# Durable nanocomposite face masks with high particulate filtration and rapid inactivation of coronaviruses | Scientific Reports – Nature.com

### Nanocomposite fabrication

Nanocomposites were created by submerging a commercially available face mask textile in a 0.5 M precursor solution of zinc salt in deionized water. The face mask materials tested were melt-blown polypropylene (Vanalay LLC) and 50/50 Cordura® nylon/cotton blend (Rockywoods Fabrics LLC). The hydrophobicity of the polypropylene mask requires that pressure be applied to ensure thorough wetting of the material. The mask, still submerged in solution, was then placed in a commercial convection oven (Model FDL 115, BINDER GmbH, Tuttlingen, Germany) at 100 °C for 4 h. After synthesis, the face masks were subjected to one wash/dry cycle following the American Association of Textile Chemists and Colorists (AATCC) LP1: Home Laundering method in 20 °C water in a machine washer (Vortex M6, SDL Atlas) followed by drying at high temperature in a tumble dryer (Vortex M6D, SDL Atlas). AATCC high efficiency liquid standard reference detergent was used for all wash cycles.

### Zinc colorimetry

Small samples of nanocomposite fabrics between 0.2–0.5 g were digested in an acid mixture of 20 mL nitric acid, 10 mL of 35% hydrochloric acid, and 10 mL of DI water. Digestion is performed by submerging the textile in the acid bath and heating in an oven at 95 °C for 2 h. Analysis of the digested sample is then performed according the USEPA Zincon method 8009 with a Hach DR300 Pocket Colorimeter44,45.

### HRIPT testing

The Human Repeat Insult Patch Test (HRIPT) was performed on human participants by Evalulabs LLC to determine skin irritation from a treated fabric. This testing protocol was performed in accordance with relevant guidelines and regulations and was carried out under the direction of a licensed dermatologist. All experimental protocols were approved by an Ethics Committee, the Evalulab LLC Independent Ethics Committee (IEC), to ensure the protection of the rights, safety, and well-being of the subjects participating in the study. Informed consent was obtained from all 50 human subjects and/or their legal guardians. For more details, please see the full report in the supplemental information.

### Virucidal testing

As recommeneded by the ASTM Guidance on SARS-CoV-2 Surrogate Selection, we used TGEV in this testing folllowing the ISO 18184 method with modifications31,46. Aliquots (75 µL) of TGEV suspension (with initial titer = ⁓ 6.5 Log TCID50/mL) were placed on the center of 2 × 2 cm2 sterile parafilm squares that were cut earlier and placed in Petri dishes. Nine squares (2 × 2 cm2) of untreated (control) and 9 nanocomposite materials (face mask and nylon/cotton) were placed separately over the surface of each parafilm square where the virus droplets were sandwiched between the tested fabric and the parafilm square. The virus droplets were absorbed immediately by the nylon/cotton specimens as they are hydrophilic while a little pressure by a pipette tip was applied on the polypropylene specimens for sample absorption. After 10 min, 30 min, and 60 min of contact, triplicate sample sets (tested specimen with the absorbed virus and the parafilm square) were withdrawn from the control as well as the nanocomposite specimens. Each sample set was then transferred into a round bottom 13 mL plastic centrifuge tubes (Falcon) containing 1 mL of the virus recovery medium (Eagle’s MEM with 4% FBS and standard antibiotics). All tubes were then vortexed for 2 min immediately after transferring to recover the viral particles from the tested specimens. In a concurrent experiment (leached NP control), nanocomposite specimens (unspiked with the virus) were transferred first into virus recovery tubes and vortexed for 2 min followed by the addition of 75 µL aliquot of the virus into each tube (without direct contact with the fabric). This was done to know whether a fraction of viral particles was inactivated by contact with nanoparticles that might have leached in the virus recovery solution following the recovery of the virus from the fabric.

The titer of surviving virus recovered in the recovery medium was performed by the 50% tissue culture infective dose (TCID50) method. Serial tenfold dilutions were prepared from the recovery medium of each sample. These dilutions were inoculated in 80% confluent monolayers of swine testicular (ST) cells, pre-prepared in 96-well microtiter plates using 3 wells per dilution (100 μL of each sample dilution/well). The infected cells were incubated at 37 °C in a 5% CO2-incubator for up to five days and examined daily under an inverted microscope for the appearance of cytopathic effects (CPE). The highest dilution of the virus, which produced CPE in 50% of the infected cells, was considered as the endpoint. The titer of the surviving virus in each sample was then calculated by the Karber method and expressed as log10 TCID50/sample47.

### Real time RT-qPCR

To gain some insights on the mode of action of virus inactivation by the nanocomposites, we quantified the viral genome copy numbers in the recovery solution after virus recovery from the control and nanocomposite specimens. Viral RNA was extracted from 140 μL of each sample using QIAamp DSP Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The RNA was eluted in 100 μL of elution buffer and stored at − 80 °C until used for viral genome quantification. For RT-qPCR, we used PCR primer set and probe shown in Table 2. The RT-qPCR primers were designed to target a conserved 146 bp region (corresponding to the region between nucleotides 370 and 515 of the TGEV S gene with reference to the sequence of TGEV-GenBank accession no.: KX900410.1). The primers and probe were manufactured by Integrated DNA Technologies (IDT, Coralville, IA). The RT-qPCR reactions were performed using AgPath-ID One-Step RT-PCR kit (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA). The reaction mixture (25 μL) consisted of 5 μL of template RNA, 12.5 μL of 2 × RT-PCR buffer, 1 μL 25 × RT-PCR Enzyme Mix, 0.50 μL of 10 μM forward primer (200 nM final concentration), 0.50 μL of 10 μM reverse primer solution (200 nM final concentration), 0.30 μL of 10 μM probe (120 nM final concentration), and 5.20 μL of nuclease-free water. The RT-qPCR was performed in the QuantStudio™ 5 PCR thermocycler system (Thermo Fisher Scientific, Applied Biosystems™). Reverse transcription was performed at 45 °C for 10 min. Taq polymerase activation was done at 95 °C for 15 min followed by 45 amplification cycles using a 95 °C/15 s denaturation step and an annealing/extension step at 58 °C for 45 s. Fluorescence was measured at the end of annealing step in each cycle. In each run of RT-qPCR, standard curve samples and no template control were used as positive and negative controls, respectively. The TGEV standard/calibration curve was constructed for absolute quantification of viral genome copy number, in which we used serial ten-fold dilutions of a 557 bp purified conventional RT-PCR amplicon of TGEV S gene (including the 146 bp target sequence of the RT-qPCR prime/probe set). The 557 bp TGEV S gene fragment was produced by conventional RT-PCR reaction using an in-house developed primer set shown in Table 2. Results were expressed as cycle threshold (Ct) values. The Ct values and standard curve were used to calculate the absolute genome copy number of TGEV, expressed as genome copies/sample.

### Statistical analysis

The virucidal testing experiment was performed twice. In each of them, triplicate samples were tested at each contact time. Hence, the results presented here are the geometric means of 6 replicates ± one gemoetric standard diviation. The presented results of real-time RT-PCR are the geometric means of dublicate samples ± one gemoetric standard diviation. The analysis of variance was pefromed by One-way ANOVA and the significance of differences between the means were performed by paired comparisons using Tukey’s test at significance = 0.05.

### Germicidal testing

The germicidal efficacy can be determined through standardized American Association of Textile Colorists and Chemists (AATCC) methods that evaluate the antibacterial effects and determine the minimum inhibitory concentrations (MICs) and minimum bactericidal concentration (MBCs) of the different metal nanoparticles on the select microorganisms. Quantitative and qualitative tests based on standardized AATCC TM100-2004 and AATCC TM147-2004 protocols were utilized, respectively48. Per these methods, Staphylococcus aureus subsp. aureus (ATCC 6538) were grown in tryptic soy broth (TSB) to get 108 cells. This was followed by serial dilution using TSB to get a final concentration of 105 for inoculation purposes. Two swatch sets of untreated fabric and one swatch set of treated fabric were each placed in a 60 mm × 15 mm petri dish. The swatches were separately inoculated with 1 mL of the bacterial strain in broth prepared as described above. Broth solutions were ensured to completely soak into each fabric. A control swatch was inoculated with 1 mL of sterile TSB as a measure of contamination. These petri dishes were sealed with paraffin plastic film and incubated for 24 h at 37 °C. For immediate elution tests, inoculated swatch samples were prepared as described above and were each transferred to tubes containing 50 mL of 0.15 M NaCl solution. The swatches were compressed against the walls of the tube and vortexed to ensure complete elution of the inoculum. After thorough elution, the swatches were removed from NaCl solution. A separate tube containing 50 mL of NaCl solution was directly inoculated with bacterial culture to serve as a control. These elution steps were repeated for the 24-h incubated swatch samples. The eluate for each sample was serially diluted once using TSB for the immediate elution samples and five times for the 24 h incubated swatch samples before plating on tryptic soy agar (TSA) and incubating for 24 h at 37 °C. After incubation, bacterial colonies were counted, and percent reduction was calculated as described in the following section.

The antimicrobial activity of nanocomposite fabrics was tested against Staphylococcus aureus (Gram-positive bacteria) as outlined in the AATCC methods. The microorganism Staphylococcus aureus is a pathogenic bacterium and was manipulated in Biosafety Laboratory Level 2 (BSL2) at the Veterinary Diagnostic Laboratory at the University of Minnesota. Quantitative methods are based on calculating the reduction percent of bacteria from 0 to 24 h contact time from both the inoculated fabric sample and control sample (with no antibacterial agent). The percentage reduction is determined as follows:

$$R\;(\% ) = \frac{A – B}{A}$$

where, R is the reduction in colony forming units (cfu), A is the number of bacterial colonies from the control textile, and B is the number of bacterial colonies from the treated textile. The qualitative assessment of antibacterial activity was performed using a parallel streak method (AATCC 147-2004), and the quantitative test was performed using AATCC 100-2004 method.

### Filtration performance testing

The experimental setup is shown in Fig. 5. The test aerosols were produced from a NaCl-water solution with a mass concentration of 0.1% by using a constant output atomizer (Model 3076, TSI Inc., Shoreview, MN). The aerosols were diluted, dried, and then homogenized in a mixing chamber. The LOG3Mask material was cut into a disc with a diameter of 37 mm and tightly pressed onto the mesh support of a filter cassette (Air Sampling Cassette, Zefon International Inc., Ocala, FL) and sealed at the edge. The size distributions of aerosols in the range of 30–600 nm upstream and downstream of the filter holder and the concentration of the mobility-classified particles were determined by a scanning mobility particle sizer (SMPS, Model 3936, TSI Inc., Shoreview MN). A portable aerosol spectrometer (GRIMM Model 11-D, Durag Inc., Mendota Heights, MN) measures the size distributions of aerosols in the 500–5000 nm range. A two-digit manometer (RISEPRO, 365BG947677, measuring range ± 13.79 kPa, 0.001 kPa resolution) was used to track the flow resistance of the materials, as the flow resistance across the filter material is a crucial component for determining the breathability of the material. The filtration efficiencies obtained from the size distributions measured by the SMPS and GRIMM, and the size-dependent filtration efficiency ($$\eta (D_{p} )$$) was calculated by:

$$\eta (D_{p} ) = 1 – \frac{{n_{o} (D_{p} )}}{{n_{i} (D_{p} )}}$$

where $$n_{o} (D_{p} )$$ and $$n_{i} (D_{p} )$$ are the particle number concentrations for each particle size measured at the outlet (downstream) and inlet (upstream) of the filter cassette. The standard deviation was calculated based on the error propagation method discussed in Hao et al.49,50.