Peter Bruggeman, University of Minnesota
Mounir Laroussi, Old Dominion University
Masaaki Nagatsu, Shizuoka University
Stephan Reuter, Leibniz Institute for Plasma Science and Technology - INP Greifswald e.V/ZIK plasmatis
Eric Robert, GREMI, CNRS/University of Orleans
G2: Interactions between Plasma and Biomaterials
Thursday PM, December 03, 2015
Hynes, Level 3, Room 305
2:30 AM - *G2.01
Controlling Properties of Plasma Activated Liquids for Life Sciences through Control of Gas Phase Plasma Sources
Wei Tian 1 Seth Norberg 2 Amanda M. Leitz 1 Mark J. Kushner 1
1Univ of Michigan Ann Arbor United States2US Military Academy West Point United StatesShow Abstract
In biomedical applications, and the treatment of tissue in particular, the delivery of plasma produced reactivity to the tissue is transformed by an overlying liquid. In most cases, the reactivity produced in the gas phase does not reach the tissue in its original form. For example, most ions when incident onto the liquid (in this case, basically water) are transformed to H3O+aq. However, a small subset of gas phase species, such as ozone, may solvate and reach the tissue in its original form, O3aq. The varying time scales for solvation of gas phase species into the liquid affords some ability to control their reactivity. OH rapidly solvates into water whereas NO slowly solvates into water - and so controlling the residence time of these plasma produced species above the liquid affords some ability to customize the liquid resident reactivity. In this presentation, methods to control the plasma produced reactivity in water layers over cells and tissue will be discussed from the perspective of controlling the properties of the gas phase plasma. Results from two modelling platforms will be discussed. nonPDPSIM is a 2-dimensional plasma-hydrodynamics model which accounts for gas and liquid phase transport and plasma chemistry, radiation transport and electrostatics. GlobalKIN is a global plasma chemistry model modified to represent gas and liquid phases, and transport of species and activation energy between the phases. Control schemes will be discussed for plasma jet and dielectric-barrier-discharge treatment of thin water layers having impurities of dissolved gases and proteins.
3:00 AM - *G2.02
Inactivation of Bioparticles in Aqueous Media by Radicals Injected from Electrical Discharge Devices
Akira Mizuno 1 Hirofumi Kurita 1 Kazunori Takashima 1
1Toyohashi Univ of Technology Toyohashi JapanShow Abstract
Recently, commercial home electric appliances with bactericidal/viral inactivation function of indoor air have been widely available. Most of these home appliances equip discharge devices which generate oxidative chemical species such as reactive oxygen species, ozone, and nitrogen oxides. However, the bactericidal and viral inactivation effects are still controversial problem. Furthermore, its safety for human should be evaluated. To evaluate the inactivation effect and the safety of these devices, we have tested the DNA fragmentation method, in which large DNA molecules is used as a biomarker. By exposing the DNA suspension to the air containing the chemical species, oxidative damage to the large DNA molecules, such as strand breaks, can be induced. We used two types of discharge devices in this study. These devices were dismounted from the original main unit. They were put inside an acrylic chamber (30 L) and operated by the main unit. DNA solution and bacterial suspension for comparison were exposed to the air containing the chemical species generated by the discharge. After the exposure, the exposed DNA were purified and stained with fluorescent dyes. Single-molecule observation that involved molecular combing was then used to measure the length of individual DNA molecules. The measured DNA length showed that the oxidative chemical species from the discharge devices caused a marked change in length of DNA molecules. These results indicated that the commercialized home electric appliances equipped with discharge devices induced strand breakage on large random-coiled DNA molecules. Bacterial inactivation was also confirmed under the same exposure conditions. The gamma ray exposure was compared with the electrical discharge devices by measuring the strand breaks of the large DNA molecules. The obtained breakage rate showed that the discharge devices could supply rather large amount of radicals to the media, or statistically 10 h exposure to the discharging device could be equal to 1000 Gy of the gamma ray exposure that is enough for sterilization. It is concluded that the discharge devices can generate oxidative radicals that could be harmful to human body when inhaled, and that our method can be applied to estimate the intensity of radical reaction induced by the discharge devices and to develop safer home electric appliances.
3:30 AM - G2.03
Generation of Micro Solution Plasma for Minimally-Invasive In Vivo Gene Transfection
Toshiro Kaneko 1 Yutaro Hokari 1 Shota Sasaki 1 Makoto Kanzaki 1 Takehiko Sato 1
1Tohoku Univ Sendai JapanShow Abstract
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. On the other hand, the micro-scale plasmas generated in solution (micro solution plasma) are desired to realize in vivo gene transfection, because most of the human body consists of water and the most of internal organs are filled with solution. Therefore, we attempt to generate the micro-scale plasma in solution  and apply it to the adherent cells for clarifying the transfection mechanism toward developing minimally-invasive in vivo gene transfection system.
In this experiment, the coaxial type electrode is used to make the micro-scale plasma, where the high voltage electrode is the tungsten rod whose tip is sharp-pointed with less than 1-mu;m curvature radius. By applying a pulse voltage (peak voltage: Vin = 1.5 kV, pulse width: Tp = 6-10 mu;s) to this electrode, we succeed in generating the micro scale plasma in phosphate buffered saline (PBS), which is irradiated to the adherent cells. After the micro solution plasma irradiation, the cell membrane permeability, which is strongly related to transfection, is evaluated using fluorescent probe YOYO-1 [2,3].
It is found that YOYO-1 molecules are transferred into the cells that have high permeable membrane by the micro solution plasma irradiation, and as a result, strong YOYO-1 fluorescence is observed around plasma irradiation area. When the pulse width is increased, the YOYO-1 fluorescence region becomes large, because the diameter of the micro solution plasma is increased with the pulse width and the plasma irradiation area is spread. Based on these results, the micro solution plasma is found to locally enhance the cell membrane permeability at the plasma irradiation area.
 H. Fujita, S. Kanazawa, K. Ohtani, A. Komiya, T. Kaneko, and T. Sato, Europhys. Lett., 105 (2014) 15003.
 S. Sasaki, M. Kanzaki, and T. Kaneko, Appl. Phys. Exp., 7 (2014) 026202.
 T. Kaneko, S. Sasaki, Y. Hokari, S. Horiuchi, R. Honda, and M. Kanzaki, Biointerphases, 10 (2015) 029531.
3:45 AM - G2.04
Possible Mechanisms of Blood Coagulation Induced by Low Temperature Plasma Treatment
Sanae Ikehara 1 Hajime Sakakita 2 Kenji Ishikawa 3 Yoshihiro Akimoto 4 Hayao Nakanishi 5 Nobuyuki Shimizu 6 Masaru Hori 3 Yuzuru Ikehara 1
1National Institute of Advanced Industrial Science and Technology (AIST) Tsukuba Japan2National Institute of Advanced Industrial Science and Technology (AIST) Tsukuba Japan3Nagoya University Nagoya Japan4Kyorin Univ School of Medicine Mitaka Japan5Aichi Cancer Center Research Institute Nagoya Japan6SANNO Hospital Tokyo JapanShow Abstract
Appearance of energy devices for hemostasis has significantly shorten time for surgery to improve operative mortality, while the use of such equips is known to be a cause for post-operative disorder (PD) due to the heat injury and following inflammatory responses. As there is still no way to treat PD, new methods to control bleeding without heat injury are requested.
Low temperature plasma in atmosphere has been pointed out to be a feasible technology for hemostasis without cauterization. Indeed, earlier reports demonstrated that plasma treatment much shortened the whole-blood clotting time comparing with the time of natural occurring coagulation. Plasma played a role to stimulate platelets to aggregate and turned on the proteolytic activities of coagulation factors, resulting in the acceleration of the natural blood coagulation process. It is worth mentioning that the shape of erythrocytes in clotted blood is kept the same as in clotted blood through the natural coagulation process.
To rationally use low temperature plasma for hemostasis, we have developed plasma source better to supply glow-like plasma based on a dielectric barrier discharge. Indeed, our device succeeded to make a clot immediately upon the contact with plasma flair with neither being over 40°  nor thermal damage commonly observed after high-frequency electro-coagulator treatment . It is striking that fibrous membrane-like structure (FMLS) was appeared to overlay on the disrupted vascular wall, which was an evidence for unnatural coagulation in formed clots treated by our device. Additionally, formed FMLS can be comparable to treatment with anti-adhesion sealant to prevent PD , from the view point of wound care.
In this symposium, we will provide an overview of current attempts to use plasma for bleeding control and the revealed mechanism on clot formation by plasma treatment. Moreover, we would like to discuss the feasibility of this technology not only for preventing PD but also improving prognosis of pancreatic cancer patients who are applied for surgical therapy. .
Acknowledgements. We thank the support provided by Ms. Mika Hashimoto in histopathology experiments. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Area (21590454, 24590498, and 24108006 to Y. I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
 H. Sakakita, and Ikehara Y., Plasma and Fusion Research S2117, 1-4 (2010).
 Y. Ikehara, H. Sakakita, et al. J. of Photopoly Sci Tech 26(4), 555-557, (2013).
4:30 AM - *G2.05
Plasma Processed Surfaces for Life Sciences
Pietro Favia 1 2 Francesca Intranuovo 1 Ilaria Trizio 1 Daniela Pignatelli 3 4 Marta Garzia-Trulli 1 Chiara Loporto 1 Vincenza Armenise 1 Giuseppe Camporeale 1 Fabio Palumbo 2 Eloisa Sardella 2 Roberto Gristina 2
1University of Bari Bari Italy2CNR NANOTEC Bari Italy3Scuola Superiore Sant'Anna Pontedera (PI) Italy4Istituto Italiano di Tecnologia, IIT Pontedera (PI) ItalyShow Abstract
Plasma processes are largely employed today in many different academic and industrial fields, resulting constantly in newer surfaces and materials. The field of biomedical materials, in particular, benefits from the use of non equilibrium plasmas at low and atmospheric pressure for tailoring the surface of materials to the best predetermined interactions with cells, bacteria, biological fluids and tissues in continuously growing number of applications. Smarter and smarter surfaces and materials are being developed nowadays by means of sophisticated plasma processes, often integrated with other modification techniques.
The ability of modifying the surface of materials with no alterations of the bulk, in a sterile medium, became soon obviously of interest in Biomaterials Sciences, a field that has experienced an impressive interdisciplinary growth in the last decades, with strong economic impact. Further, plasma processes at atmospheric pressure are also being experimented at therapeutic level for sterilization of wounds, wound healing and cancer treatment, just to mention a few fast developing fields of Plasma Medicine.
This talk will describe, from the point of view of the authors, the evolution and some recent advances of Plasma Processing Science and Technology in producing newer surfaces of potential use in the biomedical field. In particular, processes aimed at modifying external and internal surfaces of 3D biodegradable polymer scaffolds for Tissue Engineering (TE) and Regenerative Medicine (RM) [1, 2, 3] will be reviewed, as well as aerosol-assisted atmospheric pressure plasma processes utilized for the deposition of nano/biocomposite coatings embedding active biomolecules (proteins, enzymes, antibacterial compounds, drugs, etc.) in a thin polymer-like matrix [4, 5].
 J.J.A. Barry et al, Adv. Mater. 18, 1406, 2006;  F. Intranuovo et al, Acta Biomater. 7, 3336, 2011.  M. Domingos et al, Acta Biomater. 9, 5997, 2013.  P. Heyse et al, Plasma Proc. Polym. 11, 184, 2014.  F. Palumbo et al, Plasma Proc. Polym. on line 9 June 2015 DOI: 10.1002/ppap.201500039.
The following projects are acknowledged for funding: RIGENERA (Regione Puglia); LIPP (Regione Puglia; Laboratorio di ricerca Industriale Pugliese dei Plasmi, Rete di Laboratorio 51); PON RINOVATIS (MIUR; 02_00563_3448479); PON SISTEMA (MIUR, a3_00369); EC COST action MP1101 (Biomedical Applications of Atmospheric Pressure Plasma Technology). Mr. S. Cosmai (NANOTEC-CNR) and Mr. D. Benedetti (Univ. Bari) are acknowledged for technical support.
5:00 AM - G2.06
Impact of Nanoscale Surface Modification Induced by Cold Atmospheric Plasma (CAP) on Human Mesenchymal Stem Cell (hMSC) Differentiation
Mian Wang 1 Michael Keidar 2 Thomas Webster 1
1Northeastern Univ Boston United States2The George Washington University Washington DC United StatesShow Abstract
Three-dimensional (3D) printing is a new fabrication method for tissue engineering. A significant advantage of this new scaffold fabrication method is that it can precisely control scaffold architecture. However, scaffolds not only need 3D biocompatible structures, but they also require mimicking of the extracellular matrix properties of the tissue they intend to replace, such as providing a template for cell attachment, and to stimulate bone tissue formation in vitro . In order to achieve the second target, the objective of the present in vitro study was to use cold atmospheric plasma (CAP) to modify a 3D printed scaffold&’s surface roughness and chemical composition . Under the optimized CAP conditions, the water contact angles of a poly-lactic-acid (PLA) scaffold dramatically dropped from 70±2° to 24±2° after CAP treatment. On the other hand, surface roughness also changed after CAP treatment as determined via atomic force microscopy (AFM) measurements. The roughness (Rq) of the untreated scaffold and CAP treated scaffolds for 1, 3, and 5 min were 1.168 nm, 10.45 nm, 22.87 nm, and 27.60 nm, respectively. This means that the nanometric roughness had a maximum 250 % increase. Both hydrophilicity and nanoscale roughness changes represented very efficient plasma treatment. When studying CAP treated 3D printing scaffolds, results showed the above surface property modification enhanced cell attachment and bone cell functions. Such promising results suggest that CAP surface modification may have potential applications previously not thought of for enhancing bone tissue engineering applications.
 Webster, T.J., Schadler, L.S., Siegel, R.W. et al. Tissue Engineering, 2001, 7, 291.
 Hsu S.H, Lin C.H, Tseng C.S. Biofabrication, 2012, 4, 015002.
 Wang M, Cheng X, Zhu W, et al. Tissue engineering: part A, 2014, 5(20): 1060-1071.
This study was supported by Northeastern University.
5:15 AM - G2.07
Plasma Deposition and Modification of Semiconducting Thin Films for Photoelectrochemical Hydrogen Production
Yakup Goenuellue 1 Jennifer Leduc 1 Thomas Fischer 1 Sanjay Mathur 1
1Univ of Cologne Cologne GermanyShow Abstract
The PE-CVD is a unique technique for thin film deposition, since it provides a good control over several parameters (time, plasma power and reactive gas composition) and therefore over the properties of the resulting films. In addition PE-CVD processes show a high reproducibility and they are scalable for large area productions. It finds applications in growth and processing of nano-materials, such as semiconductor thin films or carbon structures like graphene, carbon nanotubes (CNT), or DLC. Since the PE-CVD is a low temperature process it is possible to deposit under mild conditions onto sensitive materials like polymers.
In this work we present the deposition and modification of semiconducting metal oxides (TiO2, Fe2O3, SnO2 ) and their multijunctions for photoelectrochemical (PEC) hydrogen production. Furthermore the as deposited films were modified using plasma techniques or supporting with graphen layer. Namely those modifications were the partial reduction of the metal oxides by hydrogen plasma, the doping with different metal cations. All this techniques allowed the selective tuning of the optical and electrical properties of the films and therefore lead to a substantially enhanced PEC performance.
5:30 AM - G2.08
Hydrophobic Coating of Expanded Perlite Particles by Plasma Polymerization in a Rotating-Bed CVD System
Mustafa Karaman 1 Mehmet Guersoy 1
1Selcuk Univ Konya TurkeyShow Abstract
Perlite is a volcanic glass mineral found extensively in volcanic rock, and it is one of the nature&’s most versatile and efficient materials. When perlite ore is rapidly heated over 870#9702;C, it expands 4 to 20 times its original size and becomes porous. Expanded perlite is an excellent choice for many applications including construction, filtration, horticulture, and insulation because of its unique characteristics of being lightweight, sterile, insulating, and fireproof. However, high water absorption capacity of expanded perlite is not desired for the applications requiring heat and sound insulations, because the material moisture content increases the heat and sound transfer coefficients. It is apparent that the surface properties of the particles play a crucial role in determining the overall function and performance of the final product. These necessitate good strategies for modification or functionalization of particle surfaces with an optimal method.
The main objective of this study is to make expanded perlite surfaces hydrophobic through an all-dry encapsulation process, namely rotating-bed plasma enhanced chemical vapor deposition (rPECVD) method. An 13.56 MHz radio frequency plasma discharge was employed to deposit thin poly(hexafluorobtyl acrylate) (PHFBA) polymeric films on expanded perlite surfaces. A rotating plasma reactor was used to provide effective agitation and mixing of the particles during the depositions. Chemical and morphological properties of uncoated and coated expanded perlites were determined by FTIR, XPS, SEM, and contact angle measurements. Surface of expanded perlite, a hydrophilic porous material, was successfully transformed into super-hydrophobic due to the highly fluorinated chain of PHFBA. Deposition rates up to 42 nm/min were measured. It was also observed that the pulsed plasma approach helps to minimize undesirable monomer fragmentation while providing better film structure. Based on results of this study, it can be concluded that rotating-bed PECVD method can be used for conformal coatings around particles having complex geometries like expanded perlite. PECVD avoided many problems associated with existing liquid-based particle coating techniques such as particle agglomeration, toxic solvents, and poor uniformity. The important plasma deposition parameters, specifically plasma power and operation mode (continuous vs. pulsed) were found to be effective in providing control of the surface hydrophobicity. The water holding capacity of expanded perlite was decreased from 70% down to around 4% upon coating on pulsing mode. These features can widen the area of use of expanded perlite especially in building industry and open up new application areas. The method of this study can be extended to functionalize many other types of particles having dimensions down to micro and even nano scale.
5:45 AM - G2.09
Spatio-Temporal Distribution of Atomic, Molecular and Ion Dynamics in Laser Produced Carbon Plasmas
Subhash C. Singh 1 2
1Allahabad University Allahabad India2Dublin City University Dublin IrelandShow Abstract
Pulsed laser deposition is well established method for the growth of diamond like carbon films, carbon nanostructures such as tubes, wires, rods etc., and nanoparticles. Morphology and hence derived electronic, optical and mechanical properties depend on the experimental growth conditions such as laser wavelength, pulse energy, focal spot size, and nature and pressure of gas. These experimental conditions drive laser produced plasma (LPP) parameters such as temperature, density, and ligh, ion, and electron emitting properties of plasma.
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 (Tvib) 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.
G3: Poster Session: Interactions between Plasma and Biomaterials
Thursday PM, December 03, 2015
Hynes, Level 1, Hall B
9:00 AM - G3.01
DNA Microarray Analysis of Gene Expression in Response to Floating-Electrode Dielectric Barrier Discharge (FE-DBD) Plasma Irradiation to Escherichia coli
Yoshihito Yagyu 1 Nobuya Hayashi 2 Takashi Yamasaki 1 Yuta Hatayama 1 Taiki Miyamoto 1 Tamiko Ohshima 1 Masahiro Koshimura 1 Takeshi Ihara 1 Hiroharu Kawasaki 1 Yoshiaki Suda 1
1National Institute of Technology, Sasebo College. Sasebo Japan2Kyushu University Fukuoka JapanShow Abstract
Deoxyribonucleic acid (DNA) is a molecule, which includes all the information required for all living organisms. In this study, influence of plasma irradiation to E. coli, which is one of the most common industrial microorganisms, was examined by microarray method to identify DNA expression of microorganisms. Floating-electrode dielectric barrier discharge (FE-DBD) as an atmospheric plasma source was generated by an AC high-voltage power source (Logy Electric Co., LHV-10AC) at 10 kVpp with 9-11 kHz. FE-DBD plasma exposed to Escherichia coli, NBRC3301, colony cultured on 802 agar medium. A few colonies were irradiated by FE-DBD plasma for 80 seconds by intervals of a second to avoid temperature increase, and thus total irradiation period of plasma was 40 seconds. E. coli was cultured in 702 liquid medium for 10 hours from immediately after plasma irradiation. Gene expression in E. coli after plasma irradiation was measured by custom oligo DNA microarray commercialized by Agilent technology, and a genetic expression pattern of each experimental condition was compared in this study. Gene groups of which the expression rises and reduced immediately after plasma irradiation was measured. Furthermore, it was found that a gene expression amount of E. coli cultured for 10 hours after plasma irradiation has tendency to reduce. Effect of plasma in response to an expression amount of gene cultured for 10 hours tended to be lower in comparison with gene measured immediately after irradiation. A group of expression gene detected immediately after FE-DBD plasma irradiation included gene associated with energy production system. It is supposed that the gene group play a major role in repairing DNA injured by plasma irradiation or promoting growth of E. coli.
Acknowledgement: We are profoundly grateful to Dr. Satoru Kuhara, Ph.D. Kosuke Tashiro and Dr. Muta (Faculty of Agriculture, Kyushu Univ.) for their valuable discussions and suggestions. This work has been partially funded by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Science, Sports and Culture of Japan.
9:00 AM - G3.02
Toward the Development of New Functional ZnO Thin Films by Dry Plasma Processing for Biosensing Application
Mihai Alexandru Ciolan 1 Iuliana Motrescu 3 Dumitru Luca 2 Masaaki Nagatsu 1
1Shizuoka University Hamamatsu Japan2Alexandru Ioan Cuza University Iasi Romania3University of Agricultural Science and Veterinary Medicine ldquo;Ion Ionescu de la Bradrdquo; Iasi RomaniaShow Abstract
Nowadays zinc oxide semiconductors are one of the most versatile materials due to their remarkable properties, both optical and electrical, at room temperature being already implemented in a wide range of commercially available applications. Development of new biosensing application based on this kind of materials attracts more and more interest and diverse processing techniques of the surface of the ZnO are reported.
In our study, we propose a novel method for the improvement of the magnetron sputtered ZnO thin films properties by employing microwave excited surface wave plasma. The advantages of dry processing with plasma are overcoming and simplifying the limitations of wet chemical methods and make the ZnO a truly biocompatible materials with specific functional properties. By grafting functionalities on the surface of the material not only that we can tune the photoluminescence in the visible range we can also put the bases for a novel biosensing chip. It is very important to understand the role of the plasma and the way on how the functionalization works and how it can be improved and in this sense, we discuss the influence of the species in our plasma analyzed by quadrupole mass spectroscopy.
Our results are showing that plasma processing of the ZnO thin films has many benefits both by inducing morphological changes in the structure of the material and by grafting functional groups on their surface and further information will be presented at conference site.
9:00 AM - G3.03
A Simple Method for Quantifying Dose of Reactive Species Generated by Atmospheric Pressure Plasmas
Takaaki Amano 1 Thapanut Sarinont 1 Giichiro Uchida 2 Toshiyuki Kawasaki 3 Kazunori Koga 1 Masaharu Shiratani 1
1Kyushu University Fukuoka Japan2Osaka University Ibaraki-shi Japan3Nippon Bunri University Oita JapanShow Abstract
Non-thermal atmospheric pressure plasmas have been widely employed in biological and medical applications because they have high oxidizability with little thermal damage to biomaterials. They can provide high density reactive species and synergetic effects induced by a combination of reactive species, ionic species, and photons generated by the plasmas. Measurements of dose of reactive species from plasmas are important for quantifying effects of plasma irradiation. However, it is difficult to measure dose of each reactive species produced by plasmas. Here we measured the absorbance of KI-starch solutions after plasma irradiation as a simple method to evaluate effective dose of reactive species from plasmas.
Experiments were carried out with a scalable dielectric barrier discharge (DBD) device . The discharge voltage and current were 9.2 kVpp and 0.2 A, respectively. The discharge power was 1.14 Wcm-2 deduced from voltage/charge Lissajous plots. The KI-starch reagent was prepared as 0.5 % starch, and 0.3 % KI solution of DI water . KI-starch reagent and DI water were prepared into quartz 96 microwell plate. The solution volume was 400 ml. We measured time evolution of the absorbance of the KI-starch solution after plasma irradiation. Plasma was irradiated at y = 3 mm below the electrode and absorbance of 850nm was measured with the plate reader (Biotek, Synergy HTX). We also measured the ESR spectra of the 0.1 M DMPO solution after plasma irradiation.
For absorbance of the KI-starch solution, the absorbance at 0, 30, 60, 120, 180 s is 0.0044, 0.0487, 0.0643, 0.0984, and 0.1323 (OD), respectively. The absorbance of the KI-starch solutions increases with plasma discharge duration. For ESR spectra measurement, the intensity of the peak for DMPO-OH at 0, 30, 60, 120, 180 s is 0.0044, 0.0487, 0.0643, 0.0984, and 0.1323 (arb. units), respectively. There is clear correlation between the absorbance of the KI-starch solutions and the peak intensity of ESR signal for DMPO-OH. Since the intensity of the peak for DMPO-OH depends on the dose of reactive species irradiated to solutions, these results indicate that the absorbance of the KI-starch solutions can be employed as dose-equivallent index of reactive species supplied by discharge plasmas.
This work was partly supported by JSPS KAKENHI grant numbers 24340143 and 24108009.
 S. Kitazaki et al., Curr. Appl. Phys. (2014) S149.
 T. Kawasaki et al., IEEE Trans. on Plasma Sci. (2014) 2482.
9:00 AM - G3.04
Deposition Kinetics of Metal Nanoparticles Produced by Discharges in Water
Takaaki Amano 1 Thapanut Sarinont 1 Kazunori Koga 1 Miyuki Hirata 1 Akiyo Tanaka 1 Masaharu Shiratani 1
1Kyushu University Fukuoka JapanShow Abstract
Assessment of bio-compatibility and toxicity of nanoparticles in living body is an emerging topics in nanotechnology. Synthesis of nanoparticles using discharge plasmas in water offers a simple way to synthesize nanoparticles in aqueous suspension which is useful for their administration to living bodies. Previously, we succeeded in producing indium-containing nanoparticles using plasmas in water, and in analyzing their transport from subcutaneous of the rats and mice . Here we report synthesis of nanoparticles using Gold (Au), platinum (Pt), and silver (Ag) electrodes to study their kinetics in living body, because Au, Pt, and Ag nanoparticles are promising nanomaterials for medical applications.
Synthesis of nanoparticles was carried out using pulsed discharge plasmas in DI water. A metal rod electrode of 1 mm in diameter and a metal plate electrode of 0.2 mm in thickness were immersed into deionized (DI) water. The discharge voltage and frequency were about 10 kV and 10 kHz, respectively. Optical emissions from the plasma were measured with a spectrometer (Hamamatsu photonics, C7473-36). After discharges, we sampled the supernatant of the solution with a pipette and desiccated the solution on grid mesh for transparent electron microscopy (TEM) measurements.
Atomic emission lines of oxygen and hydrogen were observed in the emission spectra of electrical discharge plasmas in water. The lines of Au, Ag, or Pt were also observed, when Au, Ag, or Pt was employed as the electrode materials, respectively. These results indicates that Au, Ag, or Pt atoms exist in the discharge plasmas, leading to formation of nanoparticles. The generation rate of nanoparticles of Au, Ag, and Pt were 0.47, 0.21, and 0.25 mg/min, respectively. For each composition of the electrodes, synthesized nanoparticles were observed by the TEM. The size distributions deduced by TEM images are the Gaussian one and the mean size of the primary nanoparticles of Au, Ag, and Pt were 6.72, 22.6, and 5.43 nm, respectively. We will report results of subcutaneous administration towards rats of these synthesized nanoparticles to study their effects on living body.
This work was partly supported by KAKENHI grant numbers 24340143 and 24108009.
 T. Amano et al., J. Nanosci. Nanotechnol. (2015) in press.
9:00 AM - G3.05
Basic Fibroblast Growth Factor Delivery Using a Micro-Patterned Hydrogenated Amorphous Carbon (a-C:H) Nano-Coating on the 2-Methacryloyloxyethyl Phosphorylcholine (MPC) Polymer
Kenta Bito 1 Terumitsu Hasebe 2 1 Tomoki Maeda 1 Shunto Maegawa 1 Yuya Yamato 1 Tomohiro Matsumoto 2 1 Takahiko Mine 2 Tetsuya Suzuki 1 Atsushi Hotta 1
1Department of Mechanical Engineering, Keio University Yokohama Japan2Tokai University Hachioji Hospital, Tokai University School of Medicine Hachioji JapanShow Abstract
A hydrogenated-amorphous carbon (a-C:H) film deposited by the radio frequency plasma-enhanced chemical vapor deposition (RF-PECVD) has been known as a coating material with high biocompatibility, which has clinically received a lot of attention in recent years for a stent coating. Previously, we proposed a new coating method for the drug-release stent possibly used in indwelling devices with e.g. a cell adhesive scaffold preserving the blood compatibility. The new method consisted of a micro-patterned a-C:H film coated on a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer film. Moreover, the a-C:H/MPC film with a model drug included in the MPC film presented an effective controlled drug-release capability depending on the deposition area of the micro-patterned a-C:H. In this study, we attempted to add the basic fibroblast growth factor (bFGF), a protein promoting proliferation of endothelial cells, to a-C:H/MPC platform as a new drug-eluting system. The MPC films containing bFGF were prepared from the mixture of MPC/ethanol and bFGF/water. A metallic mesh whose pore size was 60×60 µm2 was overlaid on the MPC-containing bFGF films. An a-C:H thin film was then deposited on the bFGF/MPC by the RF-PECVD, where the RF power was set at 200 W with the deposition time of 20 s. Acetylene (C2H2) was used as a process gas at 13 Pa. The structural details of the carbon were obtained by an Ar laser micro-Raman spectrometer, revealing two peaks of D-peak (1350 cmminus;1) and G-peak (1530 cmminus;1), which were the representative signals of a-C:H films. The width of the micro-patterned a-C:H blocks was approximately 60 mu;m, which actually well corresponded to the mesh pore size. The bFGF release rate from the a-C:H/MPC was measured using an enzyme-linked immune sorbent assay (ELISA), which detected the amount of unbroken proteins. The results indicated the existence of bFGF even after the plasma processing. In fact, the bFGF-released amount was well suppressed with the increase in the a-C:H-coated surface area. The endothelial cell adhesion and the cell proliferation were examined by immersing the substrates in human umbilical vein endothelial cells. The proliferation of the endothelial cells on the a-C:H/MPC containing bFGF was analyzed by the cell adhesive area from the fluorescence microscopy images. It was found that the cell adhesive area of the a-C:H/MPC with bFGF was about 1.5 times larger than that of the a-C:H/MPC without bFGF at 24 hours. It was therefore confirmed that the proliferation of the endothelial cells was enhanced by the released bFGF. The suggested a-C:H/MPC platform with bFGF could be utilized as an alternative device of conventional drug-eluting stent (DES) possibly after the in vivo testing on the anti-thrombogenicity and the promotion of the endothelialization.
9:00 AM - G3.06
Robust SERS Substrates with Plasmonic Cavities on a Large Scale Derived from Silver Nanocubes atop a Massed Silver Surface
ShuChun Cheng 1
1National Cheng Kung University Tainan TaiwanShow Abstract
Robust SERS substrates with plasmonic cavities on a large scale from nanogaps can be fabricated by self-assembling Ag nanocubes on a massed Ag surface via 1, 2-ethanedithiol monolayer as an ultra-thin spacer. X-ray spectroscopy is used to confirm the existence of nanogaps. With the binding energy shift of Ag 3d core level and feature peaks of the sulfur atoms on the substrate treated by 1, 2-ethanedithiol, we can know that Ag nanocubes were self-assembled on the massed Ag surface by Ag-S chemical bonds and the nanogaps formed between Ag nanocubes and the massed Ag surface.
The plasmonic interaction between Ag nanocubes and the massed Ag surface was corroborated by UV-Vis spectra because the frequency change of plasmon resonance indicates the coupling of Ag nanocubes and the massed Ag surface. The theoretical investigate via finite difference time domain (FDTD) method is used to obtain the simulated absorption spectra and the electromagnetic (EM) field density diagrams of substrates to show plasmonic cavities with high efficiency are formed by the propagating surface plasmons between nanocubes and the massed silver surface. Since the intensity of Raman is proportional to the fourth power of the electric field strength, the huge changes in the EM field of Ag nanocubes self-assembled on Ag surface result in the substantial increase with several orders in magnitude in SERS signals. The rhodamine 6G (R6G) is used as a model compound to confirm SERS results and adenine detection is used in real application.
With the homogeneous distribution of plasmonic cavities, the SERS results prove the supreme performance of the robust substrate by detecting 10#65293;9 M R6G solution with high sensitivity (enhancement factor 2.8×108), high reliability (6.6#65285; standard deviation from 20-sites measurements), and high precision (calibration line with 99.9#65285; correlation coefficient). on the basis of 1 cm × 2 cm area. Furthermore, the straight calibration line with 99.9#65285; correlation coefficient is obtained by using the concentration of R6G solution from 10#65293;6 M to 10#65293;9 M, confirming its practical application in quantitative analysis. In adenine detection, the standard deviation from 20-sites measurements is 4.8#65285; by using 10#65293;7 M solution, promising real applications to biomolecules.
It is very difficult to arrange nanoparticles regularly on a substrate in the general case. The substrate with high degree of regularity used for Raman quantitative analysis is usually manufactured by FIB or E-beam etching which is heavy in cost and time. Our robust substrate, prepared by self-assembling Ag nanocubes on the massed Ag surface represents the homogeneous distribution of nanocubes, possessing the high EF and the low standard deviation for the potential application in quantitative analysis.
9:00 AM - G3.07
Long-Time Stability of Catalytic Coatings on the Basis of Nanoporous TiO2 under the Influence of Corona Discharge Plasma in Air
Sergey M. Karabanov 1 Dmitriy Suvorov 1 Gennadiy Gololobov 1 Maria A. Klyagina 1 Vladimir V. Vasilev 1 Nikolay M. Vereshchagin 1 Evgeny V. Slivkin 1
1Ryazan State Radio Engr Univ Ryazan Russian FederationShow Abstract
Coatings on the basis of nanoporous TiO2 have high catalytic activity in relation to volatile organic compounds (VOCs). It is known that the decay rate of VOCs in air increases significantly at sharing catalytic coatings and corona discharge plasma.
The problem of long-term stability of catalytic coatings on the basis of nanoporous TiO2 which is affected by corona discharge plasma in air is investigated insufficiently that limits the possibility of practical use in air cleaning systems.
The paper presents the results of experimental studies of structure properties of catalytic coating on the basis of nanoporous TiO2 affected by corona discharge plasma in air at atmospheric pressure. The point-plane configuration is used as the experimental test system. The tip material is stainless steel; the rounding diameter is 15 µm. A titanic plate with the size of 5x5 cm on the surface of which the coating of nanoporous TiO2 was synthesized (pore diameter- 90 nm, pore density per surface unit - 20 pores/µm2, coating thickness - 12 microns) was used as a flat electrode. The coating was synthesized by the method of electrochemical anodizing by direct current at the voltage of 60 V (electrolyte - NH4F of 0.3% (mass), H2O of 0.5% (mass) and ethylene glycol as a basis). The corona discharge was ignited at DC voltage of both negative and positive polarity, the value of which varied within 10-15 kV, the discharge current was 75-100 µA, duration of influence of the corona discharge plasma - 200 h.
The experimental data on change of the surface morphology at various processing modes by corona discharge plasma is obtained. It is shown that at the negative corona at the voltage of 10 kV and discharge current of 70 µA the insignificant surface erosion is provided (thickness reduction is not more than by 10%). The porous coating structure remains constant. At the positive corona at the voltage of 10 kV and discharge current of 70 µA the surface erosion is about 50% higher. It is obviously caused by radically different chemistry of the positive corona discharge. The geometry of coating pores does not degrade and remains constant in all studied processing modes. The work obtained the data on thickness change and coating roughness degree in time at various polarity and corona discharge parameters.
The received results have the practical importance for application of catalytically active coatings on the basis of nanoporous titanium oxide in air cleaning systems.
9:00 AM - G3.08
The Mechanical and Tribocorrosion Properties of CoCrMo Alloys Coated with TiAlN/TiAl Multilayers
Martin Flores 1 Omar Jimenez 1 Cesar Rivera 1
1Universidad de Guadalajara Zapopan MexicoShow Abstract
The wear-corrosion phenomenon is present in biomedical alloys that are used in prosthesis to replace natural joints. This damage limits the service life of such implants. The hard coatings can improve the resistance of wear and corrosion. The multilayers of TiAlN/TiAl were deposited on CoCrMo alloys by magnetron sputtering. In this work we study the wear mechanisms of the samples coated and alone in a simulated body fluid with an ion concentration similar to that in the human blood. The mechanical properties of multilayers were studied by means of naoindentation tests. The structure of coatings was studied by means of XRD and the composition by EDS techniques. The tribocorrosion was performed using a ball on plate reciprocating tribometer, the tests were conducted at 37 °C of temperature. The loads used were between 1 N to 2N, the oscillating frequencies was 1Hz. The corrosion and tribocorrosion were studied using open circuit potential (OCP) and potentiodynamic polarizations. The potentiodynamic polarization was used to estimate the change in the corrosion rate due to wear and the potensiostatic polarization in the passive region to measure the change in the wear rate due to corrosion. In order to study the wear mechanisms, the debris, the topography and composition of worn surfaces were analyzed by means of Raman spectroscopy, SEM and EDS. The coatings improve the nanohardness, corrosion and tribocorrosion resistance of CoCrMo alloys.
9:00 AM - G3.09
Study of Tribocorrosion Behavior of ZrN Coatings Deposited by Magnetron Sputtering on Ti6Al4V Biomedical Alloys
Luis Martiacute;n Flores 1 Omar Jimenez 1 Martin Flores 1
1Universidad de Guadalajara Guadalajara MexicoShow Abstract
Ti6Al4V alloy is widely used in biomedical devices due to its excellent corrosion resistance and biocompatibility, however, this alloy has low wear resistance which is a drawback because reduces its service life. For this reason, many coating systems have been deposited on this alloy in order to improve its wear resistance. In this study we deposited ZrN coatings by magnetron sputtering technique. The thickness was measured by profilometry and SEM. The cross section images obtained by SEM were used to study the growth of the films. Wear-corrosion tests were performed using a tribometer and a potenciostat with a reciprocating sliding motion, using a 10 mm diameter Al2O3 ball with a normal load of 1 N, frequency of 1 Hz, a stroke length of 10 mm and a sliding time of 30 minutes. These tests were conducted in a simulated body fluid (SBF) at 36.5 ± 1 °C. The structure and composition of the coatings were analyzed by XRD and EDS techniques respectively. The mechanical properties and nanotribological behavior were studied using a nanoindenter. Raman spectroscopy was used to study the formation of oxides in the wear track and the adhesion was measured by scratch test. The thickness of these coatings was between of 4.5 µm. The alloys coated with ZrN coatings showed an improvement in nanohardness and tribocorrosion resistance in comparison to the uncoated substrate.
9:00 AM - G3.10
Effects of Neutral Oxygen and Nitrogen Atoms on the Inactivation of Spores in Low Pressure Surface Wave Plasma
Yang Xiaoli 1 Syuhei Hamajima 1 Masaaki Nagatsu 1
1Shizuoka University Hamamatsu JapanShow Abstract
In our previous work on the inactivation of spores using the N2/O2 gas mixture surface-wave plasmas, we found that UV radiation and the neutral radicals, such as oxygen atoms, are the important lethal factor in N2/O2 plasma. However, the specific mechanism of neutral radicals has not been completely understood.
Here we have carried out the study about nitrogen and oxygen atom&’s effects on the inactivation of spores by measuring with vacuum ultraviolet absorption spectrum (VUVAS) technique and optical emission spectroscopy in surface-wave plasmas with different gas mixture ratios. Results showed that the oxygen atomic densities agree well with the spore size etching observed at the corresponding plasma condition, that means the etching effect in N2/O2 gas mixture surface-wave plasma mainly come from oxygen atoms. However, it is noted that the gas mixture at the strongest etching did not have best sterilization. While the nitrogen atoms density decreased with the increase of O2 percentage, the highest nitrogen atomic density had low sterilization efficiency. It means the nitrogen atoms contribute directly little to the inactivation of spores in N2/O2 gas mixture surface-wave plasma. Additional sterilizing factor is the VUV emission from NO molecules observed in N2/O2 gas mixture surface-wave plasma. We will discuss the relation between these neutral species and inactivation of spores.
9:00 AM - G3.11
Enhancing Treatment of Cancer Cells by Static Magnetic Field
Xiaoqian Cheng 1 Kenan Rajjoub 2 Alexy Shashurin 1 Dayun Yan 1 Jonathan Sherman 3 Michael Keidar 1 3
1George Washington Univ Washington United States2The George Washington University Washington United States3The George Washington University Washington United StatesShow Abstract
It was reported since late 1970 that magnetic field (MF) interacts strongly with biological
systems. On the other hand, cold atmospheric plasma (CAP) has been widely studied in
various fields. In this study, we propose a novel idea to combine static magnetic field
with cold plasma as a tool for cancer therapy. Breast cancer cells and wild type
fibroblasts were cultured in 96-well plates and treated by CAP with or without static MF.
The breast cancer cells MDA-MB-231 showed a significant decrease in viability after
direct plasma treatment with magnetic field (compared to only plasma treatment). In
addition, cancer cells treated by the plasma-MF-activated media (indirect treatment)
also showed viability decrease but slightly weaker than the direct plasma-MF treatment.
The cyclotron frequency of both the ions in the biological system and the species
generated by cold plasma in the media are close to that of the plasma generator
pointing out to possible resonance. By integrating the use of magnet field and cold
plasma, we are able to discover their advantages that are yet to be utilized.
G1: Plasma Medicine
Thursday AM, December 03, 2015
Hynes, Level 3, Room 305
9:30 AM - *G1.01
Plasma-Activated Medium and Its Applications for Diseases
Masaru Hori 1 Masaaki Mizuno 1 Fumitaka Kikkawa 1 Masahiro Tanaka 1
1Nagoya Univ Nagoya JapanShow Abstract
Recently, applications of non-thermal atmospheric pressure plasma to medical field are becoming an emerging field. Plasma can directly and indirectly affect cells. Plasma-activated medium (PAM), which is categorized into an indirect way of plasma applications has received much attentions because PAM sometimes has more benefit than direct treatment of plasma to treat disseminated cancers. Non-thermal atmospheric pressure plasma with ultrahigh electron density (approximately 2 x 1016 cm-3) has been developed, and was previously applied as therapy for ovarian cancers and melanomas.
To investigate whether indirect effects on cells by plasma, we treated medium with plasma and replaced plasma-treated medium (we call this medium plasma-activated medium or PAM) with medium that culture cells. PAM selectively killed glioblastoma brain tumor cells against astrocyte normal cells. PAM has been also tested for gastric cancer cells, anticancer drug resistant ovarian cancer cells non-small lung cancer cells. PAM generally induced reactive oxygen species (ROS) and induced apoptosis on cancer cells. PAM suppressed laser-induced choroidal neovascularization (CNV) in a mouse eye and does not show retinal toxicity.
To elucidate intracellular molecular mechanisms of apoptosis on glioblastoma brain tumor cells by PAM, western blotting and immune-fluorescent imaging were performed. The most remarkable discovery is that PAM down-regulated survival and proliferation signaling molecules such as phosphor-AKT and phosphor-ERK1/2.
Cancer cells bear their specific mutations to grow continuously and inhibit apoptosis. It is important to find specific vulnerability in each cancer. PAM induced apoptosis through caspase-dependent pathway on glioblastoma brain tumor cells, while it induced through caspase-independent pathways on non-small cell lung carcinoma.
10:00 AM - *G1.02
Cold Plasma Application in Cancer Therapy
Michael Keidar 1
1George Washington Univ Washington United StatesShow Abstract
Plasma medicine is a relatively new field that outgrew from research in application of low-temperature (or cold) atmospheric plasmas in bioengineering. One of the most promising applications of cold atmospheric plasma (CAP) is the cancer therapy. Convincing evidence of CAP selectivity towards the cancel cells has been accumulated. This talk will summarize the state of the art of this emerging field presenting various aspects of CAP application in cancer such as role of reactive species (reactive oxygen and nitrogen), cell cycle modification, in vivo application, CAP interaction with cancer cells in conjunction with nanoparticles, computational oncology applied to CAP.
The efficacy of cold plasma in a pre-clinical model of various cancer types such as lung, bladder, breast, head, neck, brain and skin has been 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. 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 did not show significant differences. It was shown that reactive oxygen species metabolism and oxidative stress responsive genes are deregulated.
Effects related to plasma-activated media will be discussed. In particular, recent study shows that by altering the concentration of fetal bovine serum in Dulbecco&’s modified Eagle&’s medium and the temperature to store CAP stimulated media, controllable strategies to harness the stimulated media can be developed.
Tumor growth and its response to plasma treatment were simulated using a three-dimensional hybrid discrete- continuum model. The results compare untreated and treated tumors of varying sizes by measuring spatiotemporal data to predict trends of tumor evolution. The simulation results show that the treated tumor death, irrespective of tumor volume, follows an exponential decay and that the untreated tumor follows an expected growth pattern.
10:30 AM - G1.03
Surface Morphology Change of Lipid Bilayers Exposed by Plasma-Activated Medium
Hiroki Kondo 1 Takuya Tonami 1 Hiroshi Hashizume 1 Kenji Ishikawa 1 Hiromasa Tanaka 1 Makoto Sekine 1 Masaru Hori 1
1Nagoya Univ Nagoya JapanShow Abstract
Recently, it is reported that a medium exposed by plasma has various activities with bio materials, such as growth activation, inhibition, and so forth. One of the most important phenomena is selective killing effects for cancer cells. In such the plasma-activated medium, so-called PAM, various active species including reactive oxygen species (ROS) would be generated by plasma irradiation. According to a componential analysis by electron spin resonance (ESR) spectroscopy, a hydroxyl radical (OH) is one of essential components in PAM. However, complex phenomena induced by PAM cannot be explained only by the effects of OH. There is any other factors, which are not clarified yet. On the other hand, surface reactions of those active species on bio materials are also not sufficiently clarified yet. They primarily react with cell membrane, then, any biochemical reactions are induced on the inside of cells. It has been reported that a direct irradiation of plasma onto lipid bilayers can induce generation of pores on their surfaces. However, such kinds of surface morphology change of lipid bilayers by PAM have not been studied. In this study, we investigated such the effects of PAM on surface morphology of lipid bilayers by using high-speed atomic force microscope (HS-AFM).
0.5 mM of vesicle suspension of dioleoylphosphatidylcholine (DOPC) mixed with a fluorescence-labeled lipid [dioleoyl-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-DOPE)] was dropped on a glass slide and incubated at 45°C for an hour. 20 mu;L of PAM was added to the observation solutions by a pipette at each AFM observation. Then, surface morphologies of lipid bilayers before and after each addition of PAM was observed. Almost no change was confirmed by the repeated AFM observation without the PAM addition. After the addition of PAM below 100 mu;L, almost no morphological change was also observed on the surfaces of lipid bilayers. However, a number of pores obviously increased just after the addition of 120 mu;L of PAM. Their sizes also increased. This result indicates that the PAM has a perforation effect on the lipid bilayers.
10:45 AM - G1.04
Concentration and Extraction of Plasma-Induced Radical Species for Protein Crystallization
Yoko Yamanishi 1 Takuya Kobayashi 1
1Shibaura Institute of Technology Tokyo JapanShow Abstract
The current research reports plasma induced radical concentration microfluidic chip and radical induced protein crystallization. The novelty of our work is collection and concentration of radical species and which is confirmed by ESR (Electron Spin Resonance) measurement. Formation of bubbles which has a relatively large surface area accelerates the production of radical species at air-liquid interface and these bubbles are concentrated by the circulating flow in the microfluidic chip. It was also confirmed that the radical-induced protein crystal are produced with increasing of the circulation time. This microfluidic chip contributes to the protein crystal production and bio-medical applications.
Identifying the conformation of biological molecules such as proteins and nucleic acids at atomic resolution provides important clues to their functions and mechanisms. Recent report of plasma-assisted biological macromolecular crystallization has started to contribute to X-ray diffraction analysis to understand the structure of protein. However the most important parameters of compositions of plasma (electrons, ions, radicals, and photons.) are still unknown for crystallization. For the present study, the plasma-induced radical species are separated and amplified to expose to the protein solution to make protein crystals by using the modification of the author&’s developed chip which is the plasma bubbles (which is composed of plasma-state gas is enclosed) generation chip. For the present study, two-dimensional chip was fabricated with similar mechanism of radial amplification as a first step.
The plasma bubbles enclosing radical species have been successfully generated in microfluidic chip, and has transported and concentrated the bubbles. It was observed that the pH increased and conductivity decreased than initial condition, and this is likely due to by the reaction of radicals in bubbles with the reagent. It is thought that electric charge and ion were exchanged between the reagent and bubbles. It was also confirmed that the wave intensity of signal based on radical species increased with circulation time measured by ESR. In addition, C radical and H radical are confirmed in the reagent which was treated by the plasma bubbles. It was also confirmed that the Hydrogen peroxide after adding the ferric ion. However, mechanism and ability of radical species to the reagent is still unknown. When the plasma bubbles applied to the protein solution, protein crystal is successfully produced even lower concentration of protein, and which is unlike for the conventional vapor diffusion method. In addition, the number of the protein crystals is increased with increase of the circulation time. It is unclear that what kind of radical species are the effective to produce protein crystals. Further research is under going to identify the specific radical to produce protein crystal.
11:30 AM - *G1.05
Unraveling the Interaction Mechanisms of a Dielectric Barrier Discharge and Biological Samples
Peter Awakowicz 1 Sabrina Baldus 1 Konstantin Kartaschew 2 Daniel Schroeder 3 Friederike Kogelheide 5 Jan Lackmann 1 4 Elena Steinborn 4 Nikita Bibinov 1 Volker Schulz-von der Gathen 3 Julia Bandow 4 Martina Havenith 2 Katharina Stapelmann 5
1Ruhr University Bochum Bochum Germany2Ruhr University Bochum Bochum Germany3Ruhr University Bochum Bochum Germany4Ruhr University Bochum Bochum Germany5Ruhr University Bochum Bochum GermanyShow Abstract
Dielectric barrier discharges (DBD) are promising tools for medical applications. Clinical trials have already shown their ability to improve the wound healing process of chronic wounds . Nevertheless, the interaction mechanisms are still poorly understood. Because DBDs are ignited in ambient air, a multiplicity of reactive oxygen and nitrogen species (RONS) are produced. Some of those species are able to trigger wound healing process, while others are known to disinfect the wound. The knowledge of the exact amount of RONS which is produced by plasma is crucial to unravel the interaction processes between plasma and biological samples. Monitoring the chemical processes inside the plasma and simultaneous analysis of biological samples and their modifications after plasma treatment, will lead to better insight into the modes of action.
Here, the characterization of a DBD  is present. Electron density is an important parameter since the electrons are able to produce RONS. Hence, the spatial distribution of the electron density will be shown. Reactive oxygen species, namely ozone and atomic oxygen are measured applying optical absorption spectroscopy and two-photon laser-induced fluorescence, respectively . When it comes to the treatment of patients, the spatial distributions of these species in the discharge are important. Hence, the spatial distributions of ozone and atomic oxygen will be presented and discussed.
During wound treatment the DBD interacts with different biological samples e.g. skin cells, proteins, or bacteria. On a molecular level we investigate the changes in proteins for example RNase A , amino acids such as cysteine. Furthermore, the influence of plasma treatment on the model bacterium Escherichia coli is investigated. The connection between the impact of plasma on biological samples and the plasma parameters will be presented.
 Brehmer et al. 2015. JEADV 29, 148-155
 Kuchenbecker et al. 2009. J. Phys. D: Appl. Phys.42: 045212.
 Baldus et al. 2015 J. Phys. D: Appl. Phys. 48 275203
 Lackmann et al. submitted
12:00 PM - *G1.06
Towards a Better Comparison of Plasma Sources for Biomedical Applications: References and Safety Measurements
Kai Masur 1 Stephan Reuter 1 Kristian Wende 1 Thomas von Woedtke 1 Klaus-Dieter Weltmann 1
1INP Greifswald Greifswald GermanyShow Abstract
The new field of research Plasma Medicine is strongly growing - and with these emerging studies also a variety of different plasma sources have been developed. These devices generate cold atmospheric pressure plasma (CAP) enabling scientists to either kill or stimulate cells and tissues. However, there is the need to understand the processes of ROS /RNS generation in order to find the balance between activating or harming the biological samples . Therefore, much effort had been done in order to control the plasma components and finally modulate biological activities . Due to different physical parameters of the sources which differ with each set-up a comparison of their biological impacts is unfeasible. Therefore, we suggest an adaptation of a standard assay based on measurements of metabolic activities to analyze the impact of CAP on human cells. To assess the impact of plasma sources on mammalian cell systems the AlamarBlue assay was proposed . It allows the determination of the biological relevance of a plasma source or its parameter variations - as well as defining safety precautions.
1. Bekeschus S, Masur K, Kolata J, Wende K, Schmidt A, et al. (2013) Human Mononuclear Cell Survival and Proliferation is Modulated by Cold Atmospheric Plasma Jet. Plasma Processes and Polymers 10: 706-713.
2. Reuter S, Tresp H, Wende K, Hammer MU, Winter J, et al. (2012) From RONS to ROS: Tailoring Plasma Jet Treatment of Skin Cells. IEEE Transactions on Plasma Science.
3. Wende K, Reuter S, von Woedtke T, Weltmann K-D, Masur K (2014) Redox-Based Assay for Assessment of Biological Impact of Plasma Treatment. Plasma Processes and Polymers.
Our results with different eukaryotic cells - all treated with different plasma sources indicate the suitability of this approach as a reference method in plasma medicine. The data obtained display that this test system is easy to handle. Furthermore, the experiments also revealed that most plasma sources display differences concerning their effects on the tested cells with respect to treatment times and feed gas admixtures. Therefore, this bottom-up approach is helpful to define plasma parameters in order to improve CAP treatment of living cells.
12:30 PM - G1.07
Factors Affecting the Rate of Radical Species Production in Cold Plasma Applicators
Gregory Konesky 1
1Bovie Medical Corporation Clearwater United StatesShow Abstract
The generation of radical species by cold plasma applicators has important consequences in disinfection, wound healing and cancer treatment, for example. In the case of cancer treatment, there are prompt effects, delayed effects due to oxidative stress, and even longer term effects from activation of the body&’s immune system. However, radical species generation rates can vary widely for a given cold plasma applicator topology, depending on operating parameters and design implementation, and especially among different applicator topologies. We systematically evaluate radical species generation rates for various applicator topologies, including both direct and indirect plasma beam generation, and generation rate variations for a given topology under different operating parameters, applicator designs and carrier gas compositions. Relatively subtle modifications, such as exit nozzle diameter, are shown to have significant changes in radical specie production rates. We consider the origins of these different radical species production rates.
Peter Bruggeman, University of Minnesota
Mounir Laroussi, Old Dominion University
Masaaki Nagatsu, Shizuoka University
Stephan Reuter, Leibniz Institute for Plasma Science and Technology - INP Greifswald e.V/ZIK plasmatis
Eric Robert, GREMI, CNRS/University of Orleans
G4: Agricultural Application of Plasmas
Friday AM, December 04, 2015
Hynes, Level 3, Room 305
9:30 AM - *G4.01
R&D Status of Plasma Applications to Agriculture in National Fusion Research Institute (NFRI)
Suk Jae Yoo 1
1National Fusion Research Institute Daejeon Korea (the Republic of)Show Abstract
The traditional agriculture was innovatively replaced by the chemical agriculture with much higher productivity owing to the invention of agricultural pesticides and chemical fertilizers. Due to the pesticide residue, however, the chemical agriculture has been increasingly replaced by the inorganic agriculture which has even the disadvantage of lower productivity. Even though the inorganic agricultural products are free of the pesticide residue, they are sometimes exposed to deadly infection of harmful microorganisms bred well in the eco-friendly cultivation environment. The recent agricultural issues of the food safety and productivity can be innovatively overcome by adopting the plasma technology.
The plasma technology can be applied to the whole agricultural cyclic phases: The cultivation, post-harvest, and securing safety of agricultural products and foods. We named the whole agricultural cyclic phases with the plasma treatments as ‘Plasma Farming&’ which includes plasma cultivation for productivity promotion, plasma post-harvest for freshness preservation, and plasma safety for securing safety of agricultural products and foods.
In this paper, the concept of the plasma application to the agricultural phases, ‘Plasma Farming&’, will be introduced with some case studies of how to apply the plasma technology to the agriculture, and the planning and our strategy of how to realize the plasma farming will be given.
10:00 AM - *G4.02
Plasma Assisted Agriculture
Masaharu Shiratani 1 Thapanut Sarinont 1 Takaaki Amano 1 Nobuya Hayashi 1 Kazunori Koga 1
1Kyushu Univ Fukuoka JapanShow Abstract
The world needs to produce at least 50% more food to feed 9 billion people by 2050 . But climate change could cut crop yields by more than 25%. The agricultural productivity depends on several factors such as water limitation, nutrient limitation, and poor storage conditions. Conventional solutions are irrigation, fertilization, and crop protection, respectively. Plasma can offer novel solutions to enhance the agricultural productivity. Some of them are water treatment and purification, fertilization, soil treatment, seed treatment, insecticide, sterilization, pre- and post-harvest treatment, storage improvement. Among these potential solutions using plasmas, we are focusing on seed treatment [2-8]. Plasma seed treatment provides insecticide and sterilization of seeds, enhancement of germination, growth and crop yield, as well as Glucose amount in plants, without DNA mutations. We have confirmed long-term growth enhancement effects of 3 min. plasma irradiation to seeds for radish sprouts, arabidopsis thaliana, oryza sativa, soybean,loquat, plumeria, and so on.
This work was partly supported by JSPS KAKENHI grant numbers 24340143 and 24108009.
 S. Kitazaki, et al., Proc. IEEE TENCON 2010 (2010) 1960.
 S. Kitazaki, et al., MRS Proceedings 1469 (2012) ww06-08-1.
 T. Sarinont, et al., J. Phys. Conf. Ser. 518 (2014) 012009.
 S. Kitazaki, et al., Cur. Appl. Phys. 14 (2014) 149.
 T. Sarinont, et al., JPS Conf. Proc. 1 (2014) 015078.
 T. Sarinont, et al., MRS Proceedings 1723 (2015) mrsf14-1723-g02-04.
 T. Sarinont, et al., MRS Proceedings 1723 (2015) mrsf14-1723-g03-04.
10:30 AM - G4.03
Surface Functionalization of Rice Husk via Non-Thermal Atmospheric Plasma for Reinforced Composites
Chi-Chin Wu 1 Andres A Bujanda 1 Soha Albukhan 2 Patricia Heiden 2 Eric Jason Robinette 1
1US Army Research Laboratory Aberdeen Proving Ground United States2Michigan Technological University Houghton United StatesShow Abstract
Rice husk (RH), a low cost agricultural residue, has the potential to be used in place of wood in the particleboard industry using formaldehyde (UF) thermoset resin binder in countries that lack forest resources. However, problems exist with moisture absorption and poor interfacial adhesion between the RH and binder that leads to poor structural integrity of the composite. This work aims to modify and functionalize the surface of rice husk (RH) using atmospheric non-thermal plasma for improving interfacial interactions and water resistance for biomass-reinforced urea-formaldehyde (UF) thermoset resin composites. An L-shaped dielectric barrier discharge (DBD) reactor was employed to expose the rice husk to oxygen-containing gas plasmas at room temperature with different gas flow rates, oxygen compositions, and exposure durations. Preliminary X-ray photoelectron spectroscopy (XPS) data exhibit that the carbon bonding conditions of RH surface can be successfully changed from mostly graphitic to higher energy carbon/oxygen bonding states after plasma treatment. The plasma-treated RH surface was further modified by reacting with methyl methacrylate and acrylonitrile monomers, respectively, followed by UF to evaluate the effectiveness on moisture resistance, mechanical properties, and biological efficacy against bacteria for the polymer-grafted on plasma-treated RH as a composite binder. Organofunctional silane coupling agents were also utilized to tailor interfacial reactivity by matching the silane functional groups to the polymer matrix chemistry. The present study explores the feasibility of enhancing interfacial interactions for RH-enforced composites via plasma surface functionalization.
10:45 AM - G4.04
Patterning of Biomolecules onto Carbon Nanotube Dot Array Functionalized by Using Ultrafine Atmospheric Pressure Plasma Jet for Biochip Applications
Tomy Abuzairi 1 2 Mitsuru Okada 3 Nji Raden Poespawati 2 Retno Wigajatri Purnamaningsih 2 Masaaki Nagatsu 1 3
1Shizuoka University Hamamatsu Japan2Universitas Indonesia Depok Indonesia3Shizuoka University Hamamatsu JapanShow Abstract
The biochip, a bio-microarray device, has been extensively studied and developed to enable large-scale biomolecules analyses. Additionally, biomolecules analyses based on carbon nanotubes (CNTs) have attracted considerable attention due to their great sensitivity, good biocompatibility, and high surface to volume ratio. For successful realization of CNTs as a biochip device, it requires proper control of their surface modification. Compared to the conventional chemical modification techniques, plasma technologies have the advantages of non-polluting process, shorter reaction time, and less damage for surface modification. Plasma technologies play an important role in the modifying surface properties for wide range of applications due to their remarkable properties of plasma. However, for patterning surface of CNT, conventionally low-pressure plasmas driven by RF or microwaves require physical masks that make the process cumbersome, expensive and time consuming.
In this work, we studied the immobilization of biomolecules onto CNT dot array functionalized by using an ultrafine atmospheric pressure plasma jet (APPJ). CNT dot arrays were synthesized by a combined thermal and plasma CVD devices and constructed in dot array form for realizing the development of biochip device. For patterning surface of CNT, an APPJ with a micro-capillary of ~1µm was utilized. Plasma discharge was produced in the glass tube under the high voltage square-wave pulses of ±7.5kV at a low frequency of 5 kHz and 50 % duty ratio. The substrate was scanned automatically by computer-controlled stage for patterning hundred of CNT dot array. We used biotin-avidin system and antibody-antigen reaction of E. coli to assess the feasibility of biomolecule immobilization onto CNT dot array as a biochip device. The selective biomolecules immobilization was confirmed by chemical derivatization with the fluorescent dye visualized by fluorescent microscope. This methodology allowed introducing patterned biomolecules onto CNT dot array for the development of CNT biochip device.
G5: Synthesis and Medical Application of Nanoparticles
Suk Jae Yoo
Friday AM, December 04, 2015
Hynes, Level 3, Room 305
11:30 AM - G5.01
Highly-Sensitive Detection of Escherichia Coli Using Antibody-Immobilized, Plasma-Functionalized Carbon-Encapsulated Magnetic Nanoparticles
Masaaki Nagatsu 1 Anchu Viswan 1 Kuniaki Sugiura 1
1Shizuoka Univ Hamamatsu JapanShow Abstract
The bacteria, such as E-coli or Salmonela, are often responsible for the serious food intoxication, especially in the temperate and tropical zones. To prevent such a food poisoning caused by these bacteria, the development of faster, more sensitive and easier methods of bacteria detection is crucial.
In this study, we will present our recent results on the development of a highly-sensitive bacteria detection system using antibody-immobilized, plasma functionalized magnetic nanoparticles(NPs). So far, we have reported surface functionalization of carbon-encapsulated iron compound NPs by RF plasma processing, where it was found that amino groups were efficiently introduced onto the surface of the magnetic NPs by an RF driven Ar/NH3 gas mixture plasma. After immobilizing anti-E-coli antibody onto the functionalized surface of the NPs, then we have tested the ability of the surface-functionalized NPs to capture and concentrate E-coli by a magnet. The concentration and capture efficiency of bacteria by the magnetic NPs were compared with each other under different plasma treatment conditions. We have successfully demonstrated a rapid and sensitive detection of E-coli using antibody-immobilized magnetic NPs. The details of experimental results will be presented at the symposium.
11:45 AM - G5.02
Synthesis of Radioactive Gold Nanoparticles for Brachytherapy Treatments, Using Plasma Electrochemistry
Mathieu Bouchard 1 2 3 Marc-Andre Fortin 1 2 3 Myriam Laprise-Pelletier 1 2 3 Olivier Plamondon 1 2 3 Stephane Turgeon 2 3
1Universite Laval Quebec Canada2CR-CHU de Quebec Quebec Canada3Centre Quebecois des Materiaux Fonctionnels Quebec CanadaShow Abstract
Gold nanoparticles (Au NPs) have been suggested as radioactive sources (198Au; 2.7 days) for prostate brachytherapy procedures , . The range of the 198Au β-particle (0.9 6 MeV, ~ 11 mm in soft tissue, ~ 1100 cell diameters) is sufficiently long to provide cross-fire effects of a radiation dose delivered to cells within the prostate gland, and short enough to minimize the dose to healthy peripheral tissues. However, the efficient technological transfer of 198Au NPs into clinical brachytherapy procedures, will require the development of novel, more efficient, safer, and more compact Au NP synthesis methods. The preparation of NPs with conventional colloidal synthesis techniques comes with several manipulation steps (ligand exchange, solvent exchange, purification procedures), which represent critical radioprotection challenges. Therefore, novel bench-top technologies must be developed to facilitate the automated production of 198Au-NPs. Here we report on the use of plasma to synthesize Au NPs based on plasma-liquid electrochemistry (Fig. 1a) . An argon plasma is generated at the surface of an aqueous solution containing gold salts (AuCl4-) and surfactant molecules. This method yields a continuous production of stable Au NP suspensions directly in water. In only 45 minutes, a 50 mL solution containing 1 mM of AuCl4- can be reduced into NPs with a reduction yield of 99.3 ± 0.7 %. Thus-synthesized Au NPs are readily capped with dextran, a biocompatible molecule widely used in vascular injections media. The diameter of Au NPs can be tuned by varying the concentration of dextran in the solution. Finally, an integrated UV-visible spectrometer is used to monitor Au NPs growth kinetics and final size (Fig. 1b; plasmon peak: 531 ± 3 nm for 5 nm diameter NPs, 570 ± 4 nm for 120 nm diameter NPs), which is necessary for quality control. A comprehensive radioactive 198Au NPs synthesis was performed, to measure NP synthesis efficiency, as well Au NP ripening in solution. The fractions were measured by single photon emission computed tomography (SPECT). NPs were analysed in high-resolution TEM, UV-Vis, XPS and dynamic light scattering (hydrodynamic size). Whereas NP synthesis efficiency rates of 22% were noted in the few minutes following synthesis, NP ripening occur at room temperature, that leads to very high conversion yields within 16 hours (96%). Although the exact mechanisms by which NP grow and stabilize after application of the argon plasma are not completely elucidated, the role of reactive oxygen and nitrogen species in 198Au NP growth was explored. Overall, plasma electrochemistry could enable the efficient, on-site and upon request production of 198Au NPs for a next generation of brachytherapy procedures.
 M. K. Khan et al, Nanomedicine Nanotechnol. Biol. Med., vol. 4, no. 1, pp. 57-69, 2008.
 R. Shukla et al, Proc. Natl. Acad. Sci., vol. 109, no. 31, pp. 12426-12431, 2012.
 M.-A. Fortin et al, Patent CA2859694 A1, 2012.
12:00 PM - G5.03
Plasma-Induced Fabrication and Surface Modification of Graphite-Encapsulated Gold Nanoparticles Aiming to Multiple Biological and Biomedical Applications
Enbo Yang 1 Masaaki Nagatsu 1
1Shizuoka University Hamamatsu JapanShow Abstract
Gold nanoparticle is believed to be one of the best candidate for biological and biomedical applications due to its special physical properties, especially the optical properties. However, conventional synthesis methods for gold nanoparticle are mainly in wet chemical methods having many problems should be overcome. Graphite-encapsulated metallic nanoparticles have attracted many interests due to their combined properties of carbon and the metal core. We have developed a set of sophisticated plasma processing technology to fabricate and functionalize the graphite-encapsulated magnetic nanoparticles. Fabrication of graphite-encapsulated gold nanoparticle especially using arc discharge method has been seldom reported during last twenties years. With these concerns, we tried to fabricate the carbon layers coated gold nanoparticles by arc discharge plasma. After that, we further performed the surface modification for the as-fabricated nanoparticles by plasma processing.
The experiments started from the DC arc discharge plasma for fabricating the graphite-encapsulated gold nanoparticles which were very robust and stable. To make the particle bio-compatible, surface amination using RF plasma was consequently conducted to treat and improve the surface of the as-fabricated nanoparticles. Finally, the optical properties of the nanoparticles were confirmed which offers our gold nanoparticle the capability for further biological and biomedical applications.
To understand the role of the plasma and the interactions between plasma and nanoparticles during the surface modification for the graphite-encapsulated gold nanoparticles, characterization methods such as SEM, HR-TEM, XRD, XPS, Raman, UV-Vis, FT-IR and OES microscopic and spectroscopic analyses were perfomed evaluating the properties of the nanoparticles and the plasma itself.
To quantify the amino groups which have been introduced onto the surface of the graphite-encapsulated gold nanoparticles, two kinds of chemical derivitizations were performed on our particles. Under the specific plasma treatment conditions, selectivity of primary amino group was estimated as 62.7% out of all Nitrogen containing functionalities and the amino population was estimated as in the order of magnitude 10,000 per particle. The optical properties of the modified graphite-encapsulated gold nanoparticles were evaluated by LSPR spectroscopic study using Uv-vis spectrometer. LSPR located at 540-600 nm for the particles treated by different plasma conditions. The graphite-encapsulated gold nanoparticles owing proved optical properties should be potential nanomaterial for biological and biomedical applications as we proposed in this paper.
12:15 PM - G5.04
Bioimaging with Plasma-Functionalized ZnO Nanophosphors for Virus and Bacteria Detection System
Mihai Alexandru Ciolan 1 Kuniaki Sugiura 1 Anchu Viswan 1 Masaaki Nagatsu 1
1Shizuoka University Hamamatsu JapanShow Abstract
In this study, we are discussing the experimental results of the plasma-functionalized ZnO nanophosphors for bioimaging systems employed for the detection of different viruses and bacteria. For bioimaging applications, it is necessary that biomolecules of interest should be covalently bound with reactive sites, represented by functional groups, grafted on the surface of the nanoparticles.
The novelty of our work consists in proposing the use of ZnO for bioapplications and the functionalization of ZnO by dry plasma processing, instead of existing complex chemical wet processes. The preliminary results indicate that successful functionalization with amine groups is achieved for ZnO NPs produced by pulsed laser ablation technique. Increasing the microwave power to produce SWP results in a higher population of amine moieties, probably own to a higher plasma density. Also, by applying bias voltage on a substrate situated under the ZnO material results in an increase of functionalities number. The successful connection of the functionalities is confirmed with fluorescent dye molecules indicating the possible further usage of such biofunctionalized NPs.
The results presented in this work show that there are very good premises for further use of such ZnO nanophosphors in specific bioapplications. Furthermore, photoluminescence of the ZnO nanophosphors after capturing bacteria, such as E-coli, will be presented at the conference.
12:30 PM - G5.05
Enhancement of Anti-Bacterial Properties of Cold Atmospheric Plasma (CAP) by Combined Treatment with Silver Nanoparticles
Mian Wang 1 Michael Keidar 2 Thomas Webster 1
1Northeastern Univ Boston United States2The George Washington University Washington United StatesShow Abstract
As we all know, bacteria are central players in the development of infections, and eradicating specific pathogenic bacteria without an effect on normal mammalian tissue cells still remains a major challenge in medicine. Traditional anti-microbial applications involve using antibiotics, UV photons, or nanoparticles, but they all have shortcomings such as low efficacy for some specific bacteria, like antibiotic -resistant bacteria . Cold-atmospheric plasma (CAP) has come into the spotlight as an effective alternative to traditional antibiotics for non-systemic infections, as plasma treatment shows remarkable effectiveness against a range of microorganisms, even including antibiotic-resistant biofilm-forming strains and spores .
All four bacterial suspensions (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Staphylococcus epidermidis) at log phase were prepared in fresh medium and each was diluted to an optical density equivalent to 5*106 CFU/ml. Aliquots from each standardized bacterial suspension were exposed to the cold plasma plume for 0s, 30s, 60s, 120s, and 180s treatment. After plasma exposure, each sample was transferred to fresh PBS and the resultant suspensions were used for the surviving cell viability determination using the colony count method.
The increasing CAP exposure time resulted in a continuous reduction in the viability of bacterial cells. Any of the four bacterial species were sufficient to achieve 80% deduction after exposure to CAP for 3 min. Interestingly, the Gram-positive species like S. aureus and S. epidermidis were less susceptible to CAP mediated bactericidal activities compared with the two Gram-negative species (E. coli and P. aeruginosa). The antimicrobial of two Gram-positive species of S. aureus and S. epidermidis were 83.4% and 80% when exposed under CAP for 3 minutes, while the two Gram-negative species E. coli and P. aeruginosa were high around 95% with the same treatment. In summary, results from the present study showed that CAP is an effective tool in anti-bacterial study. The Gram-positive species were less susceptible to CAP mediated bactericidal activities compared with the two Gram-negative species.
 Ma Y, Chen M, Jones JE, Ritts AC, et al. Antimicrob Agents Chemother, 2012; 56: 5923-37.
 Vatansever F, de Melo WCM a, Avci, et al. FEMS Microbiol Rev, 2013; 37: 955-89.