470
Communication
DOI: 10.1002/ppap.200600005
Summary: A device capable of generating a relatively long cold plasma plume has recently been developed. The advantages of this device are: plasma controllability and stability, room temperature and atmospheric pressure operation, and low power consumption. These features are what is required from a plasma source to be used reliably in material processing applications, including the biomedical applications. In this communication we describe the device and we present evidence that it can be used successfully to inactivate Escherechia coli in a targeted fashion. More recent experiments have shown that this device inactivates other bacteria also, but these will be reported in the future.
Photograph of a He plasma plume launched out of the plasma pencil.
Inactivation of Bacteria by the Plasma Pencil Mounir Laroussi,*1,2 Claire Tendero,2 Xinpei Lu,2 Sudhakar Alla,1,2 Wayne L. Hynes3 1
Electrical and Computer Engineering Department, Old Dominion University, Norfolk VA 23529, USA Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk VA 23529, USA E-mail:
[email protected] 3 Department of Biological Sciences, Old Dominion University, Norfolk VA 23529, USA 2
Received: January 19, 2006; Revised: April 10, 2006; Accepted: April 13, 2006; DOI: 10.1002/ppap.200600005 Keywords: bacteria; cold plasma; plasma processing; plume; radicals; sterilization
Introduction Non-equilibrium, atmospheric pressure gaseous discharges have recently been used in emerging novel applications such as the surface modification of polymers, the absorption or reflection of electromagnetic waves, biomedical treatments, and plasma-aided combustion.[1–12] Amongst these various interesting new applications, the interaction of atmospheric pressure ‘‘cold’’ plasmas with biological media promises to open new horizons in the medical and environmental fields. One of the attractive features of non-equilibrium plasmas is their enhanced plasma chemistry without the need for elevated gas temperatures. This is because the plasma chemistry is driven by the energetic electrons, while the heavy particles remain at low energy. In these plasmas the electrons collide with the background atoms and molecules causing enhanced level of dissociation, excitation and ionization. The low temperature feature of non-equilibrium plasmas makes them the technology of choice in applications requiring medium preservation, and in those where surface chemistry is desired,
Plasma Process. Polym. 2006, 3, 470–473
but without damage or even changes to the bulk of the material under treatment. Biomedical applications are amongst these. As low temperature non-equilibrium plasmas come to play an increasing role in biomedical applications, reliable and user-friendly sources need to be developed. These plasma sources have to meet stringent requirements such as low temperature (at or near room temperature), no risk of arcing, operation at atmospheric pressure, portability, optional handheld operation, etc. A cold plasma device (the plasma pencil) that meets these requirements was recently developed and reported on by Laroussi et al.[13] This device is capable of generating a cold plasma plume several centimeters in length. It exhibits low power requirements (1–3 W) as shown by its current voltage characteristics. Using helium as the main component of the operating gas, the plasma temperature (heavy species, specifically neutrals and ions) remains at room temperature, as it is shown by both emission spectroscopy and infrared detector measurements.[13] The plasma plume can be directed manually by a user to come in contact with
ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
471
Inactivation of Bacteria by the Plasma Pencil
delicate objects and materials including skin and dental gum without causing any heating or painful sensation.
Experimental Part The plasma pencil is basically a 2.5 cm diameter dielectric tube (about 12 cm long) where two disk electrodes of about the same diameter as the tube are inserted and are separated by a gap that can be varied from 0.5 to 1 cm. Each of the two electrodes is made of a thin copper ring attached to the surface of a centrally perforated dielectric disk. To ignite the plasma, sub-microsecond square high voltage pulses at repetition rates in the 1–10 kHz range are applied between the two electrodes, and a gas mixture (such as helium and oxygen) is flown through the holes of the electrodes (flow rates in the 1–10 L min1 range). When a discharge is ignited in the gap between the electrodes, a plasma plume reaching lengths up to 5 cm is launched through the hole of the outer electrode and in the surrounding room air. Figure 1 is a photograph of the plume emitted by the plasma pencil. The length of the plume can be controlled by the gas flow rate, and by the magnitude of the applied voltage pulses. The plasma plume remains stable (length, color, remains ignited, etc.) and maintains its low temperature for extended operating times (hours). The plume can be touched by bare hands without any harm. Figure 2 shows the current-voltage characteristics of the plasma pencil. The magnitude of the voltage pulse in this case is 5 kV and the pulse width is 500 ns. The current pulses are a combination of the displacement current (capacitive coupling) and discharge current. Current peaks of up to several Amperes are present. The applied voltage originates from a DC power source that drives a pulsing unit. The power supplied by the DC source to the pulser is typically in the 15 W range. Only a small fraction (about one fifth) of this power is actually dissipated by the plasma plume. The power dissipated by the plume increases with the pulse repetition rate.
Results and Discussion Preliminary experiments testing the effectiveness of the plasma pencil to inactivate bacteria were recently carried out. In these tests, the plasma pencil was attached to a sup-
Figure 1. Photograph of the plasma plume launched out of the plasma pencil. Helium is the operating gas. Plasma Process. Polym. 2006, 3, 470–473
www.plasma-polymers.org
Figure 2.
Current-voltage characteristics.
port with the plume shooting downward. Samples, such as Petri dishes, containing pre-determined concentrations of E. coli cells evenly spread over agar, were put right under the plasma plume and on a platform/stage the height of which can be adjusted. Two sets of tests were carried out: One with helium as the operating gas, and the second with helium and a 0.75% admixture of oxygen. Our goal was mainly to test the inactivation efficacy of the plasma pencil, and if this efficacy improves with the presence of oxygen, as predicted by prior literature on plasma-based sterilization.[6,14,15] It is important here to mention that atomic oxygen emission bands at 777 nm were observed both when helium or helium/oxygen were used. Figure 3a and 3b show an example of spectra between 600 and 800 nm recorded by a half-meter spectrometer (Acton Research SpectraPro 500i) equipped with a photomultiplier (Hamamatsu R928 ). A 1 200 g mm1 grating and 200 mm slit width were used. It is interesting to note that when oxygen is added to helium, the ratio of the intensity of the helium emission lines to that of the atomic oxygen emission line decreases. Reactive oxygen species, including atomic oxygen and ozone, which are generated by the plasma plume, play an important role in bacteria inactivation processes, by oxidizing lipids and proteins of the outer membranes of cells. This can lead to irreversible damage to the cells, and, ultimately, could cause their demise. The samples that were treated by the plasma pencil were prepared as follows: E. coli was the organism of choice for this preliminary report. An overnight culture containing approximately 109 cfu mL1 was prepared (cfu: colony-forming unit). Successive ten-fold dilutions, down to 102 cfu mL1, were carried out. These dilutions were evenly spread over agar plates and incubated at 37 8C for 24 h. Plates were then inspected to determine which dilutions gave a sufficient reduction in the number of colonies (the colonies are sufficiently separate to be countable). From these results, a 105-fold dilution was selected. New suspensions were then ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
472
M. Laroussi, C. Tendero, X. Lu, S. Alla, W. L. Hynes
Figure 3. Emission spectra in the 600–800 nm wavelength range: a) for a helium plume; b) for a helium þ 0.75% oxygen plume.
prepared and appropriately diluted. 500 mL of the cell suspensions were placed in each of three wells of a six-well titer plate. After centrifuging the plate for 10 min at 4 000 rpm, the liquid was removed leaving the cells in the bottom of the
wells. One of the wells was kept as control while the other two wells were exposed to the plasma plume for two different treatment times, 30 and 120 s. The applied voltage pulses were 500 ns wide, 5.3 kV in magnitude, and with a rep-rate of 5 kHz. Helium with a 0.75% admixture of oxygen was the operating gas. After the plasma treatment, 500 mL of saline was added to the wells and the cells resuspended. 100 mL aliquots were then taken out and spread evenly over trypticsoy agar in Petri dishes. The Petri dishes were then incubated at 37 8C for 24 h. Figure 4 shows the results. It is clear from the photographs that the 120 s treatment led to a substantial decrease in the number of the surviving cells. To show a clear visual effect of the plasma pencil on E. coli and to demonstrate in an observable way the influence of a small admixture of oxygen on the inactivation efficiency, the results from a helium plume and a helium þ oxygen plume were compared. The tests were conducted as follows: 100 mL suspensions containing cell concentrations of 107 cfu mL1 were evenly spread over agar in Petri dishes. Two Petri dishes were kept as controls; two Petri dishes were exposed to a helium plume, one for 30 s and the second for 120 s, and two other Petri dishes were exposed to a helium þ 0.75% O2 plume also for 30 and 120 s. In all these tests, the applied voltage pulses had a magnitude of 5.3 kV, the pulse repetition rate was 5 kHz, and the tip of the plume was in contact with the agar (the plume was aimed at the center of the dishes and the distance between the exit hole of the plasma pencil and the Petri dishes was about 3 cm). After exposure, all the Petri dishes were incubated overnight at 37 8C. Figure 5a and 5b show the results of these tests. These pictures were taken against a black background with the Petri dishes being made of plastic transparent material. So the dark circles in the center show no-growth regions. Around these regions a dense lawn of bacterial growth covers the agar and masks the dark background. From these photographs one can conclude that: i) The area of the inactivated region increases with increasing the treatment time, and ii) The area of the in-
Figure 4. Photographs of Petri dishes containing E. coli colony forming units (CFUs) on agar: a) control (far left); b) 30 s treatment (middle); and c) 120 s treatment (far right). Operating conditions: Gas mixture: helium þ 0.75% O2, applied voltage: 5.3 kV, pulse frequency: 5 kHz, pulse width: 500 ns. Plasma Process. Polym. 2006, 3, 470–473
www.plasma-polymers.org
ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
473
Inactivation of Bacteria by the Plasma Pencil
It is important to note that, although this communication reports on the efficacy of the plasma pencil to inactivate E. coli, other bacteria such as Pseudomonas aeruginosa, Vibrio cholerae, and Streptococcus sangris have also been inactivated by this device in some preliminary tests. Effects similar to those shown in Figure 5 have been observed. Detailed studies on these bacteria and others will be published in the future.
Conclusion In this communication we have shown that a cold plasma plume generated by a pulsed plasma device, ‘‘the plasma pencil’’ can be used to inactivate E. coli effectively. Since the plume is launched in the surrounding room air, oxygenbased reactive species are generated even if the plume operating gas itself does not contain oxygen. However, adding oxygen to the plasma operating gas enhances the inactivation capability of the device. The results presented in this paper are only a first stage in our research program. In the future, we plan to test the inactivation of a variety of microorganisms, including gram-negative and gram-positive bacteria (in the vegetative and spore states). Potential applications of the plasma pencil are surface sterilization in a more targeted way than it is possible with large volume plasmas, selective surface properties modification (hydrophobic, hydrophilic, oleophyilic, oleophobic, etc. surfaces), and the inactivation of oral bacteria. Preliminary tests on these bacteria have shown some success.
Acknowledgements: This work was partly supported by the US Air Force Office of Scientific Research.
Figure 5. Photographs of Petri dishes showing the effects of the cold plume, generated by the plasma pencil, on E. coli cells for the case of: a) helium only; b) helium þ 0.75% O2. Top Petri dishes are controls, bottom Petri dishes were treated for 30 s (left) and 120 s (right).
activated region is much greater when oxygen was added to helium, especially for the longer treatment time. The first conclusion shows that for both helium and helium/oxygen plumes, the build up of reactive species with time leads to further diffusion of these away from the location where the tip of the plume hits the agar, and therefore a wider area is affected. The second conclusion is in agreement with prior findings, which also claimed that higher concentrations of oxygen-based reactive species enhance the ability of cold plasmas to inactivate bacteria.[6,14,15] Plasma Process. Polym. 2006, 3, 470–473
www.plasma-polymers.org
[1] F. Massines, C. Mayoux, R. Messaoudi, A. Rabehi, P. Segur, Proc. Int. Conf. Gas Discharges Appl., Swansee 1992, p. 730. [2] M. Laroussi, Int. J. Infrared & Millimeter Waves 1995, 16, 2069. [3] M. Laroussi, IEEE Trans. Plasma Sci. 1996, 24, 1188. [4] H. W. Hermann, I. Henins, J. Park, G. S. Selwyn, Phys. Plasmas 1999, 6, 2284. [5] J. G. Birmingham, D. J. Hammerstrom, IEEE Trans. Plasma Sci. 2000, 28, 51. [6] M. Laroussi, IEEE Trans. Plasma Sci. 2002, 30, 1409. [7] M. Laroussi, J. P. Richardson, F. C. Dobbs, Appl. Phys. Lett. 2002, 81, 772. [8] N. M. Sayama, K. Ohwaki, I. Kimura, J. Propuls. Power 1993, 9, 176. [9] I. E. Kieft, D. Darios, A. J. M. Roks, E. Stoffels, IEEE Trans. Plasma Sci. 2005, 33, 771. [10] H. Koinuma, H. Ohkubo, T. Hashimoto, Appl. Phys. Lett. 1992, 60, 816. [11] E. Stoffels, A. J. Flikweert, W. W. Stoffels, G. M. W. Kroesen, Plasma Sources Sci. Technol. 2002, 11, 383. [12] A. Scutze, J. Y. Jeong, S. E. Babyan, J. Park, G. S. Selwyn, R. F. Hicks, IEEE Trans. Plasma Sci. 1998, 26, 1685. [13] M. Laroussi, X. Lu, Appl. Phys. Lett. 2005, 87, 113902. [14] M. Moisan, J. Barbeau, S. Moreau, J. Pelletier, M. Tabrizian, L’H. Yahia, Int. J. Pharmaceutics 2001, 226, 1. [15] M. Laroussi, Plasma Proc. Polym. 2005, 2, 391. ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
