Plasma Sterilization. David Sirajuddin NERS Plasma Engineering Professor John E. Foster Nuclear Engineering & Radiological Sciences

Transcription

1 David Sirajuddin NERS Plasma Engineering Professor John E. Foster Nuclear Engineering & Radiological Sciences April 19, 2007

2 Contents 1 Abstract 3 2 Introduction 3 3 Theory Quantification of Sterilization Performance Conventional Sterilization Methods Heat Sterilization Wet Heat Dry Heat Chemical Sterilization Radiation Sterilization Parameters Choice of Gas Gas Flow Gas Pressure Power RF/MW Frequency Quantity of Media to be Sterilized Nature of Microorganism Density and Surface Layer Formation Packaging Geometrical Factors Mechanics Ionizing Radiation (IR)

3 7.2 UV/VUV Radiation Free Radicals Radiochemical Reactions Radionucleic Reactions Volatilization Methods Dielectric Discharge Barrier (DBD) Key Equations Experimental Data Inductively Coupled Plasmas (ICP) Atmospheric Pressure Plasma Jet (APPJ) Microwave (MW) Plasmas Conclusions & Future Prospects References Appendix Glossary

4 1 Abstract Plasma sterilization was assessed as a candidate for economical, and effective sterilization. Four methods of plasma sterilization were investigated: dielectric discharge barrier (DBD), inductively coupled plasmas (ICP), atmospheric pressure plasma jets (APPJ), and microwave (MW) plasmas. MW plasmas were found to possess the greatest sterilization efficacy of all the methodologies, demanding only seconds for complete elimination of microorganisms. All the methods employed for plasma sterilization yielded substantially shorter treatment times than conventional means, such as: heat, chemical, and radiation sterilization. Furthermore, it was evidenced that plasma sterilization held the greatest medium preservation out of all methods in practice, evading problems such as embrittlement and damage to the medium. The main disadvantage of plasma sterilization was its low penetrability, inability to sterilize effectively given complex geometries, and its efficacy tapering off with increasing media to be sterilized (i.e. the loading effect). Because of these considerations, it is evident that plasma sterilization cannot completely monopolize the sterilization industry; however, it does present a unique ability to effectively irradicate bacteria strains such as bovine spongiform encephalopathy (mad cow disease), and provides promise for effective elimination of endotoxins. 2 Introduction Sterilization is any process or procedure designed to entirely eliminate microorganisms from a material or medium. Sterilization is a necessity for the general health of people. Among the plethora of applications, prosthetics are needed to be sterilized before implementation in a body to eliminate the possibility of bodily rejection and infection, medical tools must be rid of microorganisms if they are to do more good than harm, food need be sterilized to inhibit disease propagation, and water must be sterilized for use in the air-conditioning industry. In recent times, the challenge for sterilization has become increasingly devel- 3

5 oped in the regard that conventional sterilization techniques have proven either insufficient or impractical. Traditional sterilization techniques include heat, chemical and irradiation treatments. These methods lack in their germicidal efficacy, and furthermore may impart undesirable properties onto the medium (e.g. embrittlement). In scenarios wherein damage to the original material is a concern, conventional methods are either insufficient, too time consuming, or impractical. Plasma sterilization provides for a promising alternative. Utilization of plasmas allows for short processing times, medium preservation, and evades impurity contamination in the material. Specifically, plasmas also allow for heat sensitive medical equipment to be effectively sterilized, and has shown unique promise in eliminating endotoxins, and heat-resistant bacteria, such as Bovine spongiform encephalopathy (Mad Cow disease). An overview of popular sterilization techniques is presented alongside a comparison to plasma sterilization. A quantitative juxtaposition of the described methods is then undertaken in order to exhibit the advantages, and disadvantages, of plasma sterilization. Specifically, emphasis is placed on the overall efficacy, applicability, and medium preservation ability of these processes. 3 Theory 3.1 Quantification of Sterilization Performance The efficacy of any sterilization technique depends on its sporicidal effectiveness in the bacterium neighborhood of a material. Traditionally, two values are invoked for quantitative measurements of a treatment, the decimal value (D-value) and the effect of sterilization. The bacteria population choice is arbitrary and dependent on the specific objectives of the experiment; however, a generic population is conventionally represented as a colony forming unit (CFU). Typical population examples include bacterial spores, specific strains of microorganisms responsible for certain diseases, and commonly found germs. With the definition of a population, the effect of sterilization can be defined as the following: 4

6 ( ) The number of population CFU before treatment Effect of Sterilization = log 10 The number of population CFU after treatment (1) That is to say, the effect of sterilization is a state quantification of the ratio of the population before treatment to that after. The greater the effectiveness of the procedure, the larger the value of this quantity will be. A logarithm is taken of the ratio due to the typical large population sizes of bacteria. The decimal, or D-value, is defined as the time required under the specified conditions to reduce the microbial viability to 1/10 of its original value (i.e. to reduce the population to one decimal). Formally, this can be represented as, D-value = Time required to reduce viability to 1/10 (2) and is measured in units of time. 4 Conventional Sterilization Methods 4.1 Heat Sterilization Heat is utilized in two forms to eliminate microorganisms: dry and wet heat Wet Heat Wet heat is applied to the medium in the form of steam, usually with the use of a steam autoclave (Figure 2). The conditions for sterilization customarily demand the use of saturated steam under a pressure of at least 15 psi so as to achieve temperatures of 121 o C (Ref. 6). 5

7 Figure 1 - A schematic of a steam autoclave is depicted. The medium to-be-sterilized is placed within the chamber, and steam is allowed to be in contact with the medium to eliminate the microorganisms and exit through an exhaust valve [adapted from Ref. 8]. An advantage of wet heat over dry heat is the high heat transfer rate between the wet steam (saturated mixture) in comparison with that of dry heat methods. The high heat transfer rate provides for the heat to be transferred to the microorganisms and cause death effectively. Treatment times on the order of approximately minutes are usually sufficient to ensure complete sterilization (Ref. 8). Wet heat is employed in situations where any damage incurred by the heat or moisture is not of top priority Dry Heat Dry heat is realized in the form of incineration, or infrared radiation. Due to the lower heat transfer rate among the heat and the material, longer treatment times are necessitated. In cases where damage due to heat is of no consequence, higher temperatures may be used to shorten the exposure time. 4.2 Chemical Sterilization Chemical agents such as EtO (Ethylene Oxide), H 2 O 2 (Hydrogen Peroxide), and O 3 (Ozone) are used to cause reactions among these agents and the cellular chemical bonds of the bacteria to deactivate and kill microorganisms. Chemical sterilization can be used when potential damage caused by heat treatment is of concern. Sterilization times need be 6

8 approximately 45 minutes to one hour. Disadvantages of chemical means is the chemical agents damage to a variety of media, including: fiber optics, electronics, and some plastics. Furthermore, chemicals can introduce contaminants/toxicity into the material. Chemicals are conventionally employed in gas form, but can also be used in liquid form (i.e. bleach and water treatments). Treatments, specifically with EtO, necessitate long post-treatment aeration times to reduce the toxicity of the medium. Concerns also arise in that EtO is flammable. Due to these factors, chemical sterilization is usually used only when many media need to be treated. 4.3 Radiation Sterilization Gamma radiation, X rays, Bremsstrahlung, microwaves, and ultraviolet radiation are the primary forms of radiation used for sterilization. Each type of radiation is garnered from appropriate sources. Gamma radiation is most commonly utilized with a high activity Co-60 source, X rays are generated via an X-ray generation tube (molybdenum being a popular source), Bremsstrahlung is employed by the use of an electron beam gun, and microwaves and UV rays are created by similar sources. The radiation is able to interact with the DNA of the microorganisms to cause physical and biochemical changes that deactivate the cells and kill the microorganisms. Gamma radiation is the most commonly used. Due to its high penetrability and effectiveness, radiation is used in a variety of applications, including the medical and food industries. A common disadvantage of irradiation treatments is the loss of ductility in the medium. The radiation can also donate its energy to the formation of free radicals, which can go onto impart changes to polymers such as chain scission and cross linking. The treatment times are typically short, but sterilization by radiation is the typically the most expensive out of all commonly used methods. Cross linking is the dominant effect of irradiation treatments in the presence of N 2 gas. Physically, it is the breaking of chemical bonds between neighboring polymer constituents, and the reattachment to another bond site. The breaking of bonds is precipitated by free 7

9 radical reactions within the polymer. This cross-linking results in embrittlement of the material. Chain scission is the primary effect of irradiation in air. Free radicals interact with the polymer bonds in a cleavage mechanism. The shortened chains are then able pack together more tightly, giving rise to a material of higher density, higher crystallinity, and lower molecular mass. The net effect is a reduced resistance to cyclic deformation and a decrease in ductility. This reduced resistance is not an amenable effect for use in the medical industry, particularly in biomedical implants. 5 The advantages of plasma sterilization over other methods is due to a synergy of mechanisms used to accomplish the process. Depending on the gas type, the plasma accomplishes elimination of microorganisms via three mechanisms: free radical chemistry, UV/VUV radionucleic/radiochemical reactions, and direct volatilization of the microbiological matter. Given a proper choice of gas, the coupling of mechanisms can be further enhanced by choosing a gas that all ready possesses germicidal properties (e.g. H 2 O 2, and aldehydes). Thus, with an appropriate selection of gas, plasmas can potentially drive the sterilization process by way of four mechanisms as opposed to one. Treatment times are generally on the order of minutes, and its medium preservation ability is unmatched. The high efficacy and delicateness of this process makes plasma sterilization a promising alternative to conventional sterilizing methods. Several methods have been developed, and are discussed in the subsequent sections; however, the mechanics and plasma parameters in relation to the sterilization process is first described. 8

10 6 Parameters It is both practical and economical to utilize plasma methods involving processes that can be performed at atmospheric pressure. Due to this constraint, the plasma methods developed commercially use RF and microwave (MW) generated plasmas. A DC plasma would necessitate costly high vacuum equipment in order to achieve breakdown at reasonable voltages without wearing out the electrodes. Furthermore, from an economic standpoint, RF and MW generated plasmas will minimize the necessary energy expenditure required for plasma ignition. Also, low temperature plasmas are sought after so as to be accommodating to thermolabile materials. The specific conditions to achieve gas breakdown affect the properties of the plasma that, in turn, militates significantly on the effectiveness of the sterilization efficacy. A diagram of the factors affecting plasmas is shown in Figure 2. Figure 2 - Flow diagram of the effect of gas breakdown conditions, their implications on plasma parameters, and these parameters effects on the plasma sterilization process. [adapted from Ref. 7]. Each of these parameters play a significant role in the sterilization behavior of the plasma. 9

11 6.1 Choice of Gas The gas chosen is the initial variable in the plasma sterilization process that, perhaps, could be said to determine the effectiveness of the treatment most significantly. The gas selection determines the types of active species present. The types of free radicals formed are a direct result of the ionized constituent gas molecules. Also, the gas dictates the intensity and wavelengths of emitted radiation (UV/VUV photons). Another factor to consider is the effect of volatilization as a plasma sterilization mechanism. Gases containing oxygen are known to be effective in this regard. Common gas choices include: O 2, CO 2, O 2 /H 2, O 2 /Ar, O 2 /CF 4, and H 2 O 2. Oxygen is often incorporated due to its efficacy in volatilization, intensity and preference of its specific UV radiation (discussed further in Section x.x), its formation of ozonolysis and thereby oxide layers via oxidation reactions. However, care should be taken in the maximization of the sterilization efficiency, as plasmas such as O 2 /CF 4, are known to be damaging to the surface of the medium (Ref. 7). This is a consequence of volatilization and oxidation being nonspecific processes. Thus, plasma parameters should be chosen in such a way to allow for a proper matching between the sterilization efficiency and the allowable damage to the medium. 6.2 Gas Flow Gas flow rate is a parameter that directly affects the efficacy of the sterilization treatment. Increasing the rate of gas flow increases the flux of active species on the medium, and is intuitively expected, and experimentally verified, to increase the effectiveness of the treatment. The gas flow rate to a certain rate incurs are higher level of sporicidal effectiveness; however, it is also intuitive that this effect may plateau at some point. It is forseeable that at some level of increase, there may be too many active species to be used effectively. It is also forseeable that a further increase could cause the residence time τ may be too short to allow for reactions to occur among the plasma constituents and the microorganisms. This trend has been verified by Lerouge (Ref. 7), and it is evident that the gas flow must be 10

12 varied accordingly with other plasma parameters in order to achieve the desired germicidal effectiveness. 6.3 Gas Pressure The gas pressure influences the volatilization rate of the plasma. Increasing the pressure can introduce competing effects in the sterilization process, such as a corresponding recombination rate. To some extent, higher pressures constitute longer residence times of the active species enabling for the efficacy to initially increase; however, further pressure increase causes the competing effects of recombination causes for the volume of active species to decrease, and the temperatures of the gas to consequentially rise. Pressure also has an effect on the electron energy distribution of the plasma. 6.4 Power The power of the RF or microwave (MW) wave used to breakdown the gas increases the electron density of the ignited plasma approximately proportionally. This increased electron density allowed for a larger volume of active species to interact with the medium. No upper bound was exhibited by either Park (Ref. 11) or Moisan (Ref. 10) in their studies in regards to sterilization efficacy and power increase. However, it is apparent that the desirable properties sought to be preserved post-treatment will place stipulations on this condition (e.g. sterilization of thermolabile materials). 6.5 RF/MW Frequency The RF/MW frequency determines the electron energy distribution function (EEDF) of the plasma. The nature of the EEDF is tantamount to the sporicidal effectiveness in the bacterium neighborhood of the medium. MW plasmas are known to have higher effectiveness in volatilization, and hence sterilization, in comparison with their RF counterparts. MW 11

13 plasmas also tend to have a more Maxwellian distribution, while RF plasmas tend to not be so. Maxwellian distributions are important to achieve due the distribution possessing more electrons in the high energy tail. The higher population of colder electrons in RF plasmas accounts for the lower concentration of effective active species such as atomic oxygen, and ozone. Due to the lower concentration of high energy electrons, and the lower efficacy of volatilization in RF plasmas, the germicidal effectiveness is necessarily less compared to that of MW plasmas. 6.6 Quantity of Media to be Sterilized An increased difficulty becomes apparent in the scenario of sterilizing large quantities of materials. This is known as the loading effect. The availability of active species in the plasma is decreased in the action of simultaneously sterilizing many media. The charged particle interactions with each medium is effectively reduced by consequence of the decrease in charged particle flux, and the overall reaction rate suffers a decrease. This complication can be compensated for by an appropriate correction of other gas parameters, such as the flow rate and pressure. 6.7 Nature of Microorganism Density and Surface Layer Formation Accessibility of the active species is dependent on the density of the microbiological matter formed on the surface of the medium and the manner in which this layer is laden upon the surface. This microbiological residue provides a physical barrier between the plasma species and the microorganisms. Microorganisms on a fresh medium exposed to a plasma will suffer spore mortality by way of free radical reactions at the most available regions of the microbiological layer to the charged particles. Due to the low penetrability of the free radicals, the microorganisms will be killed from the outermost layer to the innermost. The 12

14 mitigation of the sterilization process incurs wherein the outermost layer of microbiological film is killed, yet still present. The boundary provides for the active species to not have access to the deeper layers of microorganisms. This scenario accomplishes complete sterilization by aid of physical destruction of the organic matter (i.e. volatilization), but due to the mechanisms of sterilization being limited, the process is evenstill rendered less efficient. 6.8 Packaging The effects discussed in the previous section extend to physical layers that make up the material. The presence of so-called packaging inhibits the efficacy of sterilization in this scenario by the same means as for microbiological residue. The specific interest resides in that bacteria may form colonies within the material. The low penetrability of the plasma process is not effective in irradicating the embedded spores. Variation of gas parameters will not allow this problem to be sidestepped, and is an instance where plasma sterilization may only not be effective, but also not an option. 6.9 Geometrical Factors The geometry of the medium is known to affect the volatilization effectiveness of a plasma, a strong contributor to the overall efficacy of sterilization treatment. Geometries that are not amenable to normally incident active constituent interactions provide for a scenario where it may simply not be possible for sufficient species to contact the medium with sufficient time to cause efficient volatilization in all areas of the medium. Complicated geometries also present problems for even chemical reactions to take place. The predominant mechanism in this regime is the action of UV/VUV radiation emitted from the plasma with the material. It is then readily apparent that complex geometries inhibit sterilization mechanisms to operate, and may demand longer treatment times as a result. 13

15 7 Mechanics 7.1 Ionizing Radiation (IR) Ionizing Radiation (IR) is a blanket term used to describe any radiation capable of liberating the electron(s) of the constituent atoms in a medium. In sterilization applications, this includes charged particles, and UV/VUV photons. IR is a large contributor to the efficacy of the sterilization procedure, and interacts on the cellular, chemical, and nucleic levels of the microorganisms. When a generic form of IR imparts energy to the cell, the cell is envisaged to be able to behave in one of three manners (Figure 3). Figure 3 - Ionizing radiation causes damage to the cell, which in turn may under one of three possible reactions to the incident radiation. By consequence of the imparted damage to the cell, the cell will undergo various reaction pathways that are generalized in the above figure. Upon suffering an energy transfer from the incident IR, the cell initially senses that it has occurred. The cell then proceeds to respond in one of three ways: repair, mutation, or death. Depending on the complexity of the microorganism, several chemicals and organelles can work cohesively to recognize that damage has been done and then proceed to an attempt at repair. If the attempt at reparation fails, and some change is performed to the cell, a mutation is said to have occurred. Finally, if after cellular response, the cell is unable to recover, it results in death. This is the targeted pathway for sterilization processes. 14

16 7.2 UV/VUV Radiation The high penetrability of UV/VUV photons enables interactions with both the cellular chemical bonds of the microorganisms as well as the DNA. The effect of UV/VUV radiation on chemical bonds is indirect, in that the photons will cause ionization reactions to form free radicals which can then go onto cause free radical reactions to damage the cells. This is the predominant reaction that occurs with this radiation, occurring with a reaction rate coefficient of approximately 1.5 times that of direct radionucleic interaction. A discussion of this procedure is given in Section 7.3. The unique reaction among UV/VUV photons is the damage inflicted to the DNA. The photons can damage the DNA in a variety of manners, and the possibilities are illustrated in Figure 4. Figure 4 - UV/VUV radiation can damage DNA in several ways, the possible mechanisms are depicted above [adapted from Ref. 9]. It is possible for either base or strand damage to occur by consequence of energy deposition of the UV/VUV radiation. The photon may cause some general deterioration to the base, called base damage, or it may cleave the bonds effectively such that the base is no longer present after interaction which is dubbed abasic site damage. When two bases are damaged, and untypical bonds are formed among the bases, then an interstrand cross-link occurs. The radiation can also cause breaks in strands. This can be realized as a single strand 15

17 break, or if two single breaks occur then it is called a double strand break. All of these damages contribute to the deactivation and death of microorganisms through the consequential biophysical and biochemical changes that result from the interactions. Specifically, these changes cause DNA damage which give rise to one of four consequences that inflict death upon the cell: apoptosis, autophagy, necrosis, or mitotic catastrophe (See Glossary for more information). Research has shown that certain wavelengths of UV/VUV radiation are more effective than others. Although all microorganisms are different, the trend is generalizable in the respect that most microorganisms tend to respond well to specific wavelengths. In particular, 254 nm UV/VUV radiation has been shown to be especially effective (Ref. 6). Other authors, such as Tanino et al. (Ref. 13), and Moisan et al. (Ref. 10) have determined that the range of nm wavelengths is most amenable to volatilization, while Goldman (Ref. 4) has shown that a wavelength of 200 nm shows the most effectivness. An understanding of the UV/VUV radiation emitted from a plasma can then be used to assess the efficacy of its treatment. 7.3 Free Radicals Free radicals are compounds that have one free electron in their valence shell, rather than a pair, and are particularly reactive due to their enhanced electronegativity. Without forced ionization, these free radicals would react to form more stable molecules among the gas/air before any practical use could be extracted from them. Invoking their use in a plasma allows for them to be free radicals long enough to contact the surface of a material. Upon contacting the material, the free radicals react with either the cellular chemical bonds of the microorganisms, or their DNA to inflict sufficient biophysical and biochemical changes that deactivate and kill them. Atomic oxygen and hydroxide (OH) radicals are found to be particularly effective due to their versatility in covalent bonding with many different compounds, and their ease of generation. Safety is high due to any leakage of the plasma 16

18 simply cooling down and the free radicals will react with air to form unharmful products Radiochemical Reactions The primary reactant free radicals used in plasma sterilization is the hydroxyl radical, OH. The hydroxyl radical is an uncharged active species, that typically reacts to form more stable compounds within nanoseconds. Creating the free radical compounds in a plasma allows for the active species to reach the material surface before premature chemical reactions can take place. The free radical can then react with the microorganisms layering the surface of the material. Hydroxyl radicals target a variety of atomic sites; however, the predominant means of sterilization incur from hydrogen abstraction and the cleavage of double bonds. These two methods are illustrated in Figure 5. Figure 5 - The hydroxyl radical causes two dominant reactions in the sterilization process. In (a) hydrogen abstraction is depicted and is physically realized as the molecule extracting one electron (hydrogen) from the compound displayed to form water, H 2 O. In (b) the cleave of a double bond is shown. The double bond donates one electron to the reactive OH species. In both mechanisms, R denotes a generic organic group, and arrows denote the movement of valence electrons. In hydrogen abstraction, the hydroxyl molecule acquires stability via the extraction of electrons (i.e. hydrogen atoms) from an unsaturated hydrocarbon site. The result is the formation of water and a radical hydrocarbon site that is capable of perpetuating further 17

19 free radical reactions. The cleavage of double bonds owes itself to the high electronegativity of the free radical molecule. The hydroxyl molecules affinity for electrons is sufficient such that it can garner electrons from the pi bonds formed among alkenes (i.e. carbon-carbon double bonds). Due to the typically high concentration of alkenes within most cellular chemical bonds of microorganisms, this mechanism is the prevalent form of biochemical changes that take place within the microbes, and is a strong contributor to the sporicidal mortality of such layers. The effectiveness of the cleavage of double bonds may present a problem in the sterilization of specific media, particularly polymers. Polymers also typically contain many double bonds, and the abundance can lend itself towards actual damage of the material caused by the plasma Radionucleic Reactions Charged particles can react with the DNA of the microorganisms if they are able to penetrate the chromatin of the microbiological cells. The reactions differ from those of UV/VUV radiation in that free radicals are unable to directly cause damage to the DNA. Free radical interactions with DNA change the biochemical structure of the microorganisms. In cellular processes, the microorganisms have a defense mechanism to protect against DNA damage, and also for repair. This defense mechanism is realized as sulfhydryl compounds that act as radical scavengers. In order for damage to occur, the hydroxyl radical reaction with the DNA must have a secondary reaction that allows for a permanent change in the DNA molecule before repair can occur, this is accomplished through an oxygenation reaction. The process is depicted in Figure 6. 18

20 Figure 6 - The chemical process in which hydroxyl radicals can damage DNA is illustrated. This process is mitigated by competing reactions of sulfhydryl compound scavenging, and chemical repair processes possible. RSH denotes a generic sulfhydryl (SH) compound made up of organic group(s) (R). Before radical reaction can occur, the hydroxyl compound can be eliminated through the scavenging of a sulfhydryl compound. If this does not take place, then the hydroxyl radical is capable of creating a DNA radical compound upon interaction. The DNA radical compound can then be chemically repaired by a sulfhydryl compound. The last possibility is the interception of this reaction by DNA radical compound undergoing a subsequent oxygenation reaction. This last scenario results in DNA damage, and is the manner in which free radicals can act in the sterilization process on the nucleic level. 7.4 Volatilization Volatilization is the physical destruction of matter. This process takes place through chemical interactions of the charged particles (free radicals) and the microbiological residue and the medium. The reactions that proceed are vaporizing reactions whereby the positive ions of the plasma interact with the cellular chemical bonds to form gaseous compounds. The process is aided by the natural tendency of a sheath to form around the medium. In the case of RF plasmas, the electric field over a cycle is intuitively expected to be zero; however, due to the inherent capacitance of the medium, the electric field will be a net negative. This attracts the positive ions of the plasma and allows for volatilization to occur. 19

21 8 Methods 8.1 Dielectric Discharge Barrier (DBD) Dielectric discharge barrier (DBD) is a class of plasma generation characterized by high AC voltage induced gaseous discharges created at near atmospheric pressure. Since housing a low pressure or vacuum chamber for plasma generation would be both impractical and uneconomical, utilizing a DBD technique provides for an attractive solution. The name DBD owes itself to the utilization of dielectric layer(s) that make it possible for the plasma discharges to make contact with the electrodes. A representative diagram of a prototypic apparatus used for a DBD reactor is shown in Figure 7. Figure 7 - Representative schematic of a DBD reactor [Ref. 13]. The depiction above is of a parallel plate geometry, with a GND electrode consisting of an aluminum plate at ground and is taken from the work of Tanino et al (Ref. 13). AC voltage on the order of kv p p is applied to the mesh electrode. The voltage is typically in the khz range, with an input power of approximately W for practical application. Teflon is used as the dielectric. The medium to be sterilized is conventionally placed on a conveyor belt apparatus where it is allowed to be exposed to plasma discharges used for the sterilizing process (Figure 8). 20

22 Figure 8 - Representative schematic of a conveyor belt sterilization reactor [Ref. 1]. Adjustment of the belt speed allows for control of the sterilization, or exposure time. The sinusoidal shape of the input voltage constructs a scenario wherein discharges that extend from one electrode to the other are triggered across the length of the electrode. The character of these discharges are controllable in the respect that the input voltage has a direct effect on the discharge type. Lower input voltages give way to narrow pulses ( kv), called filaments at relatively regular spacing across the electrode. The spacing is reduced as the voltage is increased. Increasing the voltage further to intermediate values ( kv) gives rise to a pattern of alternating wide and narrow discharge bands, and even higher voltages (in excess of 1.2 kv) create a scenario where discharges occur at seeming uncorrelated positions and with such a frequency that the gap between the electrodes is essentially enveloped by the gaseous discharges (Ref. 2). Consequentially, it was also shown that varying the input voltage allows for a controlled adjustment of the electron temperature and density. Thus, high input voltages allow for a scenario in which maximum surface area is exposed to the plasma discharges, and are customarily chosen for sterilization treatments to maximize effectiveness Key Equations Equations relating to the DBD mechanisms involve an understanding of the breakdown conditions. The filament discharges can be modelled as a Paschen breakdown curve, where the breakdown voltage V is a function of the product of the pressure and the gap distance between the electrodes d. The discharges are created by a traditional Townsend avalanche 21

23 mechanism, wherein primary electrons trigger a cascade of secondary electrons from collisions within the gap. Because of the movement of electrons and positive ions with respect to the external electric field, charge separation occurs and a localized electric field is set up to oppose the external field as per Lenz s law. The end result of the presence of these electric fields is an overall reduction in the required breakdown voltage. This reduced breakdown voltage V B can be expressed as: V B = V P n e d where n is the density of electrons, d is the gap width between electrodes and V P is the corresponding paschen breakdown voltage of a gas at a given pressure and at the same gap width d. The above equation yields units of Townsend, Td, or V-cm 2. The actual avalanches that form the discharges can be approximately modelled from the typical Townsend equation: dn e n e = αdx where α is dubbed the Townsend coefficient and is for electric field strengths below the threshold energy, and tends to increases for electric field values greater than or equal to the threshold energy. the distance x is interpreted to be the track length of the electron, allowing for a one-dimensional treatment to be adopted without loss of generality. This equation is readily applied to Geiger-Mueller counters, but for DBD, in which large voltages are often applied, some changes may need to be made to the governing equation above to properly account for the physics Experimental Data Tanino examined the sterilization efficacy using DBD on a colony of Bacillus subtilus. The plasma was generated from gaseous hydrogen peroxide (H 2 O 2 ). H 2 O 2 is commonly used in chemical treatments, and is known to possess inherent germicidal characteristics. The choice 22

24 of this gas allowed for a coupling of sterilization mechanisms to take place. Furthermore, the use of this gas effectively lends itself to the formation of a large abundance of hydroxyl radicals (OH). The experimental parameters consisted of an input power of 230 W, frequency of 34.6 khz, with a gap separation of 8 x 10 6 colonies. The results are depicted in Figure 9. Figure 9 - Sterilization effect of an H 2 O 2 dielectric discharge on bacillus subtilus [Ref. 13]. The data shows that sterilization is accomplished within approximately four minutes. The high efficacy is explained by the coupling of four mechanisms: UV/VUV radiation, free radical interactions, volatilization, and the germicidal properties of the H 2 O 2 gas used in the processing. 8.2 Inductively Coupled Plasmas (ICP) Inductively Coupled Plasmas (ICP) are a class of plasmas characterized by their ignition through the use of coils that act as current carriers to induce magnetic fields. The experimental setup described is taken from Kylian et al. (Ref. 5), and the apparatus is depicted in Figure

25 Figure 10 - Experimental setup of the ICP apparatus [Ref. 5]. The apparatus is composed of two 8.5 cm radius coils situated at opposite ends, just outside of the chamber. The coils face towards the two dielectric windows of the chamber, and are connected in parallel to a common RF source of MHz. The arrangement allows for a magnetic field flux to be generated perpendicular to the substrate, and thereby an electric field that envelopes the chamber allowing for uniform effect on the contents of the chamber. The advantages of ICP is clear in that it involves no electrodes whatsoever. As indicated in the figure, a rouging pump needs to be affixed to the apparatus in order to generate the plasma at appreciable power levels of the RF supply. The pressure that need be attained is on the order of 1-3 Torr. This pressure limitation is a limitation of the ICP process in comparison with other atmospheric methods in regards to economics. The media to be sterilized are placed directly in the processing chamber via a load lock mechanism on the chamber door. In this setup, it was described that five samples were able to be simultaneously treated. If high temperatures a concern, the discharges can be adapted to pulses. In the study conducted by Kylián et al., a geobacillus stearothermophilus population of 2.5 x 10 2 spores was analyzed under varying concentrations of oxygen in a gas mixture of O 2 :N 2. Experiment revealed that the sterilization time required decreased with the increase of oxygen levels up to a certain point, but after that level has been reached, any more increase of oxygen relinquishes a decrease in sterilization time. Experimental data is given in Figure

26 Figure 11 - Experimental data by Kylián et al. shows the increase in sterilization efficacy with increased oxygen levels in the gas mixture on a population of geobacillus stearothermophilus [Ref 5]. The greatest efficiency is reached at levels of 95%-O 2 :5%-N 2. The heavy dependence on oxygen is indicative of the prevalence of volatilization reactions in the sterilization process. The study also allows for the exclusion of UV radiation as being a dominant process in sterilization. The data collected exhibits a quantitative shift of the sterilization efficiency with only the adjustment of the oxygen concentration, without regard to the intensity or wavelength of the UV radiation (Figure 12). Figure 12 - Experimental data by Kylián et al. shows the probability of sterilization changes only with the change in concentration, and is independent of the UV radiation present in the plasma [Ref. 5]. In all, the studies by Kylián reveal that the spores can be entirely eliminated on the order of minutes depending on the concentration of the gas used. Specifically, with the maximum efficiency reached in this experiment being only an exposure time of 4.35 minutes be needed. He further concluded that although UV radiation and free radical interactions may contribute, they are not the predominant mechanism in the sterilization with the use of inductively coupled plasmas. 8.3 Atmospheric Pressure Plasma Jet (APPJ) Cold plasma discharges are able to be generated in the apparatus known as the atmospheric pressure plasma jet (APPJ). The device operates on the basis of an RF-coupled 25

27 capacitive discharge formed in the flow-gas, that emits a neutral cold effluent containing a large abundance of free radicals with corresponding UV/VUV radiation. The use of an RF source constructs a scenario wherein the plasma can be generated at atmospheric pressure. The combination of the cold temperature, atmospheric pressure, radiation, and free radicals logically extends its possible applications to sterilization. The work of Cheng et al. studied the applications of APPJ to sterilization on populations of Escherichia coli (E. coli), and Bacillus subtilis (although the colony sizes were not given in the article). The apparatus used in these experiments is shown in Figure 13. Figure 13 - An APPJ is schematically shown. The insulators act as dielectrics, an AC source supplies high voltage, and gas is allowed to flow through the top opening. The plasma jet is formed in the tube and exits in the bottom [Ref. 1]. The insulators depicted act as dielectrics, and the an AC high voltage source supplies power for the plasma generation. The gas is allowed to flow through an aperture in the top of the apparatus, the plasma is formed and collimated in the form of a jet as it exits by virtue of the geometry of the jet device. A steady, homogenous plasma jet is able to be maintained at voltages of kv, and RF frequencies of 6-20 khz. The nozzle size used in the presented data is 20 mm in diameter. The scenario allows for a plasma jet on the order of o C. An argon feed gas is used, and the plasma formed is thusly a purely Ar plasma; however, as the plasma exits it interacts with the air molecule whereby hydroxyl radicals, atomic oxygen, and ozone are created via electron impact dissociation reactions (formation 26

28 of ozone requires a subsequent reaction of atomic oxygen and O 2 ). Cheng coupled the sterilization measurements with free radical/molecular formation diagnostics to confirm the presence of these molecules. Subsequent infrared (IR) spectroscopy analysis revealed characteristic peaks indicative of the postulated formation of the previously described molecules (Figure 14). Figure 14 - IR analysis of the emitted plasma evidences concentrations of atomic oxygen, O 2, hydroxide (OH), and N 2. The presence of these molecules is indicative of air contaminating the plasma jet [Ref. 1]. Furthermore, the effect of ozone in contrast to that of charged particles was able to be examined in this study. Further IR spectroscopy analysis showed that the free radical concentration sharply tapered off at a distance of 1-2 cm from the exit nozzle. This is due to the high reactivity of these molecules. The free radicals interact with air to form primarily ozone, and thus the plasma jet at and beyond this point consists of mainly Ar and O 3. By placing the bacteria sample both in and out of range of the free radical region, the effect of ozone was able to be determined by the analysis of basic survival curves. The effect of the Ar-O 3 plasma is depicted in Figure

29 Figure 15 - The Ar-O 3 plasma shows to have slow sterilization effectiveness. The population of E. coli plotted decays sharply at first, but essentially a domain of little or no decrease around S = 20%. The quantity S denotes the survival fraction of the colony [Ref. 1]. The results indicate that a plasma consisting of argon and ozone decrease the population significantly within the few minutes; however, further decrease is severely mitigated and prolonged exposure time does little to reduce the population. The sterilization efficacy was also investigated with the incorporation of charged particles. The results in this scenario revealed complete spore mortality (Figure 16). Figure 16 - The effect of charged particles is complete sterilization of the spore population. In (a) the effect on E. coli is shown, and in (b) the effect on Bacillus subtilis is given [Ref. 1]. The necessary exposure time for sterilization was different in both cases, and is reflective of different microorganisms responding differently to a given sterilization mechanism due to their differences in microstructure. In both cases, sterilization was able to be attained within minutes, and in the case of E. coli, sterilization was achieved in approximately 5 minutes. It 28

30 was observed that free radical reactions are a strong contributor to the sterilization process in this methodology. 8.4 Microwave (MW) Plasmas Microwave plasmas (MW) are created without the use of electrodes. Plasma ignition is achieved by virtue of a proper matching between the microwave frequency and the electron cyclotron resonance frequency. Park et al., used the following setup to generate an argon plasma, given in Figure 17. Figure 17 - The experimental setup of a typical MW plasma generation apparatus is shown. Argon gas flows through the inlet, and interacts with the incoming microwaves as fed through the waveguide. The plasma is produced and allowed to exit [Ref. 11]. The prototypic apparatus shown above is characteristic of MW plasma generating systems. Argon gas is depicted as flowing through an inlet at the end of a waveguide which serves as a guiding path for the microwaves emitted from the magnetron. Upon interaction with the microwaves, an argon plasma is ignited and exits below. In the setup of Park et al., a 1 kw magnetron power supply was used, a WR-284 copper waveguide with cross-sectional dimensions of 72 mm x 34 mm. The waveguide was tapered so as to increase the electric field strength and to minimize reflected waves in the vicinity of the gas region. The result was a 2.45 GHz argon plasma. Four bacterial strains were analyzed in the experiment: Bacillus subtilus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium. Additionally, two fungal strains were subjected to sterilization treatment: Aspergillus niger, and Penicillium citrinum. The colony populations were not given in the published study 29

31 by Park; however, the general procedure was described to be bacterial and fungal colonies grown on petri dish surfaces. The results of the experiment are plotted in Figure 18. Figure 18 - Bacterial and fungal populations are plotted with respect to exposure time to the MW plasma. All sources were shown to be sterilized within 20 seconds [Ref. 11]. Of interest in the above data is the efficiency of sterilization for E. coli and bacillus subtilus. E coli. was sterilized within 5 seconds, while the population of bacillus subtilus was irradicated within 10 seconds. In all, the colonies were completely sterilized in less than 20 seconds. The high efficacy of this treatment can be attributed to the synergistic mechanisms of plasma sterilization with one addition. The additional factor is that microwave plasmas are capable of being generated at a much larger density in contrast to any RF plasma. MW plasmas can be on the order of cm 3. The large density implies a large availability of the reactive species, a greater intensity of UV/VUV photons, and the greater amount of reactive species gives rise to necessarily more bombardments of the medium and hence a greater rate of volatilization. The increase in density has the net effect of amplifying the synergistic mechanisms that make up plasma sterilization. Thus, MW plasmas are known to have the highest rate of sterilization of all methodologies, and is typically on the order of seconds, in contrast to other plasma sterilization methods which hold necessary exposure times of minutes. 30

32 9 Conclusions & Future Prospects Plasma sterilization reduces sterilization time to minutes, and even seconds. The efficacy of plasma sterilization has been shown through research and actual application in the industry. It presents an effective alternative to conventional means such as heat, chemical, or radiation sterilization. Plasma sterilization provides for high efficacy, unmatched medium preservation, and does not introduce toxicity to the medium. The main disadvantage of this method; however, is its low penetrability. In the presence of packaging, plasma sterilization is significantly impeded, and may be rendered completely ineffective for sterilization purposes. The geometry of the medium also presents problems, as complicated shapes can inhibit the reaction rates of the sterilization process by virtue of the lack of regional accessibility of the plasma constituents/radiation. Finally, when used for bulk sterilization, a loading effect is introduced as a consequence of the availability of the reactive species of the plasmas. Thus, plasma sterilization can be used by and large in specific scenarios and is not as versatile as other methods of sterilization. The research of the various methods of plasma sterilization revealed sterilization times on the order of minutes, or even seconds (in the case of MW plasmas). This time interval is substantially shorter than times demanded through conventional means, which can be on the order of hours for total treatment. Although all the data collected from the different research held different scenarios in population sizes, gas mixture, etc., the data is generalizable if a comparison is done without ample specificity. Bacillus subtilus was used in the experimental procedures for the dielectric barrier discharge (DBD), atmospheric pressure plasma jet (APPJ), and microwave (MW) plasma sterilization methods. Comparing the three methods based upon this bacterial strain, MW plasmas hold the greatest efficacy of sterilization. As discussed in Section 5, this is attributed to the synergy of sterilization mechanisms in this method being amplified by the high density of the generated plasma. The MW plasma achieved complete spore mortality within 10 seconds. This is significantly shorter than the treatment times demanded by the DBD methodology which demanded a sterilization time 31