Treatment of enterococcus faecalis bacteria by a helium atmospheric cold plasma brush with oxygen addition Wei Chen, Jun Huang, Ning Du, Xiao-Di Liu, Xing-Quan Wang et al. Citation: J. Appl. Phys. 112, 013304 (2012); doi: 10.1063/1.4732135 View online: http://dx.doi.org/10.1063/1.4732135 View Table of Contents: http://jap.aip.org/resource/1/JAPIAU/v112/i1 Published by the American Institute of Physics. Related Articles Microfluidic impedance spectroscopy as a tool for quantitative biology and biotechnology Biomicrofluidics 6, 034103 (2012) Biosensors for immune cell analysis—A perspective Biomicrofluidics 6, 021301 (2012) The theory of modulated hormone therapy for the treatment of breast cancer in pre- and post-menopausal women AIP Advances 2, 011206 (2012) Selective killing of ovarian cancer cells through induction of apoptosis by nonequilibrium atmospheric pressure plasma Appl. Phys. Lett. 100, 113702 (2012) Rotating turkeys and self-commutating artificial muscle motors Appl. Phys. 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Redistribution subject to AIP license or copyright; see http://jap.aip.org/about/rights_and_permissions Treatment of enterococcus faecalis bacteria by a helium atmospheric cold plasma brush with oxygen addition Wei Chen, 1,a),b) Jun Huang, 1,a) Ning Du, 2 Xiao-Di Liu, 2 Xing-Quan Wang, 1 Guo-Hua Lv, 1 Guo-Ping Zhang, 1 Li-Hong Guo, 2 and Si-Ze Yang 1,3 1 Key Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Science, 100190 Beijing, China 2 Department of Oral Biology, Peking University School and Hospital of Stomatology, 100080 Beijing, China 3 Fujian Key Laboratory for Plasma and Magnetic Resonance, Department of Aeronautics, School of Physics and Mechanical and Electrical Engineering, Xiamen University, Xiamen 361005, China (Received 3 April 2012; accepted 30 May 2012; published online 6 July 2012) An atmospheric cold plasma brush suitable for large area and low-temperature plasma-based sterilization is designed. Results demonstrate that the He/O 2 plasma more effectively kills Enterococcus faecalis than the pure He plasma. In addition, the sterilization efficiency values of the He/O 2 plasma depend on the oxygen fraction in Helium gas. The atmospheric cold plasma brush usin g a proper ratio of He/O 2 (2.5%) reaches the optimum sterilization efficiency. After plasma treatment, the cell structure and morphology changes can be observed by the scanning electron microscopy. Optical emission measurements indicate that reactive species such as O and OH play a significant role in the sterilization process. V C 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4732135] I. INTRODUCTION In recent years, nonthermal atmospheric pressure plas- mas have been widely studied for several novel applications in biomedicine 1–16 and nanotechnology. 17,18 Among the novel applications, sterilization by low temperature atmos- pheric pressure plasmas, which are partially ionized gases, is attracting significant attention. 19–22 Very recently, a room- temperature, battery-operated, handheld air plasma jet was designed and applied to effectively inactivate multilayered Enterococcus faecalis biofilms by Lu and co-workers. 23 Enterococcus faecalis is a Gram-positive facultative anaero- bic bacterium, which has a strong adaptability to temperature and resistance to multiple antibiotics. The conventional steri- lization methods such as heat and chemical agents are not suitable to treat these notorious heat- and drug-resistant pathogens. By contrast, low temperature atmospheric pres- sure plasma is a more effective technology, which can pro- vide many chemically and biologically active species, including various atoms, ions, energetic electrons, metasta- ble particles, and UV irradiation. It has been shown that when these plasmas generated at atmospheric pressure are used for sterilization, they do not cause bulk destruction of living tissue, do not damage heat-sensitive materials, and may be touched by humans without any harm. 1 However, the available atmospheric plasma sterilization processes in cur- rent medical uses have some drawbacks that the treatment sizes from plasma jets, 24 plumes, 25 or plasma needles 26 with a diameter of a few millimeters or less are rather small. In this paper, we report an experimental study on inacti- vation of Enterococcus faecalis bacteria by means of an atmospheric cold plasma brush driven by an ac power supplier. The influences of oxygen addition into the helium atmospheric plasma brush on its sterilization capability are studied. After the plasma treatment, scanning electron mi- croscopy (SEM) is used to inspect the bacterial cell structure changes. According to the optical emission spectra of the he- lium atmospheric plasma brush with and without oxygen addition, the roles of the various plasma agents in the inacti- vation of bacteria are investigated in detail. II. EXPERIMENT A. Atmospheric cold plasma brush Figure 1(a) is a schematic diagram of the experimental setup. Our atmospheric cold plasma brush comprises of two parts: a discharge chamber and two electrodes placed outside the discharge chamber. The discharge chamber with a vol- ume of 80 Â 35 Â 1mm 3 is made of quartz. Both electrodes are made of copper foil of 10 mm wide wrapping the dis- charge chamber. The gap between the inner edges of the cop- per foil is 15 mm. The ground electrode is on the upstream side; the active electrode is on the downstream side and 10 mm apart from the chamber nozzle. A sinusoidal ac high- voltage (11 kHz, 22 kV peak to peak) is applied between two electrodes for the exci tation and sustaining of the discharges. High-purity helium and oxygen are used as the working gas, and the flow rate is controlled by the flow meter. Throughout the experimental procedure, the applied power (P) is fixed at 24 W, and the He flow rate always maintains 4500 SCCM (SCCM denotes standard cubic centimeter per minute). The separation between the nozzle and the sample is 5 mm. Fig- ure 1(b) shows the discharge photograph of the atmospheric cold plasma brush with 4500 SCCM helium (P ¼ 24 W). The visually uniform brush-shaped plasma is formed and extended out of the discharge chamber through the narrow slit at outlet. a) Wei Chen and Jun Huang contributed equally to this work. b) Author to whom correspondence should be addressed. Electronic mail: chwbetter@163.com. 0021-8979/2012/112(1)/013304/4/$30.00 V C 2012 American Institute of Physics112, 013304-1 JOURNAL OF APPLIED PHYSICS 112, 013304 (2012) Downloaded 09 Aug 2012 to 159.226.35.234. Redistribution subject to AIP license or copyright; see http://jap.aip.org/about/rights_and_permissions B. Sample preparation An overnight culture containing approximately 10 7 CFUs/ml is prepared (CFUs denotes colony-forming units). Then, with filter papers as the supporting media, 5 lL of sus- pension containing Enterococcus faecalis bacteria are dropped onto sterilized filter papers (5 mm diameter), and the papers are allowed to dry in a moderate vacuum incuba- tor at 37  C for 1 h. Afterward, the filter papers containing Enterococcus faecalis bacteria are exposed to the atmos- pheric cold plasma brush sustained with He/O 2 gas. After plasma treatment, the plasma treated filter papers are trans- ferred into a 1.5 ml centrifuge tube contain ing 1 ml physio- logical saline and mixed using a vortex mixer for 1 min. The bacterial strains are grown into the sterile Brian Heart Infu- sion agar, and the number of the living bacteria cells is counted after incubation at 37  C for 48 h. III. RESULTS AND DISCUSSION A. I–V curve Voltage is measured using a 1000:1 high voltage probe (Tektronix P6015A, maximum input voltage: DC 20 kV, bandwidth: 75 MHz). For current measurements, a magnetic core current probe (Tektronix P6021, maximum discharge current: 15 A, bandwidth: 60 MHz) is utilized. Signals from the current and voltage probes are acquired and recorded by a digital Tektronix TDS 210 oscilloscope. It can be seen from Fig. 2 that the breakdown of working gas in atmos- pheric cold plasma brush, resulting in a large number of cur- rent filaments, the so-called micro-discharges, which are randomly distributed both in time and space. In this filamen- tary mode, 27–29 the discharge starts with local gas breakdown at many points in the discharge volume. This mode is charac- terized by a periodic current constituted by many discharge pulses in each half cycle. An inverse current peak is also observed when the polarity of the applied voltage changes. B. Sterilization effect of atmospheric cold plasma brush Figure 3 shows the change of the survival curves of Enterococcus faecalis bacteria in the He with addition of dif- ferent amounts of O 2 (1%, 2.5%, 5%, 10%) into atmospheric cold plasma brush (P ¼ 24 W). As can be seen in Fig. 3,a5 log reduction of the cells required only 30 s exposure time with about 2.5% oxygen addition into the helium plasma, while 90 s were required in the pure helium plasma. It was also noted that 2.5% O 2 addition provided the faster killing speed for a complete kill of the bacteria within 60 s exposure time, while it needed 120 s to completely kill the bacteria using pure helium plasma, as shown in Fig. 3. The experi- mental data shows that sterilization efficacy of He/O 2 plasma is better than that of pure He plasma. C. Scanning electron microscopy Scanning electron microscopy (SEM) (Model S-5200, Hitachi, Japan) was used to examine the morphology and structural changes of Enterococcus faecalis after plasma ex- posure. The controlled and the plasma treated samples were placed in the fixation and then coated with a thin layer of plasma sputtered platinum. Figure 4 shows the SEM FIG. 2. Typical oscillograms of applied voltage and discharge current of a filamentary discharge in He/O 2 FIG. 3. Survival curves of Enterococcus faecalis bacteria in the He with dif- ferent O 2 additions into atmospheric cold plasma brush (P ¼ 24 W). FIG. 1. (a) Schematic diagram of the experimental setup. (b) Photograph of the atmospheric cold plasma brush with 4500 SCCM He (P ¼ 24 W). 013304-2 Chen et al. J. Appl. Phys. 112, 013304 (2012) Downloaded 09 Aug 2012 to 159.226.35.234. Redistribution subject to AIP license or copyright; see http://jap.aip.org/about/rights_and_permissions photographs of untreated control and plasma treated Entero- coccus faecalis with pure helium plasma brush and with oxy- gen addition at an amount of 1% and 2.5%, respectively. It can be seen from Fig. 4 that the plasma treatment of Entero- coccus faecalis resulted in a significant alteration in cell structure and morphologies when compared with the untreated controls. As seen from images in Figs. 4(b)–4(d), more structure damages were observed on Enterococcus fae- calis with increasing the oxygen addition amount with vol- ume percent of 0, 1%, and 2.5%. Consistent with the cell surviving curve shown in Fig. 3, He plasma with 2.5% O 2 addition provided a much faster killing of the bacteria. From Fig. 4, it was also noted that the oxygen-based plasma spe- cies were mainly responsible in improving the plasma inacti- vation efficiency and oxidation capacity of Enterococcus faecalis. D. Optical emission spectra To identify the various reactive species generated by the atmospheric cold plasma brush, the optical emission spec- trum (Stellarnet, EPP-2000 C) of He/O 2 plasma is measured in the 200–850 nm wavelength range at atmospheric pres- sure. Figure 5 shows the optical emission spectra of the plasma with 4500 SCCM He/O 2 (2.5%) taken at 5 mm bot- tom the nozzle along axis. It is well known that UV radiation in the 200–300 nm region with doses of several milliwatt per square centimeter may cause lethal damage to cells. How- ever, the UV radiation intensity in 200–300 nm wavelength range is below 50 lW/cm 2 . In addition, the inactivation effect on bact eria by ultraviolet radiation is mostly related to the DNA/RNA damage in UV-C (200–280 nm). 30 Therefore, the UV emission plays a minor role in the inactivation of the bacteria. It clearly shows that excited O lines (285 and 777 nm), OH line (309 nm), molecular nitrogen lines (C 3 G u —B 3 G g ) (316, 337, 357, 380, and 391 nm), O þ line (427 nm), H a line (656 nm) and excited He atom lines (501, 587, 640, 669, 707, and 729 nm) are presented in the plasma brush. Atomic oxygen is probably due to the direct electron impact dissociations of oxygen molecule (e À þ O 2 ! O þ O þ e À ). 31 O may also be formed through Penning ionization (N 2 þ O 2 ! N 2 þ O þ O). 32,33 We assign the fea- ture at 309 nm to the OH (A 2 R þ —X 2 G) transition; 34,35 the formation of which is attributed to the reaction of excited O with water vapor (H 2 O þ O * ! 2OH) and the electron impact dissociation (H 2 O þ e À ! H þ OH þ e À ). The pres- ence of N 2 (C!B) lines clearly reveals the air entrainment in the plasma brush. The H a emission line at 656 nm is formed by the collision between water vapor molecules and electrons (H 2 O þ e À ! H þ OH þ e À ). The formation of O 2 þ is probably due to Penning ionization (He * þ O 2 ! He þ O 2 þ þ e À ). 36 Furthermore, the addition of oxygen to the He plasma decreases He molecules’ emission, 37 but enhances intensity level of reactive oxygen radicals. This can be accounted for from that some electrons are probably consumed to produce O radicals. Others then collided with air, water vapor, and He to produce N, OH and He radicals. In fact, reactive oxygen species (ROS) play a major role in FIG. 4. Scanning electron micrographs of Enterococ- cus faecalis bacteria of (a) untreated control, (b) helium plasma treatment, (c) He þ 1% O 2 plasma treatment, (d) He þ 2.5% O 2 plasma treatment. Plasma conditions were 4500 SCCM helium flow rate and 60 s exposure time (P ¼ 24 W). FIG. 5. Emission spectra of the plasma with 4500 SCCM He/O 2 (2.5%) taken at 5 mm bottom the nozzle (P ¼ 24 W). 013304-3 Chen et al. J. Appl. Phys. 112, 013304 (2012) Downloaded 09 Aug 2012 to 159.226.35.234. Redistribution subject to AIP license or copyright; see http://jap.aip.org/about/rights_and_permissions bacterial inactivation 38,39 and lead to various biological effects in the intracellular space. 40 These reactive species could directly act on microorganisms, especially their outer membranes, and damage them because of a strong oxidation mechanism. To increase the content of OH and O radicals, increasing O 2 concentration is a useful method. IV. CONCLUSION In this study, an atmospheric cold plasma brush is designed and fabricated. The effect of oxygen on the deacti- vation of Enterococcus faecalis by an atmospheric cold plasma brush is presented. Experimental results show that oxygen addition into the plasma brush can improve the deac- tivation efficiency of Enterococcus Faecalis. The atmos- pheric cold plasma brush using a proper ratio of He/O 2 (2.5%) reaches the maximum sterilization efficiency. After plasma treatment, SEM images show obvious cell structure damages in morphology of the organisms. In addition, opti- cal emission spectroscopy clearly indicates that there are excited OH, O, N 2 , and He in the atmospheric cold plasma brush, and OH and O radicals are mainly responsible for the bacteria death. ACKNOWLEDGMENTS This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China under Grant No. 11005151. 1 X. H. Zhang, J. Huang, X. D. Liu, L. Peng, Y. Sun, W. Chen, K. C. 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Redistribution subject to AIP license or copyright; see http://jap.aip.org/about/rights_and_permissions . Treatment of enterococcus faecalis bacteria by a helium atmospheric cold plasma brush with oxygen addition Wei Chen, Jun Huang, Ning Du, Xiao-Di Liu, Xing-Quan Wang et al. Citation: J. Appl or less are rather small. In this paper, we report an experimental study on inacti- vation of Enterococcus faecalis bacteria by means of an atmospheric cold plasma brush driven by an ac power supplier inacti- vation of bacteria are investigated in detail. II. EXPERIMENT A. Atmospheric cold plasma brush Figure 1 (a) is a schematic diagram of the experimental setup. Our atmospheric cold plasma