Design of testing chamber
The plasma generated for this work was done using patented Compact Portable Plasma Reactors (CPPRs) provided by SurfPlasma. The CPPR is a self-contained plasma device for decontamination of air and surfaces withing enclosed volumes38. It uses SDBD from ambient air to produce reactive oxygen and nitrogen species. The system itself is approximately 1 inch cube in size and can operate with only an electrical power supply. A schematic diagram of the system can be seen in Fig. 3 below. For the purposes of this research, a testing chamber which includes the CPPR system was needed.
CPPR schematic. The specific components and description can be seen in the associated patent38.
An APS testing chamber was built to accommodate the goals of this study. The design goal of the chamber was to create a leak-free 30 L chamber capable of hosting up to 6 Compact Portable Plasma Reactors (CPPRs) as seen in Fig. 4. The material chosen for these prototypes was Acrylic as it is easy to modify and allows the user to clearly see inside the chamber during testing. The chamber was constructed using a prebuilt acrylic chamber with hinged lid without any outlet holes except those needed for wiring and ozone measurement. The chamber was designed such that the hinged lid opened sideways, allowing easy placement of testing coupons within the chamber. This sideways lid is held closed using a series of magnets located along the outer edge of the chamber. The use of magnets allowed the chamber to be held firmly shut without the need for drilling of holes. Three pairs of magnets were placed along the bottom, left side, and top of the chamber. A high number of magnets were chosen to ensure proper sealing of the chamber along each side. The design of the chamber was further iterated upon as the project moved forward, with the addition of increased electronics and an ozone decomposition system. However, any further chamber designed retained the 1 cubic foot chamber size, ensuring any results obtained during testing will be applicable to all future designs of this chamber size.
APS Testing chamber (left) and CPPR’s located inside chamber (right).
This chamber was used for the decontamination tests performed during the study. For the other testing performed, a separate chamber was developed which included many of the desired final electrical systems. To provide ease of use to the user of the APS system, a smart control system was developed. With the goal of a user friendly and user safe system, the following attributes were added to the system:
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1.Display – A display for the system gives user control of cycle time, time elapsed in cycle, and ozone levels inside the chamber.
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3.User Control –The APS front panel has a set of push buttons to allow users to start and stop the system and choose the decontamination cycle time. These are the minimum required options for user input.
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4.Sensors – A ozone sensor has been employed within the APS chamber that can tell the user whether the chamber is safe to open (i.e. the ozone inside the chamber has reached <0.1 ppm). Additional sensors, such as temperature and humidity, may be added in the future.
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5.Connection to the ODM – The smart system automatically activates the ODM system once the decontamination cycle ends. The system also activates the interior fans for airflow and turns on the heating element of the ODM. The system communicates with the ozone sensor to stay active until ozone levels are safe.
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6.Safety Features – To ensure user safety, an additional failsafe feature has been introduced such that plasma will not turn on if the chamber door is open.
The latest APS design used for this paper can be seen in Fig. 5 below. It has all of the features described above.
Final APS testing chamber.
Decontamination testing
Experiments were performed to determine decontamination efficacy in three locations inside the internal volume using coupons against three selected pathogens. The following test pathogens were selected:
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1.Deinococcus radiodurans (ATCC 13939): Deinococcus radiodurans (ex Raj et al.) Brooks and Murray, Type strain, BSL 1 level: is gram-positive bacteria, and it is vegetative, easily cultured, and nonpathogenic. It is some of the most radiation-resistant organisms discovered. The radiation-resistant bacterium Deinococcus radiodurans withstands harsh environmental conditions present in outer space because of metabolic stress response, but it is very sensitive bacteria on Low Earth – the orbit induces molecular rearrangement mechanisms. Due to this ability, this species is of interest in evaluating decontamination technologies for planetary protection and the development of new sterilization techniques for future space missions39.
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2.Geobacillus stearothermophilus (ATCC 7953): Geobacillus stearothermophilus (Donk) Nazina et al., BSL 1 level: is a rod-shaped, Gram-positive bacterium, and a member of the division Firmicutes, a thermophilic, aerobic bacterium, which produces heat-resistant spores. It is known as the most heat-resistant organism. G. stearothermophilus in spore form is used as a challenge microorganism to inoculate paper or stainless-steel carriers, known as Bio-indicators (BIs). These BIs can be used to establish sterilization efficacy of different decontamination technologies e.g., steam sterilization (autoclaves); gas sterilizers (hydrogen peroxide).
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3.Aspergillus fumigatus Fungi (ATCC 1022): Aspergillus fumigatus Fresenius, Type strain, BSL 2 level: This is a species of fungus in the genus Aspergillus and is one of the most common Aspergillus species to cause disease in individuals with an immunodeficiency. Aspergillus fumigatus primarily causes invasive infection in the lung and represents a major cause of morbidity and mortality in these individuals. Since this fungus is widespread, countless conidia are released from phialides and dispersed in the air every day, contaminating the environment. Thus, this fungus is chosen for establishing decontamination efficacy of APS prototype against fungus.
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Stocks of Geobacillus stearothermophilus, Deinococcus radiodurans, and Aspergillus fumigatus strains were stored at -80°C in Nutrient broth, Nutrient broth + 1% Glucose and Czapek broth, respectively, with 30% glycerol. Frozen stocks were grown: Geobacillus stearothermophilus in Nutrient broth overnight at 55°C, Deinococcus radiodurans in Nutrient broth + 1% Glucose overnight at 30°C, and Aspergillus fumigatus in Czapek broth 72 hours at 30°C. A Spectrophotometer was used for estimating the concentration of bacteria in the fresh broth culture, followed by dilution, if necessary, to get approximately 5×107 CFU (colony forming units)/ml. 10 μl of these broth cultures was used to inoculate coupons with 4 to 5 logs. Aspergillus fumigatus presence was confirmed by electron microscope. All experiments were performed in a BSL1 and BSL2 lab spaces at the Emerging Pathogens Institute (EPI) at the University of Florida according to the required protocols for the bacterial and fungal strains procured for the project. Statistical analyses were conducted using triplicate individual experiments with at least 3 replicates within each condition/strain for each experiment. The results of microbiological tests were transformed into log values for statistical analysis18.
Pre-processing of coupons and APS test chamber: Before each experiment, the test coupons were autoclaved at 121°C to ensure proper sterilization. Before each experiment, the APS test chamber was wiped with 70% isopropyl alcohol to avoid external contamination. To ensure each experiment started at the same level of ozone concentration in the chamber (ambient levels), the chamber was left open inside a ducted BYPASS fume hood – Phoenix Controls Corporation – 100 lfm for at least 10 minutes before the experiment started18.
Post-processing of coupons and APS test chamber: After exposure experiments, the coupons were mixed thoroughly in 15 ml PBS solution using a Fisher Scientific ® Mini Vortexer lab mixer. For half of the coupons for D. radiodurans and non-spore G. stearothermophilus, 100ul of this mixture (for exposed coupons) or its dilution (for unexposed coupons – Control) was plated on agar plates (with appropriate agar for each bacterium) followed by incubation. For the remaining coupons for those bacteria as well as all of the spore G. stearothermophilus and A. fumigatus coupons, after mixing they were placed in appropriate broth culture inside of falcon tubes to check for growth over a 24-hour to 72-hour period (depending on each pathogen). For the agar plates, plate counts were obtained to quantify the bacterial colonies present in the coupons. For the broth samples, ocular density (OD) and visual inspection of growth inside the broth when compared to control were used to determine when complete killing was achieved. All post-processing was conducted in a Biological Safety Cabinet (BSC) Class II, Type A2 to avoid external contamination and maintain safety protocols18.
Ozone exposure experiments: Ozone exposure for decontamination experiments is quantified by the ozone concentration C integrated over time t. For the measurement of the ozone concentration C (in Parts Per Million or PPM), a 2B Technologies Model 106-L Ozone monitor was used. To calculate the ozone exposure by CT values (concentration x time), the following equation was used.
$$text{CT value}=underset{0}{overset{t}{int }}Cdtcong {sum }_{0}^{n}{C}_{i}{t}_{s}$$
where Ci refers to the i-th sample reading given by the ozone monitor, ts refers to the sampling time of the ozone monitor and n is the total number of samples collected during a specific exposure time. For these experiments, the lowest sampling time of the ozone monitor was chosen (ts = 10 sec).
Each exposure experiment involved 12 coupons of one material inoculated with 10 μl of bacteria or fungi culture containing approximately 107 CFUs/ml of one type of bacteria or fungi. Inoculation volume of 10 μl was chosen to give approximately 105 CFUs/coupon. 9 coupons were placed in the APS at the 3 measurement points (3 per measurement point) and exposed for selected times18. The remaining 3 coupons were placed outside the chamber for the same times to act as controls. After the exposure periods, for D. radiodurans and non-spore G. stearothermophilus 8 coupons (2 from each measurement point and 2 controls) were post processed to obtain CFUs/ coupon. The following equation was used to calculate CFUs/coupon:
$$text{CFUs per coupon}=frac{text{CFUs}}{ml}*text{V}1=text{D}x*{10}^{(x*text{V}1)}$$
where V1 is the volume of PBS used to mix coupons in post-processing in milliliters and Dx = CFUs counted in the xth dilution plate. The reduction in microbial colonies obtained per coupon at each measurement point was determined from the difference in CFUs/coupon of the exposed and control (unexposed) coupons. For the remaining 4 coupons (1 from each measurement point and 1 control) for D. radiodurans and non-spore G. Stearothermphilus as well as all coupons for spore G. stearothermophilus and A. fumigatus, they were placed in broth and allowed to grow for up to 72 hours. Ocular density (OD) was measured and compared to control to determine whether growth was detected. A total of 38 iterative exposure experiments were performed with three test organisms and 1 selected material – Aluminum. The goal of the iterative experiments was to find the minimum necessary exposure time for complete killing.
D. radiodurans testing
Initial testing began with D. radiodurans. Multiple tests were performed in succession, with time in between each experiment to allow the ozone inside the camber to reach <0.1 PPM. To determine the results of each test, at least 24 hours is needed for each pathogen. Thus, results from the first test of a day cannot be applied to further tests in that day as it is not known if decontamination was achieved. Results determined from one day can be applied to the next testing date.
For exposure, the first number corresponds to the time the plasma is active (on) while the second number corresponds to the time when the plasma was off but the camber remained closed (off). For example, in experiment no. 1, the chamber was sealed after inoculation. Then, the plasma was activated for 25 min (on) then deactivated. The chamber remained sealed for an additional 5 minutes (off). After these five minutes, the chamber was opened and the coupons were removed. The purpose of this “off” time period was to allow some ozone decomposition inside the chamber before opening to help protect the users.
An additional validation step has been performed – the Optical Density (OD600) of the falcon tube in which the pathogen was prepared, was measured before each experiment on a spectrophotometer, as seen in the OD column. This was done for Cell Culture Concentration calculation, that the pathogen grew effectively, with a value greater than 0.1 indicating the presence of bacteria. After exposure and plating of the post- decontamination coupon, the coupon was placed in a falcon tube with the growth bacteria and the OD was measured after 24 hours, as seen in column “Ex br OD after 24 H”. This test was used to confirm no bacteria was remaining on the coupon, with an OD less than 0.1 indicating no remaining bacteria.
The final column “Control log/coupon” represents the CFU detected on the control coupons in log 10 format. If the colony for the exposed coupon is 0, then this is the total log reduction of CFU from exposure. To account for potential uncertainty in the testing results, three sets of coupons were tested for each test, which can be seen in the tables below. While not exhaustive, this helps to validate the results obtained.
For these tests, the inoculated coupons were aluminum coupons. The aluminum coupons were placed in the PBS and growth mediums after testing. Each set of tables represents different day tests were performed. This allowed the results from the previous tests to be analyzed before the next set of tests. For the experimental results presented in this report, D. radiodurans is abbreviated as Dr and G. stearothermophilus is abbreviated as Geo.
The exposure times for each experiment can be seen in Table 1. Agar plates and broth results can be seen in Figs. 6 and 7, respectively.
Agar plates for experiments 5. The top row are the exposed coupons, the bottom row are the control coupons.
Broth testing setup for experiment 5 (left) and OD testing setup for broth (right).
G. stearothermophilus testing
Initial G. stearothermophilus began with the non-spore samples. Multiple tests were performed in succession. Experiments 7 through 26 did not show significant decontamination results due to low exposure times or low ozone production inside the testing chamber. The results from these tests are included in the supplemental materials. Continued testing was performed for greater exposure times and higher ozone production to try to achieve complete killing. Table 2 shows the details and results the tests in which 5-log reduction in CFU was achieved, experiments 27 and 28. Figure 8 shows the results for these two experiments.
Agar plates for experiments 27 and 28. The left column is for negative control (control to demonstrate no outside bacteria from setup). The next 2 columns are for the control coupons for no dilution and one dilution, respectively. The right two columns are for the exposed coupons for no dilution and one dilution, respectively.
G. stearothermophilus spore testing
G. stearothermophilus spores were tested. The testing procedure differs slightly by simply placing the spores in the chamber on the coupons, no need for growth before exposure. The procedure after exposure remains the same. The results from the experiment are not as easily quantifiable as with the traditional G. stearothermophilus testing, allowing only binary “growth” or “no growth” based on broth culture analysis. Initially testing began with 45 minutes of exposure time (experiments 29 and 30), 40 minutes with CPPRs on and 5 minutes with CPPRs off, as it was expected that the spores would be harder to kill. Testing was also performed for 35 min on and 5 min off to match the time period for the period for complete killing of non-spore G. stearothermophilus. Table 3 shows the testing results. The results from the broth cultures for the 35 min on plus 5 min off test can be seen in Fig. 9.
G. stearothermophilus spores testing results for 35 min on plus 5 min off. The left picture represents the vials after 24 h, the right after 48 h. The left vial is for control. The next 3 vials are for the exposed coupons. The right vial is for the negative control (control to demonstrate no outside bacteria from setup).
A. fumigatus testing
Because A. fumigatus is a fungus and not a bacterium, the CFU cannot be cataloged and thus a log reduction cannot be determined. Instead, whether growth was seen was used as an indication for killing of A. fumigatus. To confirm proper growth of Aspergillus fumigatus, microscopic images were taken to confirm growth. These images can be seen in fig. 10 below.
Microscopic pictures of A. fumigatus used in experiment.
The testing periods can be seen in table 4 below. Complete killing was achieved in 40 minutes total (35 min on + 5 min off). The results from one of these experiments, experiment 34, can be seen in Fig. 11.
Results from experiment 34. The left represents agar plate results, the right represents broth results.
Ozone decomposition testing
Testing of an Ozone Decomposition Module (ODM) for integration into the APS system was performed during this study. This ODM is necessary to allow safe usage of the system by rapidly eliminating ozone inside the APS chamber post- decontamination. Testing of the ozone in the chamber was performed using a 2B Technologies Model 106-L Ozone Monitor. Testing was initially done to determine the natural ozone decay rate inside the 30 L chamber. The natural decay rate to reach levels of less than 0.1 PPM (OSHA safe level) would be over an hour. To test the ODM, a schematic for the system was developed and is described below and can be seen in Fig. 12.
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1.
Casing
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2.
Inlet port(s)
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3.
Outlet port(s)
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4.
Joule heating element
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5.
Optional ozone sensor
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6.
Powering circuit
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7.
Insulation
Schematic details for the Ozone Decomposition Module (ODM).
For the heating element, an off-the-shelf Joule heating coil is used. The heating coil is fabricated inside of a CPVC pipe located on the right side of the APS chamber. This pipe is sealed, and the inlet and exit can be seen inside the chamber. As the ODM activates, a fan located at the inlet (top) of the APS chamber creates airflow into the ODM pipe. This passes the chamber air through the heating coil, heating it locally above 300°C and causing rapid ozone decomposition. This heated air then flows back into the bottom of the chamber through the outlet. This creates a recirculating flow inside the chamber, allowing the ozonated air to continually be heated and decomposed until the ozone inside the chamber reaches the OSHA safe limit (<0.1 PPM). The period necessary to reach this limit depends on the initial ozone concentration. The ODM setup can be seen in Fig. 13 and the results can be seen in the results section (Fig. 2).
ODM connected to the APS testing chamber interior (left) and exterior (right).
Visual material compatibility testing
Testing was done on three different material types:
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1.Stainless Steel 316 (purchased from McMaster-Carr)
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2.Teflon PTFE (purchased from McMaster-Carr)
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3.FR-4 PCB board (purchased from Advanced Circuits)
To determine visual material degradation, each material was analyzed under a Scanning Electron Microscope (SEM) before and after one hour of exposure time inside the APS test chamber. One hour was chosen due to it being 1.5 times greater than the determined maximum time necessary for decontamination of the tested pathogens (40 minutes). Exposure was only performed one time for these materials. Energy Dispersive Spectroscopy (EDS) was also performed to compare elemental structure before and after exposure. Testing was done at the Nanoscale Research Facility (NRF) at the University of Florida (UF) through a service contract. The specific Scanning Electron Microscope is the Hitachi S-3000, as seen in Fig. 14, and was operated by a trained NRF Engineer.
Hitachi S-3000 Scanning Electron Microscope.
5 samples of both stainless steel and Teflon PTFE were analyzed, and 3 samples of the FR-4 PCB board were analyzed. The number of samples was chosen to help reduce uncertainty in the results. The discussion below focuses on the first sample of each material type, but the results were consistent across all results. Furthermore, the EDS analysis includes an error percentage for each element detected. Any percentage below this error percentage indicates lack of reliability in the reading, which is discussed below. the The samples can be seen in Fig. 15. For both the Teflon and PCB board, an Au-Pd coating was applied to one side so it could be properly analyzed by the SEM. This may cause some of these elements to appear on the EDS readings.
Samples of Stainless steel (top), Teflon PTFE (middle), and FR-4 PCB (bottom) used in material compatibility analysis.
A comparison of the before and after analysis for the first sample of each material type can be seen in Figs. 16, 17, and 18 respectively. Further samples of each type provided similar results. Each comparison details the visual image of each sample at a 100-micrometer scale. Each comparison is the same sample, but the exact analysis area differs slightly between before and after analysis due to difficulty in achieving the exact same location. Also included in each comparison is an EDS analysis. This analysis was also performed by the Hitachi S-3000 concurrently with the SEM analysis. One thing to note regarding the EDS analysis is the large error percentage in certain elements. This is discussed on an individual basis for each sample. Furthermore, as stated by the NRF Engineer who performed the testing, “sometimes the (EDS) tool likes to overfit the data and match more elements than should be present”, which is why some samples have discrepancies between before and after.
Stainless Steel SEM results for sample #1 before (top left) and after (top right) exposure. EDS results for sample #1 before (middle table) and after (bottom table) exposure. For EDS weight percentage values less than the error percentage, the results are generally unreliable.
Teflon PTFE SEM results for sample #1 before (top left) and after (top right) exposure. EDS results for sample #1 before (middle table) and after (bottom table) exposure. For EDS weight percentage values less than the error percentage, the results are generally unreliable.
FR-4 PCB SEM results for sample #1 before (top left) and after (top right) exposure. EDS results for sample #1 selected area 1 before (first table) and after (third table) exposure. EDS results for sample #1 selected area 2 before (second table) and after (fourth table) exposure.
For stainless steel, the weight percentage of Fe seemed to increase by 4% after exposure, and the percentage of Na by about 9%. However, the error percentage for both of these measurements is ~4% and ~12%, respectively, meaning this change is likely due to errors in measurement. There is no significant introduction of oxygen into the material.
For Teflon PTFE, the weight percentage of C and F seemed to increase by 4% and decrease by 3%, respectively, after exposure. However, this is within the error percentage for both elements, meaning this change is likely due to errors in measurement. There is no significant introduction of oxygen into the material.
For EDS weight percentage values less than the error percentage, the results are generally unreliable. The locations measured before and after are slightly different due to the nature of measurement on the SEM. However, this should not significantly affect the analysis. Likewise, the PCB board is a composite of FR material and metal connections. One section from each material type is seen in the EDS breakdown.
For FR-4 PCB, the weight percentage after exposure seems to remove oxygen from the PCB. However, this weight percentage is below the error percentage, meaning this change is likely due to errors in measurement. For the second area, any perceived changes to the elemental structure are within the error percentage, meaning this change is likely due to errors in measurement.
The results from these SEM visual analysis tests indicate initial support that the chosen materials have no visual material degradation exposure after a one-time exposure in the APS system. Further testing, including mechanical tests, needs to be done to ensure complete material compatibility and demonstration that the samples have not deteriorated. In addition, the scale of 100 microns used for these tests may not be appropriate to fully demonstrate the effect of plasma treatments on these materials. Further testing should be performed at a smaller scale using other appropriate tests such as Atomic Force Microscopy.
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- Source: https://www.nature.com/articles/s41598-024-82556-8