Medicine and Sterilization

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1. Joaquin, J. C., Kwan, C., Abramzon, N., Vandervoort, K., and Brelles-Marino, G., “Is gas-discharge plasma a new solution to the old problem of biofilm inactivation?” Microbiology 155, 724 (2009). Abstract: Conventional disinfection and sterilization methods are often ineffective with biofilms, which are ubiquitous, hard-to-destroy microbial communities embedded in a matrix mostly composed of exopolysaccharides. The use of gas-discharge plasmas represents an alternative method, since plasmas contain a mixture of charged particles, chemically reactive species and UV radiation, whose decontamination potential for free-living, planktonic micro-organisms is well established. In this study, biofilms were produced using Chromobacterium violaceum, a Gram-negative bacterium present in soil and water and used in this study as a model organism. Biofilms were subjected to an atmospheric pressure plasma jet for different exposure times. Our results show that 99.6 % of culturable cells are inactivated after a 5 min treatment. The survivor curve shows double-slope kinetics with a rapid initial decline in c.f.u. ml(-1) followed by a much slower decline with D values that are longer than those for the inactivation of planktonic organisms, suggesting a more complex inactivation mechanism for biofilms. DNA and ATP determinations together with atomic force microscopy and fluorescence microscopy show that non-culturable cells are still alive after short plasma exposure times. These results indicate the potential of plasma for biofilm inactivation and suggest that cells go through a sequential set of physiological and morphological changes before inactivation.

2. Hong, Y. F., Kang, J. G., Lee, H, Y., Uhm, H. S., Moon, E., and Park, Y. H., “Sterilization effect of atmospheric plasma on Escherichia coli and Bacillus subtilis endospores,” Lett. Appl. Microbiol. 48, 33 (2009). Abstract: Recently, atmospheric plasma created by the electrical discharge of a gas has received much attention as a potential physical agent for biological decontamination and sterilization. Low-temperature radio frequency (RF) plasma sources at atmospheric pressure exhibit many characteristics of a low-pressure glow discharge and have been developed for practical applications in industry (Jeong et al. 1998;Babayan et al. 2001; Kang et al. 2003). As an atmospheric plasma source operates at atmospheric pressure, processing and treatments can be implemented continuously, without the need for costly vacuum equipment. This method can be used to destroy bacterial endospores and has attracted much interest for the decontamination of potential biological warfare agents (Schütze et al. 1998; Sato et al.2006). The destructive efficiency of various gas plasma sources and temperatures on Bacillus spores was compared, and an oxygen-based plasma was found to be more efficient than a pure argon plasma (Hury et al. 1998). Purevdorj et al. (2003) reported that the highest mortality of Bacillus pumilis spores was obtained when air containing water vapour was used as the plasma carrier gas, and suggested that hydroxyl free radicals play a significant role in the inactivation of spores. Recent bacterial inactivation studies using RF atmospheric pressure plasma have demonstrated that plasma can be employed to reduce or sterilize bacterial cells and spores on contaminated surfaces (Rahul et al. 2005; Sharma et al. 2006).

3. Simon, A., Anghel, S. D., Papiu, M., and Dinu, O., “Diagnostics and active species formation in an atmospheric pressure helium sterilization plasma source,” Nucl. Instrum. Methods Phys. Res., Sect. B 267, 438 (2009). Abstract: Systematic spectroscopic studies and diagnostics of an atmospheric pressure radiofrequency (13.56 MHz) He plasma is presented. The discharge is an intrinsic part of the resonant circuit of the radiofrequency oscillator and was obtained using a monoelectrode type torch, at various gas flow-rates (0.1–6.0 l/min) and power levels (0–2 W). As function of He flow-rate and power the discharge has three developing stages: point-like plasma, spherical plasma and ellipsoidal plasma. The emission spectra of the plasma were recorded and investigated as function of developing stages, flow-rates and plasma power. The most important atomic and molecular components were identified and their evolution was studied as function of He flow-rate and plasma power towards understanding basic mechanisms occurring in this type of plasma. The characteristic temperatures (vibrational Tvibr, rotational Trot and excitation Texc) and the electron number density (ne) were determined.

4. Iza, F., Kim, G. J., Lee, S. M., Lee, J. K., Walsh, J. L., Zhang, Y. T., and Kong, M. G., ” Microplasmas: sources, particle kinetics, and biomedical applications,” Plasma Process. Polym. 5, 322 (2008). Abstract: Thanks to their portability and the non-equilibrium character of the discharges, microplasmas are finding application in many scientific disciplines. Although microplasma research has traditionally been application driven, microplasmas represent a new realm in plasma physics that still is not fully understood. This paper reviews existing microplasma sources and discusses charged particle kinetics in various microdischarges. The non-equilibrium character highlighted in this manuscript raises concerns about the accuracy of fluid models and should trigger further kinetic studies of high-pressure microdischarges. Finally, an outlook is presented on the biomedical application of microplasmas.

5. Schuerger, A. C., Trigwell, S., and Calle, C. I., “Use of non-thermal atmospheric plasmas to reduce the viability of Bacillus subtilis on spacecraft,” Surf. Intl. J. Astrobiol. 7, 47 (2008). Abstract: Atmospheric pressure glow-discharge (APGD) plasmas have been proposed for sterilizing spacecraft surfaces prior to launch. The advantages of APGD plasmas for the sterilization of spacecraft surfaces include low temperatures at treatment sites, rapid inactivation kinetics of exposed microbial cells, physical degradation and removal of microbial cells, physical removal of organic biosignature molecules, and short exposure times for the materials. However, few studies have tested APGD plasmas on spacecraft materials for their effectiveness in both sterilizing surfaces and removal of microbial cells or spores. A helium (He)+oxygen (O2) APGD plasma was used to expose six spacecraft materials (aluminum 6061, polytetrafluoroethylene (PTFE), polycarbonate, Saf-T-Vu, Rastex, and Herculite 20) doped with spores of the common spacecraft contaminant, Bacillus subtilis, for periods of time up to 6 min. Results indicated that greater than six orders of magnitude reductions in viability were observed for B. subtilis spores in as short of time as 40 s exposure to the APGD plasmas. Spacecraft materials were not affected by exposures to the APGD plasmas. However, Saf-T-Vu was the only material in which spores ofB. subtilis adhered more aggressively to plasma-treated coupons when compared to non-plasma treated coupons; all other materials exhibited no significant differences between plasma and non-plasma treated coupons. In addition, spores of B. subtilis were physically degraded by exposures to the plasmas beginning at the terminal ends of spores, which appeared to be ruptured after only 30 s. After 300 s, most bacteria were removed from aluminium coupons, and only subtle residues of bacterial secretions or biofilms remained. Results support the conclusion that APGD plasmas can be used as a prelaunch cleaning and sterilization treatment on spacecraft materials provided that the biocidal and cleaning times are shorter than those required to alter surface properties of materials.

6. Li, S. Z., and Lim, J. P., “Comparison of sterilizing effect of nonequilibrium atmospheric-pressure He/O2 and Ar/O2 plasma jets,” Plasma Sources Sci. Technol. 10, 61 (2008). Abstract: The sterilizing effect of the non-equilibrium atmospheric pressure plasma jet by applying it to the Bacillus subtilis spores is invesigated. A stable glow discharge in argon or helium gas fed with active gas (oxygen), was generated in the coaxial cylindrical reactor powered by the radio-frequency power supply at atmospheric pressure. The experimental results indicated that the efficiency of killing spores by making use of an Ar/O2 plasma jet was much better than with a He/O2 plasma jet. The decimal reduction value of Ar/O2 and He/O2 plasma jets under the same experimental conditions was 4.5 seconds and 125 seconds, respectively. It was found that there exists an optimum oxygen concentration for a certain input power, at which the sterilization efficiency reaches a maximum value. It is believed that the oxygen radicals are generated most efficiently under this optimum condition.

7. Kolb, J. F., Mohamed, A. A. H., Price, R. O., Swanson, R. J., Bowman, A., Chiavarini, R. L., Stacey, M., and Schoenbach, K. H., “Cold atmospheric pressure air plasma jet for medical applications,” J. Appl. Phys. Lett. 92, 241501 (2008). Abstract: By flowing atmospheric pressure air through a direct current powered microhollow cathode discharge, we were able to generate a 2 cm long plasma jet. With increasing flow rate, the flow becomes turbulent and temperatures of the jet are reduced to values close to room temperature. Utilizing the jet, yeast grown on agar can be eradicated with a treatment of only a few seconds. Conversely, animal studies show no skin damage even with exposures ten times longer than needed for pathogen extermination. This cold plasma jet provides an effective mode of treatment for yeast infections of the skin.

8. Vandervoort, K. G., Abramzon, N., and Brelles-Marino, G., “Plasma interactions with bacterial biofilms as visualized through atomic force microscopy plasma science,” IEEE Trans. Plasma Sci. 36, 1296 (2008). Abstract: Bacterial biofilms are microbial communities that are less susceptible to standard killing methods than free-living bacteria. Gas-discharge plasmas were used to treat biofilms for various exposure times. After 5-min plasma exposure, 90% of culturable cells were removed. Atomic-force-microscope images that reveal the sequential changes in cell morphology occurring during plasma treatment are presented.

9. Weltmann, K. D., Brandenburg, R. Von., Woedtke, T., Ehlbeck, J., Foest, R., Stieber, M., and Kindel, E., “Antimicrobial treatment of heat sensitive products by miniaturized atmospheric pressure plasma jets (APPJs),” J. Phys. D: Appl. Phys. 41, 194008 (2008). Abstract: The technological potential of non-thermal plasmas for the antimicrobial treatment of heat sensitive materials is well known. Despite a multitude of scientific activities with considerable progress within the last few years, the realization of industrial plasma-based decontamination or sterilization technology remains a great challenge. This may be due to the fact that an antimicrobial treatment process needs to consider all properties of the product to be treated as well as the requirements of the complete procedure, e.g. a reprocessing cycle of medical instruments. The aim of this work is to demonstrate the applicability of plasma-based processes for the antimicrobial treatment on selected heat sensitive products. The strategy is to use modular, selective and miniaturized plasma sources, which are driven at atmospheric pressure and adaptable to the products to be treated.

10. Perez-Martinez, J. A., Pena-Eguiluz, R., Lopez-Callejas, R., Mercado-Cabrera, A., Valencia, R. A., Barocio, S. R., Benitez-Read, J. S., and Pacheco-Sotelo, J. O., “An RF microplasma facility development for medical applications,” Surf. Coat. Technol. 201, 5684 (2007). Abstract: Microplasmas represent, by their physical nature, a considerable potential for medical applications given their highly accurate action and extremely controllable penetration on the surface of biological tissue. This plasma modality is today a powerful tool in practical applications such as the elimination of necrotic cells or the sterilization of dental cavities. As we start up a research line into this technology, we have constructed a plasma needle capable of producing non thermal plasmas, within a 1 mm radius, produced by a typical 13.56 MHz RF generator. The plasma is developed out of helium and argon. Initial electrical tests show that the plasma needle can operate at comparatively low voltages (peak to peak 100–250 V) and low power consumption. With a view to optimizing the medical applicability of the system, a study of the effects of plasma temperature and power variation at different distances is presented.

11. Deng, X. T., Shi, J. J., Chen, H. L., and Kong, M. G., “Protein destruction by atmospheric pressure glow discharges,” Appl. Phys. Lett. 90, 051504 (2007). Abstract: It is well established that atmospheric pressure glow discharges are capable of bacterial inactivation. Much less known is their ability to destruct infectious proteins, even though surgical instruments are often contaminated by both bacteria and proteinaceous matters. In this letter, the authors present a study of protein destruction using a low-temperature atmospheric dielectric-barrier discharge jet. Clear evidences of protein removal are presented with data of several complimentary experiments using scanning electron microscopy, electron dispersive x-ray analysis, electrophoresis, laser-induced fluorescence microscopy, and protein reduction kinetics. Considerable degradation is observed of protein fragments that remain on their substrate surface after plasma treatment.

12. Abramzon, N., Joaquin, J. C., Bray, J., and Brelles-Marino, G., “Biofilm Destruction by RF High-Pressure Cold Plasma Jet Plasma Science,” IEEE Trans. Plasma. Sci. 34, 1304 (2006). Abstract: Biofilms are bacterial communities embedded in a glue-like matrix mostly composed of exopolysaccharides and a small amount of proteins and nucleic acids. Conventional disinfection and sterilization methods are often ineffective with the biofilms since microorganisms within the biofilm show different properties from those in free planktonic life. The use of the gas discharge plasmas is a novel alternative since the plasmas contain a mixture of charged particles, chemically reactive species, and UV radiation. The four-day-old single-species biofilms were produced using Chromobacterium violaceum, a gram-negative bacterium commonly present in soil and water. The gas discharge plasma was produced by using an Atomflo 250 reactor (Surfx Technologies), and the bacterial biofilms were exposed to it for different periods of time. Our results show that a 10-min plasma treatment is able to kill almost 100% of the cells. The results show a rapid initial decline in the colony forming units per milliliter (phase one) that is followed by a much slower subsequent decline (phase two) of the D-values that are longer than the inactivation of the planktonic organisms, suggesting a more complex inactivation mechanism for the biofilms. Two hypotheses are offered to explain this biphasic behavior. Optical emission spectroscopy was used to study the plasma composition, and the role of the active species is discussed. These results indicate the potential of plasma as an alternative way for biofilm removal.

13. Gaunt, L. F., Beggs, C. B., and Georghiou, G. E., “Bactericidal action of the reactive species produced by gas-discharge nonthermal plasma at atmospheric pressure: A review,” IEEE Trans. Plasma. Sci. 34, 1257 (2006). Abstract: Biological decontamination using a nonthermal gas discharge at atmospheric pressure in air is the subject of significant research effort at this time. The mechanism for bacterial deactivation undergoes a lot of speculation, particularly with regard to the role of ions and reactive gas species. Two mechanisms have been proposed: electrostatic disruption of cell membranes and lethal oxidation of membrane or cytoplasmic components. Results show that death is accompanied by cell lysis and fragmentation in Gram-negative bacteria but not Gram-positive species, although cytoplasmic leakage is generally observed. Gas discharges can be a source of charged particles, ions, reactive gas species, radicals, and radiation (ultraviolet, infrared, and visible), many of which have documented biocidal properties. The individual roles played by these in decontamination are not well understood or quantified. However, the reactions of some species with biomolecules are documented otherwise in the literature. Oxidative stress is relatively well studied, and it is likely that exposure to gas discharges in air causes extreme oxidative challenge. In this paper, a review is presented of the major reactive species generated by nonthermal plasma at atmospheric pressure and the known reactions of these with biological molecules. Understanding these mechanisms becomes increasingly important as plasma-based decontamination and sterilization devices come closer to a wide-scale application in medical, healthcare, food processing, and air purification applications. Approaches are proposed to elucidate the relative importance of reactive species.

14. Goree, J., Liu, B., Drake, D., and Stoffels, E., “Killing of S-mutans bacteria using a plasma needle at atmospheric pressure,” IEEE Trans. Plasma. Sci. 34, 1317 (2006). Abstract:  Streptococcus mutans (S. mutans) bacteria were killed using a low-power millimeter-size atmospheric-pressure glow-discharge plasma, or plasma needle. The plasma was applied to a culture of S. mutans that was plated onto the surface of an agar nutrient in a Petri dish. S. mutans is the most important microorganism for causing dental caries. A spatially-resolved biological diagnostic of the plasma is introduced, where the spatial pattern of bacterial colonies in the sample was imaged after plasma treatment and incubation. For low-power conditions that would be attractive for dentistry, images from this biological diagnostic reveal that S. mutans was killed within a solid circle with a 5 mm diameter, demonstrating that site-specific treatment is possible. For other conditions, which are of interest for understanding plasma transport, images show that bacteria were killed with a ring-shaped spatial pattern. This ring pattern coincides with a similar ring in the spatial distribution of energetic  electrons, as revealed by Abel-inverted images of the glow. The presence of the radicals OH and O was verified using optical emission spectroscopy.

15. Xu, L., Liu, P., Zhan, R. J., Wen, X. H., Ding, L. L., and Nagatsu, M., “Experimental study and sterilizing application of atmospheric pressure plasmas,” Thin Solid Films 400, 506 (2006). Abstract: The atmospheric pressure surface barrier discharge (APSBD) in air has been used in killing Escherichia coli. We have developed two similar dielectric barrier discharge (DBD) structure types of nonthermal plasma jets (PJ) driven by 5–20 kHz audio-frequency power at atmospheric pressure. At a flow rate of 200 L/h (argon), a stable, arc-free discharge was produced. At 1.5 cm from the nozzle, the gas temperature was kept at 47 °C for PJ-1 source and 38 °C for PJ-2 source. Some research on sterilization has been carried out and results show that such a plasma jet source as PJ-2 is very effective in the disruption of E. coli.

16. Stoffels, E., Flikweert, A. J., Stoffels, W. W., and Kroesen, G. M. W., “Plasma needle: a non-destructive atmospheric plasma source for fine surface treatment of (bio) materials,” Plasma Sources Sci. Technol. 11, 383 (2002). Abstract:  A non-thermal plasma source (‘plasma needle’) generated under atmospheric pressure by means of radio-frequency excitation has been characterized. Plasma appears as a small (sub-mm) glow at the tip of a metal pin. It operates in helium, argon, nitrogen and mixtures of He with air. Electrical measurements show that plasma needle operates at relatively low voltages (200–500 V peak-to-peak) and the power consumption ranges from tens of milliwatts to at most a few watts. Electron-excitation, vibrational and rotational temperatures have been determined using optical emission spectroscopy. Excitation and vibration temperatures are close to each other, in the range 0.2–0.3 eV, rotational gas temperature is at most a few hundred K. At lowest power input the source has the highest excitation temperature while the gas remains at room temperature. We have demonstrated the non-aggressive nature of the plasma: it can be applied on organic materials, also in watery environment, without causing thermal/electric damage to the surface. Plasma needle will be used in the study of plasma interactions with living cells and tissues. At later stages, this research aims at performing fine, high-precision plasma surgery, like removal of (cancer) cells or cleaning of dental cavities.

17. Chu, P. K., Chen, J. Y., Wang, L. P., and Huang, N., “Plasma-surface modification of biomaterials,” Mater. Sci. Eng. Rep. 36, 143 (2002). Abstract: Plasma-surface modification (PSM) is an effective and economical surface treatment technique for many materials and of growing interests in biomedical engineering. This article reviews the various common plasma techniques and experimental methods as applied to biomedical materials research, such as plasma sputtering and etching, plasma implantation, plasma deposition, plasma polymerization, laser plasma deposition, plasma spraying, and so on. The unique advantage of plasma modification is that the surface properties and biocompatibility can be enhanced selectively while the bulk attributes of the materials remain unchanged. Existing materials can, thus, be used and needs for new classes of materials may be obviated thereby shortening the time to develop novel and better biomedical devices. Recent work has spurred a number of very interesting applications in the biomedical field. This review article concentrates upon the current status of these techniques, new applications, and achievements pertaining to biomedical materials research. Examples described include hard tissue replacements, blood contacting prostheses, ophthalmic devices, and other products.

18. Herrmann, H. W., Selwyn, G. S., Henins, I., Park, J., Jeffery, M., and Williams, J. M., “Chemical warfare agent decontamination studies in the plasma decon chamber,” IEEE Trans. Plasma. Sci.30, 1460 (2002). Abstract: A “plasma decon chamber” has been developed at Los Alamos National Laboratory (LANL), Albuquerque, NM, to study the decontamination of chemical and biological warfare agents. This technology is targeted at sensitive electronic equipment for which there is currently no acceptable, nondestructive means of decontamination. Chemical reactivity is provided by a downstream flux of reactive radicals such as atomic oxygen and atomic hydrogen, produced in a capacitively coupled plasma. In addition, the decon chamber provides an environment that accelerates the evaporation of chemical agents from contaminated surfaces by vacuum, heat, and forced convection. Once evaporated, agents and agent byproducts are recirculated directly through the plasma, where they undergo further chemical breakdown. Preliminary studies on actual chemical agents were conducted at the U.S. Army Dugway Proving Ground, Dugway, UT. Exposures were conducted at a system pressure of 30 torr, exposure temperature of 70°C, plasma-to-sample standoff distance of 10 cm, and 10% addition of oxygen or hydrogen to a helium balance. This exposure condition was based on optimization studies conducted at LANL on agent simulants. The agents studied were VX and soman (GD) nerve agents and sulfur mustard (HD) blister agent, as well as a thickened simulant. All agents were decontaminated off aluminum substrates to below the detection limit of ∼0.1% of the initial contamination level of approximately 1 mg/cm2. For VX, this level of decontamination was achieved in 8-16 min of exposure, while only 2 min were required for the more volatile HD and GD. Evaporation and subsequent gas-phase chemical breakdown in the plasma appears to be the dominant decontamination mechanism for all of the agents. However, an observed difference in the decontamination process between oxygen and hydrogen indicates that chemical reactivity in the liquid phase also plays an important role.

19. Herrmann, H. W., Henins, I., Park, J., and Selwyn, G. S., “Decontamination of chemical and biological warfare, (CBW) agents using an atmospheric pressure plasma jet (APPJ),” IEEE Trans. Plasma. Sci. 6, 2284 (1999). Abstract: The atmospheric pressure plasma jet (APPJ) [A. Schütze et al., IEEE Trans. Plasma Sci.26, 1685 (1998)] is a nonthermal, high pressure, uniform glow plasma discharge that produces a high velocity effluent stream of highly reactive chemical species. The discharge operates on a feedstock gas (e.g., He/O2/H2O), which flows between an outer, grounded, cylindrical electrode and an inner, coaxial electrode powered at 13.56 MHz rf. While passing through the plasma, the feedgas becomes excited, dissociated or ionized by electron impact. Once the gas exits the discharge volume, ions and electrons are rapidly lost by recombination, but the fast-flowing effluent still contains neutral metastable species (e.g., O2∗, He) and radicals (e.g., O, OH). This reactive effluent has been shown to be an effective neutralizer of surrogates for anthrax spores and mustard blister agent. Unlike conventional wet decontamination methods, the plasma effluent does not cause corrosion and it does not destroy wiring, electronics, or most plastics, making it highly suitable for decontamination of sensitive equipment and interior spaces. Furthermore, the reactive species in the effluent rapidly degrade into harmless products leaving no lingering residue or harmful by-products.

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