Plasma-generated reactive water mist for disinfection of N95 respirators loaded with MS2 and T4 bacteriophage viruses

Plasma system

The prototype was made from a 53 L electric dishwasher (Fig. 1). A 20 kHz 3000 V surface DBD was installed outside the dishwasher and connected to the chamber inside. A 750ml container of water was attached to the side of the dishwasher and was connected to the ultrasonic nebulizer which was connected to the DBD reactor.

Figure 1

Diagram of the plasma system. It consists of three chambers: a large open space of 53 L for PPE, the plasma chamber (No. 1 to 6) and the heating chamber (No. 7 to 9): 1. A fan forcing the air in the plasma chamber. 2. Exit the plasma chamber where droplets, mixed with plasma-generated reactive oxygen and nitrogen species (RONS), are introduced into the area with the PPE. 3. Plasma is generated with a 20 kHz, 3000 V dielectric barrier discharge. 4. An ultrasonic nebulizer is used to generate micro-droplets of water with an average diameter of approximately 5 µm. 5. The micro water droplets form a dense mist which is produced immediately after the plasma zone and mixed with the RONS. 6. Each cycle consumes 1ml of DI water or other solutions and our current prototype has a 750ml container. 7. For the heated air cycle, air (containing residual RONS and moisture) is drawn from the chamber along with the PPE. 8. Air-RONS-moisture is heated to 50°C to aid in removal of residual reactive chemistry and drying of PPE. 9. Warm air is forced into the room with the PPE by a fan.

A predefined program was installed in the system via Arduino. The program was used to control the running time of the DBD reactor, ventilator, nebulizer and drying system. In this study, we defined the disinfection process in 3 steps for a total of 20 min. The first stage was set to 5 min of DBD reactor, fan and nebulizer on; the second stage was a 5 min DBD reactor and ventilator running without the nebulizer; and only the drying system is turned on during the third stage for 10 min. During the drying cycle, the temperature at the exit of the heating zone was raised to 50°C by circulating air using a standard computer fan at approximately 30 CFM over a coil of resistive heating. The coil voltage was monitored by an Arduino microcontroller and ramped up or down to maintain the chamber temperature at the preset 50°C.


The working solution of two bacteriophage viruses, MS2 (ATCC 15597-B1) and T4 (ATCC 11303-B4) was stored at 4°C before testing. Stock concentration was quantified by plating appropriate dilutions by the overlay plate assay method on LB Agar22. Briefly, underlay agar plates were made from LB agar (MP Biomedicals). The upper agar was made from the same type of medium but with a lower agar concentration. The mixture of 100 μL MS2 or T4 stock, 100 μL host (Escherichia coli MG1655) and 5 ml of molten top agar was smeared on top of the underlying agar. Once the top agar solidified, the plates were incubated at 37°C overnight.

Decontamination test

Two commonly used N95 respirators (3M 1804 and 3M 1860) were tested in this study. Two sides of 3M 1860 were tested as they were made of different materials. N95 respirators were cut into 1 × 6 cm coupons. 100 μL of virus stock was inoculated on each coupon. The coupons were left in a laminar flow safety cabinet until dry (about 3 h) before processing.

Then, the inoculated coupons were processed by our system and analyzed for microbial inactivation. Three to six coupon replicas were processed for each condition. Hydrogen peroxide (7.8% and 10%) or deionized (DI) water were used to generate the fog. Different concentrations of hydrogen peroxide were diluted from 35% hydrogen peroxide (Arkema). A negative control was also performed in each experiment.

Phage recovery from coupons and quantification

After treatment, T4 or MS2 was recovered from face mask materials by shaking or vortexing in 10 or 15 mL of sterile phosphate-buffered saline (PBS). The N95 outer layer 1860 and coupon 1804 were extracted with 15 ml of PBS; The inner N95 1860 layer was extracted with 10 mL of PBS. Agitation for 20 min at 200 rpm provided higher recovery efficiency than vortexing for 1 min, so agitation was used in most experiments in this study (Supplementary Material Efficiency recovery of MS2 and T4 from N95 respirator material).

A double agar overlay plate assay was used to quantify viable virus recovered in terms of plaque forming units (PFU). No processing controls were also performed to account for any non-processing associated loss of viable virus (PFU) in our system.

The decontamination efficiency was determined using the following equation, where Ntreated, medium is the average of the PFU/coupon of the coupons traded, and Nuntreated, medium is the average PFU/coupon of untraded coupons.

$${text{Decontamination efficiency }} = {text{ log }}left( {{text{N}}_{{{text{untreated}},{text{avg}}} } / {text{N}}_{{{text{treaty}},{text{avg}}}} } right)$$


Plasma Activated Fog Chemical Properties and Chamber Conditions

The chemical properties of PAM were determined by measuring pH (Hydrion, range 0–3 and range 0–6), NO3 (Quantofix, 10-500 mg/L range), NO2 (Quantofix, range 1–80 mg/L) and total peroxide (Quantofix, range 50–1000 ppm) via commercial test strips. The test strips were placed in the middle of our system’s chamber during a decontamination cycle. After the cycle was completed, the test strips were read by comparing the color chart provided by the manufacturers. Condensed water was collected by covering aluminum foil at the outlet of the plasma chamber for one treatment cycle (#2 in Fig. 1), then measured pH, NO3 and no2 through test strips.

Temperature and humidity were measured by a data logger (Elitech GSP-6G). The probe was held in the middle of the chamber during operation. Ozone was measured using narrow wavelength UV absorption of 254 nm (Model 106-M Ozone Monitor, 2B Technologies, Boulder).

Mask Filtration Efficiency Test

The filtration efficiency of N95 respirators was performed before and after different numbers of treatment cycles (1 or 20 cycles) in our system. The particles were generated in a 400 L stainless steel chamber, and by counting the particle concentrations alternately between the sampled air (I) directly from the chamber and (ii) through the filter media. The removal efficiency of N95 respirators was calculated using Equation. (1):

$$eta = 1 – frac{{C_{{{text{filter}}}} }}{{C_{{{text{room}}}} }}$$


where η is the removal efficiency of the filter; and VSbedroom and VSfiltered (#/cm3) are the particle concentrations in the chamber air and through the filter media, respectively.

Particles smaller than ~1 μm were generated using aerosolized sodium chloride (NaCl) via an aerosol generation system composed of these elements in series: an atomizer with a solution of DI water and chloride sodium (NaCl) to generate NaCl particles (TSI Aerosol Generator 3076); a diffusion dryer to remove water from the air stream (TSI Diffusion Dryer 3062); and a neutralizer to apply a standard equilibrium charge distribution (TSI Aerosol Neutralizer 3077A). The air in the chamber was mixed with fans so that the concentrations were uniform. The tested filter media were held in a 3D printed filter housing. The flow rates were designed so that the face velocity through the filter media is 10 cm/s. Particles were counted with a TSI Fast Mobility Particle Sizer (FMPS) 3091, which measures size distributions every second using 32 channels between the diameter size range of 0.0056 and 0.56 μm. The result of this procedure, for each filter media tested, is a determination of the removal efficiency of the filter for total particles and as a function of particle size (with diameters between 0.0056 and 0.56 μm ). The results of this procedure allow comparison with those of an N95 test, which tests for a removal efficiency of 95% of particles 0.3 μm in diameter.

statistical analyzes

Datasets were analyzed using SPSS. P value

Comments are closed.