfection rate and cell survivability and the other is the issue of reproducibility. We solved these problems by miniaturizing the plasma (~1 mm discharge gap) and localizing its irradiation area (Oslash;1 mm) using a micro capillary electrode (Oslash;70 mu;m). With this configuration we can achieve 60% transfection rate of pCX-EGFP to COS 7 cells with low cell damage.

Non-equilibrium atmospheric pressure plasmas are recently utilized for gene transfection which is expected to play an important role in molecular biology, gene therapy, creation of induced pluripotent stem (iPS) cells, and so on. However, the conventional gene transfection using the plasma [1] has some problems that the cell viability is low and the genes cannot be transferred into some specific lipid cells, which is attributed to the unknown mechanism of the gene transfection using the plasma. Therefore, the spatiotemporally-controlled atmospheric pressure plasma is generated using low frequency (LF) (frequency: 10 kHz, voltage: 7-12 kV) with He gas flow and irradiated to the living-cell suspended solution mixed with genes for clarifying the transfection mechanism toward developing highly-efficient and minimally-invasive gene transfection system [2]. In this experiment, first, fluorescent dye YOYO-1 is used as the simulated gene and LIVE/DEAD Stain is simultaneously used for cell viability assay. By these fluorescence images, the transfection efficiency and cell viability are calculated as the ratio of the number of transferred and surviving cells to total cell count, respectively.
The transfection efficiency is measured as a function of plasma irradiation time, where the diffusion distance is 35 mm (short diffusion) and 73 mm (long diffusion). It is clarified that the transfection efficiency is strongly dependent on the plasma irradiation time and the diffusion distance, and the high transfection efficiency of 53% is realized together with the high cell viability (> 90%) in the short diffusion case. This result indicates that the physical effects such as the electric field on the cell membrane associated with charged particles enhance cellular activity together with the chemical effects associated with plasma-induced products in solution, and as a result, the less-invasive transfection with high efficiency is realized by these synergistic effects on the living cells. Furthermore, the actual genes such as siRNA and plasmid DNA are also found to be transferred into the living cell by reducing the plasma diffusion distance to zero, which means that the physical effects of the irradiated plasma dominantly act on the efficient gene transfection.
[1] Y. Ogawa, et al.: Biotechnol. Bioeng., 92 (2005) 865.
[2] S. Sasaki, M. Kanzaki, and T. Kaneko: Appl. Phys. Exp., 7 (2014) 026202.

Jens Riedel, Berlin/Germany, Jacob T. Shelley, Kent/OH/USA, Carsten Engelhard,
Siegen/Germany
Jens Riedel, BAM Bundesanstalt für Materialforschung und -prüfung,
Richard-Willstätter-Straszlig;e, 12489, Berlin
Dielectric-Barrier-Discharge (DBD) sources are well suited for surface modifications induced by desorption, chemical passivation or ozonolysis of the primary boundary layer. Due to portability, low power consumption and flexible use, especially miniaturized DBD sources are promising candidates for applied atmospherical pressure and rough vacuum applications.
These sources are already widely used as direct desorption/ionization sources in ambient mass spectrometry (MS). However, the underlying physical and chemical mechanisms are far from being understood. Although the applied power, and thus also the formed plasma, is pulsed, most studies concentrate on temporally integrated studies.
For a deeper insight into the fundamentals of ion formation, a homebuilt miniature DBD source was designed and characterized under ambient conditions. The study includes spatially and temporally interrogation of the resulting plasma by both, optical emission spectroscopy and for the first time also a direct detection of the formed ions by MS. The time resolved detections schemes allow a characterization of the impact of the driver frequency on the plasma properties.
The obtained knowledge was directly translated into an improved design of a miniature DBD source. This source was successfully used for in-situ ozonolysis of a fatty acid monolayer on a liquid gas interface.
J. T. Shelley, A. Stindt, J. Riedel and C. Engelhard, J. Anal. At. Spectrom., 2014, 29,
359-366.

Optical emission spectroscopy (OES), optical imaging (OI) of plasma plume, and Langmuir probe diagnosis of laser produced graphite plasmas were done in order to investigate vibrational temperature (T_vib) of C₂ and CN plasma, expansion dynamics of C2 and CN molecules, and ion dynamics respectively. Fundamental wavelength of pulsed laser beam from the nanosecond Nd:YAG laser operating at 600 mJ/pulse energy, 10 ns pulse width was focused on the graphite target mounted on horizontal X-Y translational stage at the centre of spherical stainless steel chamber under vacuum and ambient atmosphere. Optical emission spectra of C₂ swan and CN violet bands were recorded spatially and temporally by collecting emissions at the tip of fiber coupled grating monochromator equipped with CCD detector. Plume images were also recorded at different times to diagnose expansion velocities of C2 and CN species. C2 molecules were differentiated from other species of the plasma using a bandpass filter centred at 500 nm that allows passing band of zero sequence bands of C2 molecules. Langmuir probe (LP) ion signals were recorded to diagnose ions in the plasma by mounting a cylindrical probe perpendicular to the direction of plume expansion. Temperature and density of single charged carbon ions were diagnosed using LP data.

Monitoring bone quality can be a very important and useful clinical tool when wanting to determine how a fracture is healing or how a bone disease is progressing in a particular person. Bone quality is often assessed through radiographic techniques such as X-ray and CT which provides visualizations based on densities of bone mineral. But these conventional methods are expensive and take long time for analysis.
In this work, monitoring of bone biomarker was performed by using electrochemical analysis and commercial screen printed Au electrode. Alkaline phosphatase (ALP) was used for anlytes that is an enzyme to stimulate mineralization by these bone forming osteoblast cells. Anti-ALP antibody was used for capturing ALP, and Electrochemical impedance spectroscopy (EIS) was used for calculating ALP concentration. Among these advantages are obtaining a faster result, lowering costs through the use of commercially available sensors, and utilizing a simpler assay that does not require any variation of labelling. Additionally, commercial electrodes are cheap and disposable which is a necessity when working in a clinical setting. Futhermore, we also performed monitioring ths ALP biomarker in serum for clinical purpose. It&’s limit of detection (LOD) was 20-fold higher than pure buffer, and 100-fold lower than cut-off value.
This work is important because it exemplifies the detection of proteins in serum is available by using utilizing electrochemical methods with commercial electrode. Futhermore, this detection is non-labeling method, it is cheap, fast anlalysis with high reliability and sensitivity. We expect that this electrochemical bone biomarker monitoring is useful strategy for diagnosis and prediction of a patient&’s bone health.

Coagulation is a complex process essential to wound hemostasis. It is initiated through a series of reactions that lead to thrombin activation, fibrinogen cleavage to fibrin, and the formation of a filamentous fibrin network replete with bound/activated platelets. Although required for hemostasis, aberrant coagulation within blood vessels or vascular stents correlates not only to restenosis but to catastrophic thrombosis. Within the stent clotting appears to be initiated by interactions between the E and D regions of intact fibrinogen and the hydrophobic device surface. These interactions induce changes in fibrinogen conformation that expose the αC domain required for fiber formation; normally exposure of this domain requires fibrin formation. Here we present an investigation on platelet activation mediated by surface-induced fibrinogen fibrilogenesis.
To generate the test surfaces, polymers (polystyrene, poly(4-vinylpyridine), polylactic acid, poly(methylmethacrylate) and polybutadiene) were spun cast into films approximately 120 nm thick on glass substrates and annealed at 130°C in a high vacuum oven. Fibrinogen coatings were formed on the spun cast films by incubation with fibrinogen solutions (0.1 mg/ml and 4 mg/ml) at room temperature for different times (10 min, 20 min, 2 h, 12 h and 24 h). Atomic Force Microscopy was used to check the morphology of fibers on these surfaces and the Pierce BCA protein assay was used to measure the amount of protein adsorbed. Platelet rich plasma was placed at a uniform density on each of the spun cast substrates and incubated at 37°C for 1 h. Platelet activation was then monitored as a function of morphology using Scanning Electron Microscopy and as a function of the increased expression of CD41 (platelet glycoprotein IIb). Fibrinogen fiber formation was monitored using fibrinogen beta chain antibody. Platelet distribution was visualized using a Leica TCS SP2 laser scanning confocal microscope.
The results indicated that surface chemistry can induce fibrinogen fiber formation, and thus influent on platelets adhesion and activation. The platelet activation domain is present on the γ chain of fibrinogen molecule. It&’s not available in fibrinogen monomers prior to thrombin cleavage, even though the αC domains are exposed, and becomes available in fibrin fibers. The domain does appear to be available in the fibers formed by fibrinogen in the absence of thrombin cleavage. Hence the surface chemistry and composition can be a contributing factor in thrombosis in the absence of thrombin.

For successful realization of miniaturized biochip devices, surface functionalization which provides sites to immobilize biomolecules onto the chip is one of the most important steps. For the surface functionalization, plasma treatment is used as one of effective methods compared to other conventional chemical methods, such as carboxylic acid, nitric acid, high temperature vapor, etc. Plasma treatment has the advantages of low temperature treatment, little damaging effects, and providing a wide range of different functional groups depending on the plasma discharge conditions.
For the development of multi-functional biochip device, a normal low pressure plasma processing might be difficult to modify the surface of substrate with different functional groups using a lithographic technique. Therefore, we propose here to use the atmospheric pressure plasma jet with a nano-capillary for micro-scale surface modification of the substrate. The atmospheric pressure plasma has many advantages, that is, no need of expensive vacuum equipment, fast reaction process, and great potential for surface functionalization. To the best of our knowledge, spatially-selective, ultrafine functionalization of the biochip substrate by atmospheric pressure plasma jet has not been reported so far.
In this work, a nano-size capillary atmospheric pressure plasma jet technique has been developed to functionalize amino or carboxyl groups selectively on the polymer or CNT dot-array substrate. Selective surface functionalization has been demonstrated by atmospheric plasma pressure jet with two stages: pre-treatment stage by atmospheric pressure plasma jet with a negative biasing for activation and post-treatment stage for functional group modification. The analysis results of fluorescent microscopy with the fluorescent dye indicate that pre-treatment and post-treatment period time strongly affect the fluorescent intensity and area of surface functionalization. Increasing the pre-treatment and post-treatment time end in decreasing the intensity of fluorescent dye and correspond to decreasing the concentration of functional groups on the target area. The successful line patterning of amino or carboxyl group functionalization on CNT dot array substrate was demonstrated. The experimental results using the biotin-avidin system to simulate the virus concentration experiment will be also presented at the conference.

Continuously released in nature via sewage, antibiotics have become ubiquitous in surface waters, including those intended for drinking water production. Present in sub-inhibitory concentrations, antibiotics notably induce the emergence of antibiotic-resistant bacteria. Numerous methods, including chlorination, ozonation or electrochemical processes, have been investigated for the removal of these persistent pollutants from water and wastewater [1]. However, current water treatments are not fully efficient in the elimination of antibiotics and can even generate more toxic secondary metabolites. The use of enzymes is an alternative or a complement to existing water treatments commonly used in water treatment plants (WTP). Wide varieties of enzymes that can degrade antibiotics, encoded by the antibiotics resistance genes of bacteria, have been reported. However, free enzymes can quickly lose their activity due to autolysis, physico-chemical and biological degradation or aggregation. The immobilization of enzymes on surfaces can prevent such phenomena, as well as increase their lifetime and degradation activity. Enzymes immobilization can be achieved by encapsulation, formation of an enzyme network or by grafting on functionalized surfaces. Atmospheric pressure plasma-enhanced chemical vapor deposition (AP-PECVD) processes have been deeply investigated for the deposition of functional coatings [2]. Recently, the use of ultra-short square pulses to initiate a dielectric barrier discharge was shown to provide a simple one-step atmospheric pressure and room temperature process for the deposition of homopolymer layers with functional group densities as high as the ones achieved by traditional wet methods [3].
In this work, we report the deposition of epoxy group-rich layers of polyglycidyl methacrylate from an easily up-scalable AP-PECVD method employing ultra-short square pulses. The epoxy group-rich layers, deposited on Kaldnes® biochips commonly used in wastewater treatment moving bed reactors, are used for the covalent grafting of enzymes able to degrade antibiotics. The developed system being intended for wastewater treatment systems, the bioactive surfaces are further saturated with Tween 20 in order to prevent adhesion of microorganisms that could be detrimental to the enzymes activity. Enzymatic activity is observed for a period in excess of 696 h for the prepared bioactive surfaces against only 48 h for the free-enzyme form. The quantities of antibiotics degraded are also shown to be 2 to15 times higher for the immobilized enzymes. In addition, erosion tests show that after exposure to a laminar flow water of 30 kmmiddot;h^-1 for 240 h, the prepared surfaces remain active.
[1] Homem and co., J. Environ. Manage. 2011
[2] Mauchauffé and co., J. Mater. Chem. B 2014
[3] Boscher and co., Plasma Process. Polym. 2014

As a survival mechanism, bacteria tend to adhere to surfaces in many environments including a living host, medical devices or industrial systems, leading to severe health and economic problems. Indeed, bacteria and the resulting biofilm formation are known to induce implant-associated infections, accelerate corrosion of metal surfaces and reduce the heat transfer and operating efficiency of industrial equipment.
Accordingly, various antibacterial coatings have been developed based on the immobilization or release of bactericidal substances such as metal nanoparticles, antibiotics, quaternary ammonium or other cationic compounds. Despite promising results, these strategies suffer from their limited efficiency, their toxicity or their role in the emergence of multi-resisting pathogens. [1] Natural antimicrobial peptides (AMPs) are promising alternative agents because of their wide spectrum of activity, high efficiency at very low concentrations and low propensity for developing resistance. [2]. Hence, a new generation of antibacterial coatings based on AMPs surface immobilization has recently emerged.
As an environmentally friendly technique, a low pressure plasma polymerization process has been exploited mainly for the deposition of adherent functional thin films carrying amino, carboxylic or epoxy groups for AMPs covalent bonding. [3]
In previous work [4], we recently highlighted the potentialities of an Atmospheric Pressure Dielectric-Barrier-Discharge (AP-DBD) process for the elaboration of robust bio-inspired antibacterial coatings through a three-step procedure.
Considering both environmental and industrial issues, the present research work focuses on the deposition of a new kind of highly chemically reactive interlayer allowing a fast one-step AMPs immobilization.
First, the plasma method for the deposition of a bio-inspired reactive interlayer (for which a patent is pending) will be described and the layers chemical and morphological properties will be reported in detail.
Secondly, the immobilization conditions of two different antimicrobial biomolecules, namely nisin and dispersine B for bactericidal and antibiofilm activities respectively, will be presented.
Finally, the antibacterial properties of the developed surfaces evaluated according to normalized tests will be presented and discussed.
[1] A. Glinel et al.Acta Biomaterialia, 2012, 8, 1670.
[2] F. Costa et al.Acta Biomaterialia, 2011, 7, 1431.
[3] C. Vreuls et al. J. Mater.Chem.2010, 20, 8092.
[4] R. Mauchauffe et al.J. Mater. Chem. B, 2014, DOI: 10.1039/c4tb00503a.

Poly(2-hydroxyethyl methacrylate) (PHEMA) is a desired hydrogel for biomedical applications because of its nontoxicity and biocompatibility. PHEMA based thin films have been used in various biological applications including drug delivery, cell adhesion, biosensing, protein adsorption and enzyme immobilisation. Although thin films of PHEMA and similar methacrylic polymers can be formed on different surfaces using either solution based or vapor based techniques, the desired technique should allow for rapid coatings with a high degree of retention of chemical functional groups and the method should be applicable for fragile and geometrically complex substrates. The solvents used in solution based methods are not compatible for most of the fragile substrates and it causes major environmental problems. Initiated plasma enhanced chemical vapor deposition (i-PECVD) method, on the other hand,is a novel dry technique that can produce well defined defect free polymeric films on many different substrates with low energy inputs. The chemical species called the initiator can easily be dissociated into reactive chemical species at low energy inputs due to their weak bonds. The basic reason behind the use of initiator in PECVD is to keep the plasma power low enough just to break the weak initiator bonds, so that the undesired monomer fragmentation ad chemical functionality lost would be avoided.
In this study thin films of PHEMA were deposited on silicon wafer and tissue paper substrates in a home built reactor. The plasma (RF: 13.56 Mhz) was inductively coupled into the reactor by a planar-coil antenna through a quartz window and a plasma is generated in the chamber. The reactor was 16 cm in diameter and 15 cm in height. Pressure in the reactor was maintained at 0.2 torr and the substrate stage is maintained at 25^oC using water cooling. The use of tert butyl peroxide (TBPO) as an initiator allowed coatings at very low plasma powers. Monomer and initiator species were vaporized at separate jars and fed to the reactor at different inlet ports. The effects of plasma power, substrate temperature and reactant flowrates on the deposition rate, chemical and morphological properties of deposited films were investigated. Deposition rates up to 30 nm/min were observed depending on the deposition conditions. FTIR analysis of the deposits indicated very high retention of functional groups at low plasma powers. The deposition at low plasma powers is also suitable for the functionalization of fragile tissue paper surfaces. SEM investigation of tissue papers before and after coatings indicated that plasma modification at low plasma powers did not change the surface geometry of the paper surfaces. Being a dry, low-cost, reliable and environmentally friendly process, the i-PECVD technique developed in this study can be used to deposit functional PHEMA and similar methacrylic thin films on many industrially important surfaces.

Atmospheric pressure plasma jet has been widely employed in biomedical applications because such plasmas induce little thermal damage to biomaterials. Especially dielectric-barrier-discharge (DBD) plasma jet, where the dielectric prevents the formation of the high temperature arcs, is the most common atmospheric pressure discharge system. The DBD non-equilibrium plasma has relatively high electron temperature and low gas temperature, and the high energy electrons can produce chemically rich gas-phase environments with reactive O species around room temperature. The atmospheric DBD plasma jet can be easily generated by the application of high pulse voltage with low frequency in the order of kHz in open-air condition. However, the control of chemical reactions in DBD plasma jet is quite difficult because the DBD discharge is transient, and self-extinguished within the order of msec after discharge initiation. Therefore, it is an essential issue to understand and control complicated chemical reactions in the transient DBD plasma jet in order to realize the biomedical applications of plasma.
Our interest has been concerned with the production of highly reactive O species in the DBD plasma jet, which are most desired in many plasma material processing, for example, the hydrophilic processing and so on. In this study, we investigate temporal behavior of excited O atoms in He DBD plasma jet by using optical emission spectroscopy (OES). The OES, in which only emission spectrum is captured from plasma jet, is quite useful to roughly estimate the existence of excited O atoms. There have been many works on the OES from plasma jet, and basic knowledge of plasma jet has been experimentally studied for the past decade. Especially, Q. Xiong et al. have reported temporal resolved OES concerning to O optical emission in DBD plasma jet [1]. However, He gas flow rate was fixed to be 2 slm in open air, and the effect of air-gas mixing in He gas flow was not investigated. On the other hand, the main purpose of this paper is to make clear the characteristics of O optical emission in the DBD plasma jet for realizing the efficient production of excited O atoms. A focus is on the analysis of He gas flow rate (R_{He-gas flow}) dependence of O optical emission, where R_{He-gas flow} is one of the most important parameters for the plasma-jet processing, because R_{He-gas flow} changes not only jet-length, but also the complicated chemical reactions under the open-air condition. Our measurements clearly show drastic effect of RHe-gas flow on the O-emission characteristics in the plasma jet. The DBD plasma jet is quite sensitive to RHe-gas flow, and an increasing RHe-gas flow leads to weak discharge with lower plasma density.
This work was partly supported by The Grant-in-Aid for Scientific Research on Innovative Areas “Plasma Medical Innovation” from The Ministry of Education, Culture, Sports, Science and Technology.
[1] Q. Xiong et al., J. Appl. Phys. 106, 083302 (2009).

Diamond-like carbon (DLC) films are the hydrogenated amorphous carbon films, which is composed of a mixture of sp²- and sp³-bonded carbon. Since this films have excellent material properties in high wear resistance, high hardness, low friction, and chemical stability, the films have been widely used for many technological applications, such as automotive, semiconductors, medical devices, and so on [1-3]. Recently, silicon-containing DLC (Si-DLC) films have been investigated, since the friction coefficient of the Si-DLC films is lower than that of DLC films [4]. However, the effect of silicon in Si-DLC films on friction properties has not been clearly understood. Therefore, the understanding of fundamental properties in tetrametylsilane (Si(CH₃)₄) plasmas, which are ion and radical source of Si-DLC film deposition, is strongly necessitated. In this paper, the fundamental properties in capacitively-coupled radio-frequency tetrametylsilane plasmas have been simulated using a one-dimensional fluid model, which is composed of the continuity equations for electron and sixteen tetrametylsilane-derived ion species, the Poisson equation, and the electron energy balance equation. The influence of extrenal parameters (e.g. gas pressure, input power) on the plasma properties is discussed. From simulation results, it is clarified that dominant ion species in tetrametylsilane plasmas are Si(CH₃)₃⁺. The other simulation results will be presented at the conference. This work was partly supported by KAKENHI (No. 26420247), and a "Grant for Advanced Industrial Technology Development (No. 11B06004d)" in 2011 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. [1] S. Aisenberg and R. Chabot, J. Appl. Phys. 42 (1971) 2953. [2] B. Bhushan, Diamond Relat. Mater. 8 (1999) 1985-2015. [3] J. Robertson, Mat. Sci. Eng. R37 (2002) 129-281. [4] H. Mori and H. Tachikawa, Surf. Coatings Technol. 149 (2002) 225-230.

Gold nanoparticle (AuNP) has attracted much attention for enzyme immobilization. However, because of its difficulty handling, deposition of the particles on the support is essential. In this study, cellulose biopolymer was selected as a support. The AuNPs of uniform size could be deposited directly onto a cellulose fiber by the solution plasma process (SPP). For the synthesis, 1 mM HAuCl₄ aqueous solution was mixed with 1 %wt cellulose before the plasma discharge. Discharge was continued for 10, 20 or 30 min. It was observed that color of the cellulose powder was changed from white to purple after the SPP and the color was more intense when the discharge time was prolonged. SEM analysis revealed that the observed purple color of the sample was caused by the deposition of the AuNPs on the cellulose fibers. Furthermore, the plasma condition was varied in order to investigate its effect on the size of the deposited AuNPs. The cellulose fiber/AuNP composite is expected to be an effective matrix for enzyme immobilization.

Among different types of atmospheric gas discharge plasma sources, the warm plasma source is intermediate between cold and thermal plasmas with characteristic gas temperature of ~3000 K and electron energy on the magnitude of 1 eV, indicating a significant non-equilibrium feature [1]. Such plasma source may become an optimized process for fuel conversion or combustion with the control of the gas temperature and the reduced electric field [1]. Although some research works on warm plasmas driven by the kilohertz high voltage power supplies have been reported [2, 3], the volume of the plasmas are usually small which, to some extent, restrict their actual applications. In this study, a hybrid gas discharge warm plasma is produced by connection of a kilohertz AC power supply and a DC power supply to the plasma torch. The experimental measurements show that, with the addition of the DC component after gas breakdown driven by the kilohertz AC power supply, the discharge volume can be enlarged obviously. The electrical and optical emission characteristics of this hybrid gas discharge warm plasma system are studied.
Acknowledgement: This work has been supported by National Natural Science Foundation of China (No. 11035005).
References
[1] Gutsol A, Rabinovich A and Fridman A (2011) Combustion-assisted plasma in fuel conversion. J Phys D - Appl Phys 44: 274001
[2] Wang Z-B, Chen G-X, Wang Z, Ge N, Li H-P and Bao C-Y (2011) Effect of a floating electrode on an atmospheric-pressure non-thermal arc discharge. J Appl Phys 110: 033308
[3] Fulcheri L, Rollier J-D and Gonzalez-Aguilar J (2007) Design and electrical characterization of a low current-high voltage compact arc plasma torch. Plasma Sources Sci Technol 16: 183-192

In the last decade, atmospheric pressure plasma (APP) has been widely used in various ways for medical or agricultural applications. Many groups developed sterilization methods using APP and reported that reactive oxygen species (ROS) and reactive nitrogen species (RNS) irradiated from APP have important physical and chemical effects on the biological tissues, such as gene transfection [1], growth promotion [2], and selective killing of cells [3]. However, there are unexplained matters; such as identification of reactive species dominating the sterilization effect for each plant pathogenic fungus. In this work, we focus on the sterilization effect of hydroxyl radical (OH*), one of the ROS, on Botrytis cinerea.
A plasma jet using air and water has been developed. Influences of two parameters; irradiation length L and total irradiation time T on sterilization were investigated. Botrytis cinerea cultivated under 25 °C on agar plate is cut to 5 × 5 mm, and put on a new agar plate for irradiation. After the plasma irradiation, Botrytis cinerea is cultivated under 25 °C. The sterilization is defined as no enlargement of the colony of Botrytis cinerea after 14 days of cultivation. The sterilization probability of Botritis cinerea is increased with the increase of the plasma irradiation time. To understand the role of the OH* on the sterilization, the concentration of OH* in the plasma irradiation region was measured using terephthalic acid [4,5]. Concentration of OH* is higher at longer T, and lower at larger L. Since both the total dose of OH* and the sterilization probability are increased with the plasma irradiation time, there is a correlation between the sterilization and the measured OH* concentration. Botrytis cinerea was totally sterilized under the total irradiation time of 45 min (15 min × 3 days) at L = 100 mm, and the total irradiation time of 105 min (15 min × 7 days) at L = 200 mm, corresponding to the total dose of OH* irradiation of 0.44, 0.58 nmol respectively. In this experiment, the minimum total dose of OH* to sterilize the Botrytis cinerea in the 5 × 5 mm mycelium is found to be approximately 0.4 nmol. Investigations on the other plant pathogenic fungi, such as Glomerella cingulata, and Fusarium oxysporum is undergoing.
[1] S. Sasaki, M. Kanzaki, and T. Kaneko : Appl. Phys. Express 7 (2014) 026202.
[2] D.P. Park, K. Davis, S. Gilani, C.-A. Alonzo, et.al.: Curr. Appl. Phys. 13 (2013) S19.
[3] S. Iseki, K. Nakamura, M. Hayashi, et al.: Appl. Phys. Lett. 100 (2012) 113702.
[4] X. Fang, G. Mark, and C. von Sonntag : Ultrason. Sonochem. 3 (1996) 57.
[5] S. Kanazawa, H. Kawano, S. Watanabe, et.al.: Plasma Sources Sci. Technol. 20 (2011) 034010.

Non-equilibrium atmospheric-pressure discharges have been widely investigated with great attentions to be utilized for a variety of applications from industrial surface processing of materials to advanced medical treatments of biomedical tissues (plasma medicine) to cure diseases including cancer. For development of innovative plasma sources for the plasma medicine, it is of great significance to investigate the basic characteristics of atmospheric-pressure discharge plasma. Our interest has been concerned with the control of the atmospheric-pressure discharge plasma, and a focus is on the driving-frequency dependence of the discharge plasma. The discharge driving frequency is one of the most important parameters to control characteristics of atmospheric-pressure discharge plasma such as plasma density, radical density, and so on. In this presentation, the characteristics of atmospheric-pressure discharge plasma have been investigated with (1) DC pulse voltages in the repetition rate from 1 kHz to 30 kHz, and (2) RF and UHF voltages in the frequency region from 13.56 MHz to 100 MHz.
First, He atmospheric dielectric-barrier-discharge (DBD) plasma jet was investigated under the open-air condition in the frequency range from 1 kHz to 30 kHz. Our experiments demonstrate drastic effect of driving voltage frequency on the discharge characteristics of DBD plasma jet. The DBD plasma jet is quite sensitive to the driving voltage frequency, and an increasing driving frequency induces a weak pulse discharge with a short plume-length. Furthermore, we carried out absorption spectroscopy to directly evaluate number density of reactive O atoms, and observed that the low-frequency operation can generate a large amount of reactive O atoms. The driving voltage frequency is useful parameter to control the number density of reactive excited O atoms in the DBD plasma jet.
Then, we performed experiments on He atmospheric-pressure RF dischages in the frequency region from 13.56 MHz to 100 MHz. The gas-breakdown voltage decreases with incresing dicharge-voltage frequnecy, and the low-voltage operation of discharges below 150 V is realized in the UHF region under the atmospheric pressure condition. Also, the spatial emission-profile drasicallty changes with increasing discharge-voltage frequnecy, where strong emisson is highly localized in the front of the power- and ground-electrode in the UHF dischrages. The lowering brakdonw voltage and the localized emission-profile are well explained by the effect of the charged-particle confinement with increasing discharge-voltage frequency.
This work was partly supported by The Grant-in-Aid for Scientific Research on Innovative Areas “Plasma Medical Innovation” from The Ministry of Education, Culture, Sports, Science and Technology.

Parylene-C film has been widely used for various applications by using its chemical resistance, electric isolation, water-proof, and transparency. Parylene-C film is commonly deposited by thermal deposition under vacuum with solid di-para-xylylene (DPX). In detailed process, the solid is converted to the dimer gas phase by sublimation. And then, the vapors are pyrolyzed in next step in a higher temperature zone, resulting in intermediate monomer. Finally, the monomer gas enters the deposition zone where the polymer film is formed onto substrate surfaces. In this work, we describe a new type of deposition method by using sublimated di-para xylylene plasma with Ar gas. The pyrolysis of the sublimated DPX dimer gas is achieved by RF plasma, which can be activated the DPX dimer. The advantage of this method was described as follows: (1) tailoring of parylene film properties, (2) tuning of the film composition such as bonding ratio of sp² and sp³, (3) compatibility to the other preparation methods of dielectric thin films. We investigated the surface morphology of parylene-C dimer by using SEM and AFM. The physicochemical properties of the parylene-C film by the plasma deposition were analyzed and compared with the parylene-C film from the conventional thermal deposition. The chemical composition of parylene-C film was analyzed by using XPS, which was estimated to be similar for the parylene-C films prepared by thermal and plasma deposition methods. The signature absorption peaks of parylene-C film (at 2852, 2925 and 2018 cm-1) were analyzed to be identical for the parylene-C films prepared by thermal and plasma deposition by FT-IR spectroscopy. We have demonstrated plasma-assisted deposition of parylene-C film, which can be tailoring of parylene film properties.

Diamond-like carbon (DLC) films are the hydrogenated amorphous carbon films, which is composed of a mixture of sp2- and sp3-bonded carbon. Since this films have excellent material properties in high wear resistance, high hardness, low friction, and chemical stability, the films have been widely used for many technological applications, such as automotive, semiconductors, medical devices, and so on. The numbers of radicals in the gas phase considerably affect to the composition of the functional film, resulting in the characteristics of films and devices. The metastable Ar atom plays an important role in the sputtering mechanism and the film property of DLC and. It is important to understand and monitor the behavior of species in gas phase and on a surface in order to control the plasma processes precisely. The absorption spectroscopy is the powerful technique for measuring absolute density of species.
In this study, we have measured the density and translational temperature of metastable Ar atom by diode laser absorption spectroscopy. The film property and diagnostics of RF magnetron sputtering plasma will be discussed.
DLC film was synthesized by the conventional RF magnetron sputtering. The RF power of 13.56 MHz was applied to the lower electrode. Pure carbon was used as the target and Ar gas was introduced into the chamber. We used a diode laser with narrow line width for absorption measurement. The center wavelength was tuned to the (4p²[3/2] - 4s²[3/2]^#9675;) transition of Ar atom at 811.36 nm and the laser scan width was 0.021 nm. The width of laser line was about 1 MHz. The electrode distance was 75 mm and the laser beam passed through 15 mm above the carbon target.
The absorption profile fits to the Doppler profile in the range from 10 to 30 Pa. The metastable Ar density decreased from 3.0 x 10¹⁰cm^-3 to 1.3 x 10¹⁰ cm^-3 with increasing the pressure. The translational temperature slightly increased from 690 K to 710 K with the pressure.
This work was partly supported by the NAGAI foundation for science and technology.

GREMI develops from years the Plasma Gun non thermal plasma (NTP) source for its application in therapeutic treatments, with a special attention to cancer treatment with NTP alone or in combination with chemotherapeutic drug [1], and for its potentialities for endoscopic protocols [2]. Besides the demonstration of antitumor action both in vitro and in vivo on various tumor bearing mice, recent studies reveal the critical roles of both plasma interaction with the target (tissue or cell culture media) and of the strong influence of plasma-generated species with the rare gas (He/Ne) flow flushed across the PG discharge reactor and emerging in the ambient air gap a few millimeters above the target under treatment. Plasma impingement over conductive targets, e.g. conductive culture media/organ or skin surfaces/metallic samples, results in the generation of secondary plasma following primary plasma stream delivery through ionization wave sustained processes, leading to drastic modifications of the reactive species generation in comparison with the free jet expansion. Such secondary plasma generation reveals the key role of the persisting plasma tail or plasma column produced during primary plasma propagation which was initially not considered with sufficient care when the “plasma bullet” moniker was suggested. The second main issue concerns the strong interplay between the rare gas flow and the plasma species generated during plasma jet ionization wave propagation. Drastic modification of the rare gas flow features have been recently evidenced and characterized through Schlieren visualization and ICCD imaging [3]. The consideration of the two processes was recently shown to be especially relevant not only for the production of hydroxyl radical over conductive targets but also for the comprehensive description of OH spatial distribution using the combination of different experimental diagnostic tools (optical emission spectroscopy, laser induced fluorescence, Schlieren visualization and ICCD imaging) [4].
This work is supported by the APR Region Centre PLASMEDNORM and ANR 2010 BLANC 093001 PAMPA.
References
[1] Brulle L, Vandamme M, Riès D, Martel E, Robert E, Lerondel S, Trichet V, Richard S, Pouvesle J M and Le Pape A., 2012, Plos one 7 (12) e52653.
[2] Robert E., Vandamme M., Brullé L., Lerondel S., Le Pape A., Sarron V., Riès D., Darny T., Dozias S., Collet G., Kieda C. And Pouvesle J.M., 2013 Clin. Plasma Medicine, 1 8-16.
[3] E. Robert, V. Sarron, T Darny, D. Riès, S. Dozias, J Fontane, L. Joly and J.M. Pouvesle, 2014 Plasma Sources Sci. Technol. 23 0120003.
[4] Riès D., Dilecce G., Robert E., Ambrico P.F., Dozias S. and Pouvesle J.M., 2014 J.Phys.D:Appl. Phys. 47 275401.

We have focused on the effects of neutral oxygen species on the inactivation of microorganisms. We measured the densities of oxygen radicals such as ground-state atomic oxygen [O(³P_j)] and singlet oxygen molecule [O₂(¹Δ_g)], and showed that O(³P_j) was the dominant species responsible for the inactivation of Penicillium digitatum spores quantitatively.[1][2] Besides, the inactivation occurred without major morphological changes.[3] In this study, we will discuss the inactivation process, including the inhibition of the function of cell membranes, the oxidation process of the spores, and nanostructural changes, on the basis of the dose of O(³P_j). To eliminate the influence of atmospheric gases, the radical source and the sample were enclosed with a plastic cover. The spore suspension of 1 mu;l was spotted on a #981;35 mm dish and dried. The samples were exposed to oxygen radicals 10 mm downstream from the radical head at a O₂/(Ar+O₂) flow rate ratio of 0.6% with a total flow 5 slm from 1.5 to 7 min, which correspond to O(³P_j) dose from 2.1×10¹⁹ to 9.8×10¹⁹ cm^-2, respectively, under the flux of 2.3×10¹⁷ cm^-2 s^-1.[2] The spores were observed using confocal laser fluorescent microscopy and transmission electron microscopy (TEM). From the results, we elucidated the inactivation mechanism of P. digitatum spores by oxygen radicals on the basis of dose of O(³P_j). Oxygen radicals, mainly O(³P_j), inhibit the function of cell membranes of spores without major morphological changes at low dose of O(³P_j) of 2.1×10¹⁹ cm^-2. Then, the degree of oxidation in spores increased until O(³P_j) dose of 2.1×10¹⁹ cm^-2, in which most spores were inactivated. Finally, intracellular nanostructures were degraded by excess oxygen radicals over O(³P_j) dose of 7.0×10¹⁹ cm^-2.
References
[1] S. Iseki et al., Appl. Phys. Express, 4, 116201 (2011).
[2] H. Hashizume et al., Appl. Phys. Lett., 103, 153708 (2013).
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Plasma irradiation to plants normally suppresses plant growth because of irradiation of ROS and RNS, whereas effects of atmospheric discharge non-thermal plasma irradiation to plant seeds on growth of the plants depend on the irradiation duration or “plasma dose” [1-3]. Low dose plasma irradiation has no effects on plant growth. Middle dose plasma irradiation enhances plant growth by 10-150%. High dose plasma irradiation suppresses plant growth. Two questions arise. Do plants have memory of plasma irradiation in their former generation? Are there any methods to enhance further plant growth? To answer these questions, here we report multigeneration study of effects of plasma irradiation to seeds of Arabidopsis thaliana and Zinnia. In the first generation, we compared plant growth using two kinds of seeds: seeds with and without plasma irradiation. Under appropriate irradiation conditions, plasma irradiation enhances plant growth by 10-150%. In the second generation, we compared plant growth using four kinds of seeds: seeds with plasma irradiation in the first and second generation, seeds with plasma irradiation in the first generation, seeds with plasma irradiation in the second generation, and seeds without plasma irradiation. Plasma irradiation in the first and second generation induces by two times strong enhancement compared to plasma irradiation in the first generation and plasma irradiation in the second generation, whereas plasma irradiation in the first generation and plasma irradiation in the second generation enhances plant growth by 10-150% compared to without plasma irradiation. These results indicate that plants or plant seeds have memory of plasma irradiation in their former generation, and plasma irradiation to seeds in multigeneraion can enhance further plant growth compared to conventional plasma irradation.
Acknowledgements
This work was partly supported by JSPS.
References
[1] T. Sarinont, et al., J. Phys. Conf. Ser. 518 (2014) 012009.
[2] S. Kitazaki, et al., Cur. Appl. Phys. (2014).
[3] T. Sarinont, et al., JPS Conf. Proc. 1 (2014) 015078.

Biomolecules have been traditionally immobilised onto surfaces using wet chemical techniques for various medical applications. Recent decades have seen plasma methods being used to prepare these surfaces through various forms of surface modification, but the direct exposure of biomolecules to plasma has been avoided due to fears that the molecules would be denatured by the energetic plasma species. Recent results are now demonstrating that direct plasma deposition of biomolecule coatings can be achieved. This creates the possibility to directly modify the surface of implants without any form of surface pre-treatment and this opens up the possibility to alter the healing processes. Materials such as collagen, chitosan, catalase and heparin can be effectively deposited onto surfaces with minimal impact on biological performance and without any chemical binders, linkers or impurities. The performance of these materials has been characterised using both in vitro and in vivo methodologies. In a further step, the results of a preclinical trial are presented which reveal that direct deposition of biomolecules onto open wounds can also be achieved and the impact of this on wound healing is measured in an immunocompromised animal model. A non-thermal plasma device was used to deliver collagen on to chronic wounds and the treatment was shown to promote wound closure in a rabbit wound healing model. Although a simple plasma treatment also reduced inflammation and enhanced wound healing, the collagen component produced significantly enhanced wound closure rates.

Carbon nanowalls (CNWs) are self-assembled, free-standing, few-layered graphene nanostructures. They have the high aspect ratio over 100 and high specific surface area. In addition to such the unique morphologies, they also have unique and excellent electrical properties. Therefore, they have attracted much attention to be applied to various types of devices, such as electrical devices, electrochemical sensors, biosensors, and so forth, and scaffold of cell culturing. To realize such the applications, not only control of their morphology, but also chemisorbed species on their surfaces were important. Recently, establishing a radical injection plasma-enhanced chemical vapor deposition (RI-PECVD) system, we realized highly reproducible growth of CNWs, control of their morphology and semiconducting properties. In this study, we investigated the surface wettability of CNWs with emphasis on the chemisorption effect by post-growth surface modification using plasma treatments.
The surface of as-grown CNWs fabricated employing CH₄/H₂ mixture was hydrophilic. By Ar atmospheric pressure plasma treatment for 30 s, the contact angle of water droplet on the CNWs drastically decreased from 51#730; to 5#730;. On the other hand, by CF₄ plasma treatment at low pressure, the contact angle value drastically increased from 51#730; up to 147#730;. X-ray photoelectron spectra indicated oxidation only at edges and surface defects. The wide-range control of surface wettability of CNWs was realized by the post-growth plasma treatments.
Using surface-modified CNWs as electrodes, the cyclic voltammetry (CV) were measured in PBS (pH 7) containing bovine serum albumin (BSA). Using the as-grown CNW electrode without Ar atmospheric pressure plasma treatment, weak oxidation and reduction peaks were observed in anode peak potential at 0.2 V and cathodic peak potential at -0.3 V, respectively. On the other hand, in the case of typical surface-oxidized CNW electrode, a broad oxidation wave was recorded with an anode peak potential of 0.2 V. A high peak reduction current was also observed in the cathodic peak potential at -0.75 V. In the case of surface-oxidized CNW electrode of low height, the CV profile exhibited small peak currents compared with the case using typical CNW-oxide electrode, due to the decrease of immobilized BSA on the reduced surface area. Furthermore, the dependence of the HeLa cell culturing rate and morphological change on the surface wettability of CNWs scaffolds was systematically evaluated. The cell culturing rates were significantly dependent on the CNW densities, although the surface wettability of the CNWs was not significantly dependent. Morphological changes of the cells were not significantly dependent on the density of CNWs. These results indicate that the ability of CNWs as a platform for nano-bio applications.

Graphene, an atomically thin two dimensional sheet of carbon, has attracted enormous attention due to its unique physical and chemical properties, such as its high Young&’s modulus, relativistic charge transport and very high transparency. Whilst initially applications focussed on harnessing it&’s electrical properties (e.g., in ultrafast computing and energy-storage), Graphene is now drawing increased interest in a wide range of fields including bio-medical applications such as drug delivery, anti-cancer therapy and cell culture substrates. These substrates are critical for: (1) investigations into the early stage development of cells and/or diseases, (2) the testing of new drugs and (3) scaffolds for tissue engineering. One of the benefits of GRM for these applications is that they can be produced as a mono or multi layer and with different structural morphology (i.e. horizontal and vertical) each having different useful properties. Each of these can be produced on (or transferred to) a plethora of substrates, an important feature as it has been demonstrated that the properties of substrates has a strong influence on the fate of cells. Concerning graphene these surface properties can be modified and tuned via plasma or chemical treatment, which decorate the surface with specific functional groups.
Successful applications of graphene-based materials found in literature for bio-med applications are predominantly produced via chemical methods, generating flakes that can be used to coat glass. When produced via Chemical Vapour Deposition, the transfer to the desired substrate involves chemical treatment, potentially contaminating the graphene. This work uses unique plasma produced graphene transferred to glass via a chemical-free process as cell culture substrates. The influence of reactive species (Ar⁺/H radicals) during growth in the plasma is investigated. The morphology of our films before and after transfer was characterized with Scanning Electron Microscopy. It is shown that the density of structures before transfer is influenced by the presence of argon. A collapsing effect is observed after transfer forming a graphene film on glass. Raman Spectroscopy was used to characterize graphene structure and quality. It is found that the number of defects and/or edges increases with the transfer whilst there is no specific trend to the reactive species.
For its use as a cell platform for biological applications, the graphene films must undergo a range of toxicity tests, which is the objective of this work. Their biocompatibility was tested using lung cancer fibroblasts cells, cultured on the films for 5 days. It is shown that our films are non toxic, regardless the graphene structure. Cells morphology and proliferation were examined by Optical Microscopy on the first and third day, and indicates similar growth among all samples and the control. There appears to be some organization related with the amount of defects and edges found on the surface.

Articular cartilage is a nanostructured tissue notoriously hard to regenerate due to its extremely poor regenerative ability and complex stratified architecture. Although various biomaterials and tissue engineering approaches to address cartilage defects have been investigated, it is still very challenging to repair it. Thus, the objective of this work is to develop a biomimetic nanostructured cartilage scaffold with cold atmospheric plasma (CAP) modified cell favorable surface and sustained bioactive factor (TGF-β1) nanoparticles embedded inside for improving stem cell chondrogenesis and cartilage regeneration. For this purpose, a random biocompatible polycaprolactone (PCL) fibrous scaffold was electrospun and further coated with TGF-β1 loaded poly(lactic-co-glycolic) acid (PLGA) nanospheres which were prepared via w/o/w double emulsion solvent extraction method. After dry in the air, another layer electrospun PCL fibers mat was deposited followed by one nanosphere layer coating and achieved a controllable layered nano scaffolds. Moreover, the nanostructured scaffolds were treated with CAP for 0, 1, 3 and 5 min in order to create a cell favorable scaffold surface for improved cell attachment and growth. Scanning electron microscope showed the nanospheres have been effectively loaded inside the electrospun scaffold. Furthermore, the nanospheres embedded scaffolds can keep a long-term TGF-β1 release when compared to controls with bare TGF-β1. More importantly, CAP treatment can create a more hydrophilic surface and greatly enhance human bone marrow mesenchymal stem cells 5 day proliferation and chondrogenic differentiation (such as glycosaminoglycan synthesis) in vitro. In conclusion, this study suggested the novel CAP treated nanostructured scaffolds can be potentially used as a biomimetic and bioactive construct for improved cartilage regeneration.

Despite recent technological advances, sepsis remains a major cause of mortality (10^th most common cause of death in US) and its incidence is increasing along with increased rates of antibiotic resistance amongst the causative microorganisms. Sepsis is defined as the presence of systemic inflammatory response syndrome (SIRS) due to infection, and represents a potentially life-threatening condition through resultant multi-organ failure.¹ Prompt diagnosis allows early treatment, which has a major impact on mortality. However, diagnosing sepsis can be challenging, especially in critically ill patients who often have signs of systemic inflammation, which can be hard to differentiate from sepsis. This impacts on patient outcomes as delayed antibiotic treatment worsens outcomes. Yet if antibiotics are prescribed too readily, then bacterial resistance rates rise, as well as costs and other complications.
Recently, leukocytes have been shown to release trigger-dependent microvesicle sub-populations in response to bacterial exposure, hence may be utilised to diagnose sepsis.² Microvesicles are small membrane-bound fragments, released from cells under both physiological and pathological conditions. ^3-4 Importantly, the microvesicle composition varies depending on the cell of origin as well as the eliciting stimlus.^2-6
This presentation will discuss the potential of novel microvesicle activity assays for sepsis diagnosis, focusing on plasma-derived microvesicles. We will show a range of microvesicle-based assay designs to measure procoagulant, inflammatory and agglutination activity in plasma samples and present first pilot study data on the diagnostic performance of these assays in a relevant intensive care unit patient cohort, including sepsis patients and patients suffering from non-infectious SIRS. Advantages as well as limitations of microvesicle-based assays will be discussed with regard to potential future clinical applications.
1. Sepsis and Non-infectious Systemic Inflammation. Edited by J.-M. Cavaillon and C. Adrie, 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
2. C. I. Timar, A. M. Lorincz, R. Csepanyi-Koemi, A. Valyi-Nagy, G. Nagy, E. I. Buzas, Z. Ivanyi, A. Kittel, D. W. Powell, K. R. McLeish and E. Ligeti, Blood, 2013, 121, 510-518.
3. C. M. Boulanger and F. Dignat-George, Arteriosclerosis Thrombosis and Vascular Biology, 2011, 31, 2-3.
4. V. L. Reid and N. R. Webster, British Journal of Anaesthesia, 2012, 109, 503-513.
5. M. Baron, C. M. Boulanger, B. Staels and A. Tailleux, Journal of Cellular and Molecular Medicine, 2012, 16, 1365-1376.
6. S. Oehmcke, J. Westman, J. Malmstrom, M. Morgelin, A. I. Olin, B. Kreikemeyer and H. Herwald, Plos Pathogens, 2013, 9, e1003529.