Electroceutical fabric lowers zeta potential and eradicates coronavirus infectivity upon contact

Coronavirus with intact infectivity attached to PPE surfaces pose significant threat to the spread of COVID-19. We tested the hypothesis that an electroceutical fabric, generating weak potential difference of 0.5 V, disrupts the infectivity of coronavirus upon contact by destabilizing the electrokinetic properties of the virion. Porcine respiratory coronavirus AR310 particles (105) were placed in direct contact with the fabric for 1 or 5 min. Following one minute of contact, zeta potential of the porcine coronavirus was significantly lowered indicating destabilization of its electrokinetic properties. Size-distribution plot showed appearance of aggregation of the virus. Testing of the cytopathic effects of the virus showed eradication of infectivity as quantitatively assessed by PI-calcein and MTT cell viability tests. This work provides the rationale to consider the studied electroceutical fabric, or other materials with comparable property, as material of choice for the development of PPE in the fight against COVID-19.

. Contact potential mapping on electroceutical fabrics. (a) Schematic of the contact potential mapping measurement. A gold-coated probe tip mounted on a 3-axis linear actuator system was raster scanned over the sample area and recorded the contact potential at each probed position with reference to the unprinted fabrics. The potential was read by a high precision voltameter, followed by data analysis and finite element modeling (FEM) simulation. www.nature.com/scientificreports/ Fig. 2). The only peak that was present other than C and O was that of Au used for coating the fabrics for SEM imaging ( Supplementary Fig. 2a). Proximity of Ag and Zn on polyester fabric forms a redox couple and is capable of driving electrochemistry when wet in an aqueous ionized environment including any body fluid (Fig. 1). Three ionized aqueous media were used to test potential difference between adjacent Ag and Zn deposits. NaCl solution (0.85% w/v), cell culture medium and tap water (of practical value to end users of PPE) were tested at room temperature. The potential difference between the two electrodes rapidly increased and achieved a steady state after the first 15 s (Fig. 3). The potential difference between adjacent Ag and Zn dots was also sustained in presence of different body fluids ( Supplementary Fig. 3).
Physical characterization of the coronavirus. SEM (150,000×) revealed the morphological features of the porcine corona virus (CoV) particle ( Supplementary Fig. 4a). Following spotting on the silicon wafer, the purified virus was fixed and subsequently dehydrated. A thin (2-5 nm) layer of carbon was sputtered on the sample to make the specimen conductive. The size of the virus ranged between 75-125 nm. Nanoparticle tracking analysis (NTA) revealed poly-dispersed peak ( Supplementary Fig. 4b). The electrokinetic property, as represented by the zeta(ζ) potential, of the viral particles is a parameter that determines adsorption and stability of the particle in any given dispersant medium. For practical purposes, viral particles are expected to be suspended in water droplets either aerosolized or resting on a surface. The average ζ potential of four different preparation of CoV was determined to be − 25.675 mV ( Supplementary Fig. 4c). All four-preparation demonstrated comparable ζ potential distribution and phase shift ( Supplementary Fig. 4d-e). The average electrophoretic mobility distribution was determined to be − 2μmcm/Vs ( Supplementary Fig. 4f).
Electroceutical fabric attenuated the ζ potential of coronavirus upon contact. Quantification of the viral particles after spotting on f e yielded 44.29% and 23.73% recovery from the fabric when exposed for 1 min or 5 min, respectively (Fig. 4a). Nanoparticle tracking analysis demonstrated that unlike the purified CoV that showed a single peak around 75 nm, the recovered CoV showed additional peaks suggesting aggregation of the viral particles upon contact with the fabric (Fig. 4b). Analysis of ζ potential showed significant graded attenuation of this electrokinetic property upon contact with the f e (Fig. 4c). Such lowering of average ζ potential of www.nature.com/scientificreports/ CoV, applied and recovered from f e , has been plotted ( Fig. 4d). Unlike 1 min exposure to the f e , 5 min exposure showed an appreciable difference in the phase plot of the viral particles (Fig. 4e).

Loss of coronavirus infectivity upon contact with electroceutical fabric.
To assess changes in the infectivity of CoV following contact with the electroceutical fabric, a cytopathic assay was employed. Infected cells were monitored for appearance of cytopathic effects (CPE; cell rounding and sloughing) until post-infection day 7. Overt CPE was observed on day 7 in response to CoV infection ( Fig. 5b; Supplementary Fig. 5aii).
Comparable CPE was noted in response to treatment of cells with CoV recovered from sham control fabric f s ( Fig. 5c; Supplementary Fig. 5aiii-iv). In contrast, CoV recovered from f e did not cause any CPE indicating loss of its infectivity ( Fig. 5d; Supplementary Fig. 5av-vi). Cells treated with f e -recovered CoV particles appeared as healthy as the uninfected cells ( Fig. 5a; Supplementary Fig. 5ai). Objective assessment of cell viability was performed using calcein/PI fluorescence assay. Only live cells with intracellular esterase activity hydrolyze the acetoxymethyl ester in non-fluorescent Calcein AM converting it into green fluorescent Calcein. Dead cells or cells with damaged or compromised cell membranes include PI stain, which is otherwise impermeant to live cells. Fold-change increase in PI/Calcein signal as shown indicates loss of cell viability in response to infection. Infection of cells with CoV caused marked loss of cell viability (Fig. 5b). Such cytopathic effect of CoV was completely absent once the virus was exposed to f e (Fig. 5d, e). The sham fabric did not afford such protection (Fig. 5c, e). The cytopathic effects of CoV and the protective effects of f e (versus f s ) was corroborated by the standard MTT assay commonly used for testing cell viability (Fig. 5f). The cytopathic effects of CoV were primarily due to increase generation of H 2 O 2 in the ST cells that were abrogated when the virus particles were exposed to f e ( Supplementary Fig. 5b). The protective effects of f e were further supported by cellular respirometry www.nature.com/scientificreports/ assay that showed increased basal respiration, maximum respiration and ATP production in cells exposed to CoV recovered from f e (Supplementary Fig. 5c-e) .

Discussion
Previous work from our laboratory has established the effectiveness of electroceutical principles as an alternative to pharmacological approaches in managing planktonic microbial pathogens and complex polymicrobial biofilms 18,20,21,23,24 . Viruses are known to rely on electrostatic interactions for optimal virion assembly and attachment 13,25 . For instance, structural proteins in coronaviruses, negatively charged amino acid residues in the nucleocapsid facilitates assembly with the membrane protein 26 . Additionally, the coronavirus envelope protein is known to generate ion conductive pores across membranes which are voltage dependent 27 . Leveraging these viral characteristics to achieve viral inactivation remains largely unexplored and has been attempted in this work. Electroceuticals have generated renewed interest in the health care industry 28 . The fabric tested in this work consists of only silver and zinc dots on polyester fabric that forms a redox couple [18][19][20][21][22][23] . Zeta potential of a particle determines its electrostatic interactions in particle dispersions and, as such, is an important determinant of the stability of viral particles 29 . Contact of CoV with the electroceutical fabric studied rapidly lowered the zeta potential demonstrating a direct effect of the fabric on the electrokinetic properties of the viral particle. Any change of zeta potential towards zero is viewed as increase in electrical instability of the particle. The observation that in contact with the electroceutical fabric eliminates infectivity of the virus leads to the hypothesis that the observed lowering of zeta potential may have caused defects in the structural integrity of the virus. Study of changes in the capsid-RNA structure following exposure to the weak electric field generated by the fabric is thus warranted.
CoV is a nanoparticle. Nanoparticle tracking analysis determines the hydrodynamic diameter of the analyte by applying the Stokes-Einstein equation after measuring the Brownian motion of individual nanoparticle 30 . NTA . Zeta potential and nanosight tracking analysis of the purified coronavirus following contact with and retrieval from the electroceutical fabric. (a) Absolute quantification of viral particles recovered from the fabric after treatment with f e . A two-fold and four-fold reduction in the recovered viral number was observed after 1-and 5-min treatment, respectively. (b) Viral particle number was quantified using NTA. One hundred microliters of the purified CoV were spotted on 1.5 cm discs of electroceutical fabric and recovered using incomplete EMEM, after 1 or 5 min of contact with the fabric. (c-e) Changes in viral zeta potential and affiliated parameters after contact with the fabric for 1 min or 5 min. Data are mean ± SEM. www.nature.com/scientificreports/ www.nature.com/scientificreports/ was utilized to estimate absolute viral particle number and size distribution in not only pure CoV but also in CoV recovered from the fabric. Observed changes in particle number and size distribution support the aforementioned hypothesis that exposure to the weak electric field causes damaging structural alterations to the virions. Cells in culture routinely display a small fraction of dead or dying cells. Cytopathic effects of viral infection are tested to examine whether exposure to the infectious particle adds to the basal cell death burden of the culture. Longterm observations, i.e. days versus hours, ensure the recording of the eventual fate of the affected cells. Reporting of short-term data alone, while sometimes may be encouraging with respect to effect of the intervention, may simply reflect results representing postponement of death from the insult and not a true rescue. In CPE studies of this work, cell rounding, and sloughing were evident in day 4 post-infection. During this time, cells treated with virus pre-exposed to the electroceutical fabric closely resembled cells that were unchallenged by exposure to the virus. In standard cell culture, the growth medium is changed every other day to wash off floating dead cells and to replenish nutrition. Under conditions of infection by virus, such frequent change of cell culture medium is not made. Cells grow in the same spent media until day 7 post-infection. Maintenance of cells without any change in culture media for seven days is expected to marginally increase basal cell death burden as shown.
Textiles evaluated for use in PPE such as masks are subject to specific FDA 510(k) requirements expecting stringent viral filtration tests to demonstrate 99.9% reduction of 1.1-3.3 × 10 4 plaque forming units of standard phiX174 bacteriophage. The phiX174 is widely used as a model organism because of it being a standardized test. However, it is important to note that unlike SAR-CoV-2 which is a RNA virus, phiX174 bacteriophage is a DNA virus with numerous contrasting physical, chemical as well as biological properties. Furthermore, this bacteriophage is much smaller in size than SAR-CoV-2. The non-enveloped icosahedral morphology of phiX174 are aerosolized with a mean particle size of 3.0 ± 0.3 µm 31 . This is in direct contrast with the coronaviruses that cause diseases in animals and humans which are ~ 100 nm in diameter and are aerosolized as respiratory droplets with sizes > 5 µm 32,33 . Importantly, phiX174 cannot infect mammalian cells. It infects and forms visible plaques on a lawn of Escherichia coli (Migula) Castellani and Chalmers strains. In the context of COVID-19 pandemic, this work studies the coronavirus and tests cytopathic effects on mammalian cells. Testing methods such as AATCC TM100 recommends a textile contact time of 24 h for both enveloped and non-enveloped viruses. We report results on contact time that is much shorter and more relevant to PPE usage in the context of COVID-19.
This work presents first evidence demonstrating that the physical characteristic features of CoV may be exploited to render it non-infective following exposure to weak electric field generating electroceutical fabric. The observation that lentiviral infectivity is also eliminated following contact with the electroceutical fabric contributes to the rigor of our central finding. Lowering of zeta potential of the CoV particles following exposure to the electroceutical fabric constitutes direct evidence supporting the contention that electrokinetic stability of the viral particle is weakened. Additional studies are necessary to characterize specific structural changes in response to exposure to the electroceutical fabric, and to connect such changes to loss of infectivity. In the meanwhile, this work provides evidence supporting the rationale to consider the studied electroceutical fabric, or other materials with similar property, as material of choice for the development of PPE in the fight against COVID-19.

Methods
Electroceutical fabric. An FDA cleared wireless electroceutical dressing was used as a source of weak electric field for the current study and is referred to as electroceutical fabric (f e ). This fabric, co-developed by our laboratory [18][19][20][21][22] , has been commercialized by Vomaris Inc. (Phoenix, AZ) was provided to us by the manufacturer. It is made of polyester fabric printed with alternating circular dots of Ag and Zn metals (ø2 mm and 1 mm, respectively), generating electric fields. A polyester fabric without any metal deposition (hence unable to generate electric field) was used as an experimental control and is referred to as sham fabric (f s ).
Contact potential mapping for dressings. Two-dimensional maps of the surface potential of the electroceutical dressing were collected by the modified version of a method reported previously 34 . Briefly, a goldcoated probe tip (100 µm diameter) was mounted on a microprobing system consisting of miniature linear actuators (Zaber Technologies Inc., Vancouver, Canada) with independent control in the XYZ dimensions. The dressings were moistened by EMEM media and mounted on the sample stage. With the microprobing system, the probe tip raster scanned to contact the sample surface for potential measurement at each pixel. The scan area was 5 × 5 mm in 20 × 20 pixels covering 2 Ag dots, 2 Zn dots and empty dressing. The potential at the probe tip was referenced to the empty dressing, as measured by a high precision and high input impedance voltmeter (EMF 16, Lawson Lab Inc., Malvern, PA, USA) at each pixel. The collected data were plotted and processed by Gwyddion 35 . The finite element model was constructed and analyzed by COMSOL Multiphysics 5.4.

Respiratory coronavirus infection and propagation. ST cells were cultured in complete EMEM till
they attained a confluency of 80-90% followed by washing monolayers with 5 ml of 1X PBS. Coronavirus stock (ATCC VR-2384™; USDA permit 141794) aliquot was diluted with 3 ml of incomplete culture medium (without FBS and antibiotic-antimycotic solution) to attain a Multiplicity of Infection (MOI) of 1. This diluted viral stock was added to the washed monolayer and incubated at 37 °C, humidified 5% CO 2 in air atmosphere. Flasks were rocked gently for 2 min at intervals of 30 min, to re-distribute viral inoculum. After 2 h infection, viral adsorption was terminated by adding 10 ml of complete culture medium to the monolayer.
Coronavirus purification. Coronavirus purification was performed as described previously 36 . In brief, culture medium from flasks with infected cells was harvested at 10,000×g for 20 min at 4 °C. Viral precipitation from this supernatant (12 ml) was done by addition of polyethylene glycol and NaCl. PEG precipitated proteins and virions were pelleted at 10,000×g for 30 min at 4 °C and the pellet was dissolved in 100 µl of ice-cold 1X HEPES-saline buffer (10 mM HEPES -Sigma H7523 + 0.9% w/v NaCl, pH 6.7). Dissolved pellet was then loaded on a discontinuous sucrose gradient (10-20-30%, 800 µl each; in 1X HEPES-saline) and subjected to ultracentrifugation at 100,000×g for 90 min at 4 °C.
Nanoparticle tracking analysis. Viruses were diluted in EMEM or 18.2 MΩ water. Mean particle diameter and concentration of viral particles were analyzed by NanoSight NS300 with a 532 nm laser and SCMOS camera (Malvern) as previously described [37][38][39] . Standard 100 nm latex spheres were run at 1000:1 dilution in mil-liQ to test optimal instrument performance. Data were analyzed using NTA 3.0 software (Malvern Instruments).
Zeta potential analysis. Zeta (ζ) potential measurement of viral particles was determined by Zetasizer (Nano-Z, Malvern Instruments Ltd., UK) as described previously [37][38][39] . All samples were dispersed in doubledistilled water and tested in volume-weighted size distribution mode in folded capillary cells (Fisher Scientific NC0491866). An average of three readings (60 s) were recorded.
Scanning electron microscopy. Viral particles were suspended in ddH2O with 2.5% glutaraldehyde or other buffer and dropped onto clean silica wafers. After drying, samples were desiccated in a vacuum chamber for at least 12 h before analysis. Images were obtained after carbon sputter coating using a field emission scanning electron microscope (JEOL 7800F, JEOL Japan) at a beam energy of 5 or 10 kV. For the SEM images of the fabric, gold sputter coating was used.
Energy dispersive X-ray (EDX) microanalysis. For elemental detection, the EDX microanalysis associated with SEM was used 40 . The x-ray emissions at different wavelengths were measured using a photon-energysensitive detector. The EDX detector system performed a simultaneous display of all mid-energy (1-20 keV) x-rays collected during any individual analysis period.
Coronavirus infectivity. ST (coronavirus) cells were seeded at densities of 10,000 /well and 1000/well in 24-well and 96-well cell culture plates, respectively. Seeded plates were incubated at 37 °C, 5% CO 2 humidified incubator for 18 h. One hundred microliter (10 5 particles) of aqueous suspension of viruses (10 6 /ml of AR310) were placed on 1.5 cm diameter discs of f e and f s at room temperature and time was allowed for the droplet to be fully absorbed into the respective fabric. As soon as that was achieved, a timer was started for either 1 min or 5 min of contact time followed by recovery of the particles. For 10 5 AR310 viral particles that were placed in direct contact with the fabric, 2.5-4 × 10 4 particles were recovered from the fabric. Serum free medium (100 µl) was used to rinse each fabric for recovering viral particles from the fabric. NTA was performed, as above, to estimate viral recovery efficiency. Recovered VR-2384 viruses were diluted with serum free medium and used to infect ST cells at MOI of 10 (10 5 viruses). Parallel sets of cells infected with untreated viruses (at the same MOI as that of treated viruses) were used as positive control while uninfected or non-transduced host cells were accounted as negative control.
Cell viability staining by calcein AM and propidium iodide. Viability of ST cells, infected as above, was assessed by dual staining with Calcein AM and Propidium iodide (PI) 41,42 . Media from wells with ST cells (uninfected or infected with untreated or fabric-contacted viruses) was washed briefly with 1 ml of 1X PBS (per well) for 1 min, followed by addition of 250 µl of freshly-prepared staining solution in 1X PBS (Calcein AM; final concentration 1 µg/ml, Catalog no: C1430, Invitrogen™) and PI (Sigma-Aldrich). Cells were incubated under dark conditions at 37 °C for 15 min and then observed under a Confocal Laser Scanning Microscope using a 10× objective. The ratio of PI:calcein signal was normalized with the average PI intensity of untreated cells to obtain fold-change compared to non-viable cells (basal cell death) in untreated cells.