Transparent and Sprayable Surface Coatings that Kill Drug-Resistant Bacteria within Minutes and Inactivate SARS-CoV-2 Virus

Abstract

Antimicrobial coatings are one method to reduce the spread of microbial diseases. Transparent coatings preserve the visual properties of surfaces and are strictly necessary for applications such as antimicrobial cell phone screens. This work describes transparent coatings that inactivate microbes within minutes. The coatings are based on polydopamine (PDA) adhesive, which has the useful property that the monomer can be sprayed, and then the monomer polymerizes in a conformal film at room temperature. Two coatings are described: (1) a coating where PDA is deposited first and then a thin layer of copper is grown on the PDA by electroless deposition (PDA/Cu), and (2) a coating where a suspension of Cu₂O particles in a PDA solution is deposited in a single step (PDA/Cu₂O). In the second coating, PDA menisci bind Cu₂O particles to the solid surface. Both coatings are transparent and are highly efficient in inactivating microbes. PDA/Cu kills >99.99% of Pseudomonas aeruginosa and 99.18% of methicillin-resistant staphylococcus aureus (MRSA) in only 10 minutes, and inactivates 99.98% of SARS-CoV-2 virus in 1 hr. PDA/Cu₂O kills 99.94% of Pseudomonas aeruginosa and 96.82% of MRSA within 10 minutes, and inactivates 99.88% of SARS-CoV-2 in 1 hr.

Graphical Abstract

1. INTRODUCTION

Many diseases can spread to humans by fomite transmission.¹ Some of these are bacterial and some are viral. Among bacterial diseases, the ones caused by antibiotic-resistant bacteria are particularly problematic. An example of antibiotic-resistant bacteria is methicillin-resistant Staphylococcus aureus (MRSA). MRSA, a Gram-positive bacterium, was declared a “Serious Threat” by the United States Center for Disease Control (CDC) in 2019.² It has been the cause of both healthcare-associated infections (HAIs)³ and community-associated infections (CAIs).⁴ MRSA causes a wide range of conditions, such as skin infection, pneumonia, and blood infections.^5–6 Pseudomonas aeruginosa (P. aeruginosa), a Gram-negative bacterium, is another causative agent of HAIs and has also been labelled by the CDC as a “Serious Threat”.⁷ This is one of the more difficult bacteria to kill because it has both intrinsic and acquired resistance to many antibiotics, and it can adapt to the environment to reduce the effectiveness of antibiotics.⁸ P. aeruginosa causes many diseases, such as blood, lung, or skin infections.^{6, 9}

Both P. aeruginosa and MRSA can survive on solids for a prolonged period of time (even up to months),¹⁰ and are known to be transmitted via direct contact, e.g. touching contaminated skins or surfaces.^11–12 To hinder such transmission, it would be desirable to kill the bacteria as soon as they land on surfaces.

The global COVID-19 pandemic has severely impacted life: from early 2020 to July 2021, more than 185 million infections and 4 million deaths have been attributed to COVID-19,¹³ and estimates based on excess deaths suggest that the actual number of deaths may be much higher.¹⁴ The financial cost to the U.S. alone is estimated to be $16 trillion.¹⁵ This disease is caused by the virus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The World Health Organization states that “fomite transmission is considered a likely mode of transmission for SARS-CoV-2”.¹⁶ Unpublished experimental work shows that a substantial number of SARS-CoV-2 virus can be transferred to skin from contaminated surfaces.¹⁷ A hamster study found that direct inhalation of infectious droplets is the main mechanism of transmission, but that 1/3 of the animals were infected when exposed to fomites only.¹⁸ Mathematical modelling showed that fomite transmission is responsible for up to 25% of COVID-19 transmission during lockdown.¹⁹ SARS-CoV-2 also remains infectious up to 7 days after a droplet of virus-suspension is placed on solid surfaces.^20–21 The SARS-CoV-2 virus remains infective for up to 9 hours (on a dead human skin model²²) or up to 4 days (on dead pig skin²³) at room temperature. As a result, there has been significant fear of touching communal objects, and health authorities recommended frequent hand-washing to avoid getting the disease.²⁴

The use of common disinfectants, such as 70% ethanol, to decontaminate surfaces is useful for reducing infection, but one significant problem is that contamination can reoccur shortly after the ethanol has evaporated. An alternate approach is to utilize surface coatings that provide an ongoing or continuous kill, and to apply these coatings to everyday solid objects. Some coatings have been developed to meet this objective, both for SARS-CoV-2^25–29 and bacteria,^30–31and have been reviewed recently.^32–34 In addition, considerable research has investigated the antimicrobial properties of metals and metal oxides,^35–41 textured antimicrobial surfaces,^42–44 and antimicrobial additives to face masks.⁴⁵

An important issue with existing metal and metal oxide coatings is that they are opaque. In some applications this destroys the functionality of the object (e.g., a computer or phone touch screen) and in others it destroys the aesthetic appeal. To this end, we have developed transparent antimicrobial coatings. To achieve a transparent coating, one needs to avoid both scattering and adsorption of light. Here we are focused on copper and copper oxides because of their known antimicrobial properties. Bulk copper metal is opaque, but we have used a very thin film to achieve transparency⁴⁶, and electrodeless deposition for ease of use in applications. Cuprous oxide (Cu₂O) is an excellent antimicrobial agent, but it has a red-brown color. For a transparent coating, we simply use a thin, sparse layer of particles. As one decreases the density of particles to obtain transparency, one expects to ultimately lose antimicrobial properties. In fact, we found that a layer that is sufficiently sparse to obtain transparency maintains antimicrobial activity.

We describe two transparent surface coatings that very rapidly inactivate microbes. The adhesive element in the coatings is polydopamine (PDA),^47–48 which polymerizes from aqueous solution at low temperature (<100 °C) in a conformal manner, meaning that PDA takes the shape of the surface. This makes the coatings easy to prepare. Our first coating, designated by PDA/Cu, consists of PDA and a very thin layer of copper that was deposited onto PDA by electroless deposition. Our second coating, designated by PDA/Cu₂O, is made by simultaneously polymerizing PDA and depositing Cu₂O particles in a way that the PDA forms menisci between the Cu₂O particles to hold them on a solid and to bind together the particles. PDA/Cu₂O is sprayable in a single step, which makes it easy and rapid to apply to objects with different shape. The coatings are thus easy to prepare and also consist of inexpensive and relatively safe materials.

These coatings have outstanding antimicrobial activity, and are transparent. The PDA/Cu coating reduces the number of viable MRSA by 99.18% and the number of viable P. aeruginosa by >99.99% compared to uncoated glass within only 10 minutes. The PDA/Cu₂O coating reduces the number of viable MRSA by 96.82% and the number of viable P. aeruginosa by 99.94% compared to uncoated glass within 10 minutes. The PDA/Cu and PDA/Cu₂O coatings inactivate 99.98% and 99.88%, respectively, of SARS-CoV-2 virus within 1 hour, compared to uncoated glass.

2. RESULTS and DISCUSSION

2.1. The antimicrobial coatings are transparent

The antimicrobial coatings are transparent and allow different colors to be clearly seen when the coatings on glass are held in front of a computer screen. The visible spectra data (Figure 1) confirms that there is little variability for transmission of different colors. The PDA, which forms the basis of both coatings, is already somewhat opaque, absorbing 20–40% of the light. The two coatings were made in different ways so have slightly different transmission. Addition of copper to PDA decreases the transmission, with slightly more adsorption at longer wavelength as expected⁴⁶, which counteracts the trend for PDA to give a more consistent transmission than PDA alone. The PDA/Cu₂O coating also transmits less light than PDA-alone, presumably because of scattering and absorbance by the particles. (See Figure S2 for particle size distribution.)

Figure 1.

A) and C) photographs of PDA/Cu and PDA/Cu₂O coatings, respectively, held in front of a computer monitor. Both coatings are transparent and allow all colors to be seen in transmission. B) and D) visible spectra of the coatings. B) Transmission spectra of PDA, and the PDA/Cu coating. Data for PDA is from the PDA that was prepared in the first step of making the PDA/Cu coating (designated as PDA_Cu). D) Transmission spectra of PDA, and the PDA/Cu coating. The PDA was prepared by the same method as the PDA/Cu₂O coating, but without adding the Cu₂O particles (designated as PDA_Cu2O). The spectra of the two PDAs (i.e. PDA_Cu and PDA_Cu2O) are similar. To ensure that coatings formed on one side only for the optical measurements, the PDA_Cu and PDA/Cu coatings were prepared with tape covering one side of the glass. The tape was removed prior to spectrometry. The background for the visible spectra is glass.

2.2. Other characterization of the coatings

For antimicrobial activity, the active ingredients, i.e., copper or Cu₂O, should be accessible from the surface, and we verified this with SEM imaging and XPS, which is sensitive to the chemistry in the outer few nanometers of a surface. Considering first the PDA/Cu coating, the copper in the PDA/Cu coating was grown from the surface of a PDA coating. That PDA coating is rough (Figure 2A), by design, to present a large surface area on which Cu can form. After deposition of the Cu, some depressions in the PDA coating have been filled in (see Figure 2) and additional small-scale roughness is visible (see Figure S3 E & F in Supporting Information). SEM images show that the copper layer is approximately 22 nm thick and the PDA is about 55 nm thick (see Figure S3 E & F). The presence of Cu in the PDA/Cu coating is also obvious from XPS: the PDA/Cu coating is about 37% copper (See Figure S5 in Supporting Information for XPS spectra). High resolution XPS (Figure S5) is consistent with an oxidized outer layer consisting of both Cu(I) and Cu(II) species, as observed previously.^49–50 Consistent with existing literature on electroless deposition,^{47, 51–54} in this paper we will refer to the coating as copper or Cu, even though the oxidation state of the inner part of the 22 nm layer film is not clear from the XPS spectra (See Figure S6).

Figure 2.

Plan view SEM images of the coatings. A) PDA-only from an intermediate step in fabrication of the PDA/Cu coating. B) PDA/Cu coating. The copper coating is apparent from the slight roughening of the coating as well as the filling of depressions compared to A. C) plan view PDA/Cu₂O coating. D) 50-degree tilt of magnified view of PDA/Cu₂O coating. The PDA meniscus that holds Cu₂O particles is shown with a white arrow. Further images are shown in Supporting Information Figure S3 & Figure S4.

The PDA/Cu₂O coating utilizes Cu₂O particles that have a mean size of about 5 μm (see Figure S2 for Cu₂O particles size distribution), which is a much larger scale than the thickness of the Cu coating in the PDA/Cu coating. In the preparation of PDA/Cu₂O coating, we incorporated a heat treatment step at 80° C for 30 minutes. XPS shows that both Cu(I) and Cu(II) are present on the outer 1 nm or so of the coating (see Figure S5). However, The duration and temperature of heat-treatment is not sufficient to oxidize Cu₂O, consistent with our previous work.²⁵

The Cu₂O particles clearly protrude from the PDA (Figure 2 D). Our goal was a coating with good transparency, so knowing that the Cu₂O particles scatter light, we have deliberately made a sparse layer of Cu₂O (Figure 2 C&D). XPS data shows that the PDA/Cu₂O coating contains only 5.7% percent copper (see Figure S5 in Supporting Information). Closer inspection of the SEM image that was taken on a tilt angle reveals the formation of a PDA meniscus that helps to adhere the Cu₂O particles to the substrate.

Advancing and receding water contact angles for PDA/Cu are 72 ± 22 and 10 ± 7 degrees (the number after “±” sign is the 95% confidence interval from measurements of independent samples). The PDA/Cu₂O is hydrophilic, with both advancing and receding angles being <10°.

2.3. Inactivation of microbes

2.3.1. Both transparent coatings kill bacteria in 10 minutes

Figure 3 shows very low survival of P. aeruginosa and MRSA on each of the transparent coatings, even after only ten minutes. Survival is a comparison between the input titer and the titer at a later time and is calculated from:

$$\text{log}\hspace{0.33em}\text{survival}=\text{mean }\left[{\text{log}}_{\mathbf{10}}\left(\frac{\text{sample}\hspace{0.33em}\text{titer}}{\text{units}}\right)\right]-\text{mean}\left[{\text{log}}_{\mathbf{10}}\left(\frac{\text{input}\hspace{0.33em}\text{titer}}{\text{units}}\right)\right]$$ (Eq. 1)

$$\text{\%}\hspace{0.33em}\text{survival}=\left[1-{10}^{-\text{log}\hspace{0.33em}\text{survival}}\right]\times 100$$ (Eq. 2)

Figure 3.

Time course of survival of P. aeruginosa (PA) and methicillin-resistant staphylococcus aureus (MRSA) on transparent PDA/Cu (left) and PDA/Cu₂O (right) coatings. Survival was calculated with Equation (3) so zero represents the CFU in the initial droplet. For the coatings, each symbol represents the average of 3 separate samples, the error bar is the comparison interval from ANOVA, and the detection limit for each bacterium is shown with a dotted line. Compared to glass control, within 10 minutes, the PDA/Cu coating caused a Reduction of more than 99.99% of P. aeruginosa and 99.18% of MRSA, and the PDA/Cu₂O caused a Reduction of more than 99.94% of P. aeruginosa and 96.82% of MRSA. We performed a 3-way ANOVA with each of the coatings: factor 1 = surface type (glass vs each coating), factor 2 = time, factor 3 = bacteria type (P. aeruginosa and MRSA). The p-values for surface type were < 10⁻¹⁰ for PDA/Cu and < 10⁻⁹ for PDA/Cu₂O. Each data point is tabulated in Supporting Information.

Microbes can become inactivated with time on inanimate surfaces, and bacteria can die, even without an active ingredient, so we also measured survival of the microbes on the uncoated glass as a control. Figure 3 shows that survival is very high on uncoated glass. Our performance metric is $\text{“Reduction”}$, which is a comparison of the titer on the coated and uncoated samples:

$$\text{log}\hspace{0.17em}\text{Reduction}=\text{mean }\left[{\text{log}}_{\mathbf{10}}\left(\frac{\text{uncoated}\hspace{0.17em}\text{glass}\hspace{0.17em}\text{titer}}{\text{units}}\right)\right]-\text{mean}\left[{\text{log}}_{\mathbf{10}}\left(\frac{\text{sample}\hspace{0.17em}\text{titer}}{\text{units}}\right)\right]$$ (Eq. 3)

$$\text{\%}\hspace{0.17em}\text{Reduction}=\left[1-{10}^{-\text{log}\hspace{0.17em}\text{Reduction}}\right]\times 100$$ (Eq. 4)

The %Reduction by PDA/Cu is 99.99% (4-logs) for P. aeruginosa and 99.18% (> 2-logs) for MRSA within only 10 minutes (Figure 3). The Reduction on PDA/Cu₂O is 99.94% (> 3-logs) for P. aeruginosa and 96.82% (~ 2-logs) for MRSA in 10 minutes. This compares favorably to typical United States Environmental Protection Agency (EPA) standards of 99.9% (3-logs) in 1 hour of exposure.⁵⁵

We also measured antimicrobial properties of PDA alone and found that PDA did not have antimicrobial activity against bacteria or viruses (see Supporting Information, Figure S7 and Figure S8), confirming that copper and Cu₂O are the active ingredients in the two coatings.

2.3.2. Both coatings rapidly inactivate SARS-CoV-2

PDA/Cu and PDA/Cu₂O coatings reduce the infectivity of SARS-CoV-2 virus by 99.98% and 99.88% within 1 hour, respectively compared to a glass control (see Figure 4). PDA alone does not inactivate SARS-CoV-2 (see Figure S8), demonstrating that the Cu and Cu₂O are the active ingredients.

Figure 4.

Time course of SARS-CoV-2 titer for transparent PDA/Cu (left) and PDA/Cu₂O (right) coatings. Each marker shows the average of 3 independent samples and the error bars are the comparison interval from ANOVA. The detection limit was 90 TCID50/mL (shown with dotted line). When data was below the detection limit, the point was plotted at the detection limit. PDA/Cu reduces the infectivity of SARS-CoV-2 by 99.98% and PDA/Cu₂O reduces the infectivity by 99.88% in 1 hour. We performed a 2-way ANOVA with each of the coatings: factor 1 = surface type (glass vs each coating), factor 2 = time. The p-value for coating was < 10⁻⁸ for PDA/Cu and < 10⁻⁶ for PDA/Cu₂O. Each data point is tabulated in Supporting Information, Table S6.

The PDA/Cu₂O results can be compared to our previous results for Cu₂O in a polyurethane coating.²⁵ The earlier coating used a much higher loading of Cu₂O particles, such that the coating was completely opaque, and it inactivated ~99.9% of SARS-CoV-2 after one hour. The similarity of the results for the two coatings shows that there is little benefit to the greater solids loading. On the other hand, the lower solids loading confers the significant advantage that the coating is transparent and therefore can be used on touch screens, monitors, etc., and maintains aesthetic appeal of solids. Additionally, the similarity of two results supports the idea that the killing is a surface effect. The infectivity of SARS-CoV-2 over time was previously measured on a copper sheet. In that work, the virus remained infectious for 4–8 hr²⁰, while here ~99.9% of it is inactivated within 1 hr. on our coatings. Note that in the work on copper²⁰, the starting titer was about log(TCID50/ml) of 3.7, which is about 2.5 logs lower that in this work. The coatings therefore work more rapidly than copper, even with a greater initial dose of virus

2.4. Mechanism of Action

The mechanism of action of copper or Cu₂O against microbes is one or combination of 3 routes: contact killing, ion release, and generation of reactive oxygen species (ROSs).^{32, 35, 56} In the contact killing route, the microbe needs to physically make contact with the active ingredient. Ion release from copper can be in the forms of simple ions (Cu⁺ and Cu²⁺)³⁵, and possibly hydroxides and/or oxides of copper ions⁵⁷ where the composition of copper species depends on the presence of other ions⁵⁸. These dissolved species are typically cationic and can interact with anionic biomolecules such as DNA⁵⁹, RNA, some lipids, parts of proteins etc. For example, copper species may inactivate metalloenzymes by replacing native metal ions.³² Another mechanism is that Cu₂O is thought to produce ROSs, including superoxide $\left({\text{O}}_2{}^{-}•\right)$, hydroxyl radicals $\left(\text{•OH}\right)$, and hydrogen peroxide (H₂O₂), which can oxidize biological material.³⁵ Typically this oxidation begins with superoxide and/or hydrogen peroxide, which are produced by bacteria but not viruses. The current literature appears to propose that the generation of ROSs does not play a role in inactivation of viruses.^{35, 60}

2.5. The PDA/Cu₂O coating is sprayable

It is desirable that a coating is easy to deploy, and spraying is one of the fastest techniques for coating. The microbial results above are for drop-casted coatings. We made a second set of PDA/Cu₂O coatings by spraying glass with a suspension of Cu₂O suspension in aqueous dopamine (see Experimental section), and subsequently dried the coated slides at 80°C in an oven. The sprayed coating was characterized with UV-Vis, SEM, and XPS (see Figure S9 in Supporting Information). The results were similar to the drop-cast coating, i.e., the coating was transparent with wavelength-independent transmission (Figure S9D), XPS shows a similar copper composition and SEM shows that the particles protrude from the PDA.

Spraying was typically done within about 4–5 minutes after the suspension was prepared. At this time the particles were still suspended. We found that the particles eventually sedimented, but after sedimentation, the particles could be resuspended simply by shaking and then sprayed.

2.6. Other Aspects of the Coating

The polymerization of dopamine is achieved in water, which is an environmental-friendly and non-toxic solvent. Likewise polydopamine is non-toxic: polydopamine nanoparticles injected in a mice have a median lethal dose (LD₅₀) in the range 400 – 585 mg/kg.⁶¹ Although copper has some toxicity to aquatic species, the US EPA allows its use in antimicrobial coatings.⁶² Also, since ancient times, copper has been in wide circulation as coins that are already frequently handled by the public. The cost of copper in 2021 is about $6 per kg, which is not a substantial factor in a coating that is very thin. In some applications, surfaces are wiped periodically with disinfectant. We tested the antimicrobial efficacy of the coating after it was abraded by a sponge and found that the coating still caused a Reduction of ~99.8% (see Table S1). Finally, the process of coating is simple, using spraying or dip coating, with low temperature curing, and no need for expensive equipment.

3. CONCLUSION

Two rapid methods are described for preparing transparent antimicrobial coatings based on PDA using inexpensive and relatively safe materials. These coatings are very effective at killing bacteria and inactivating the SARS-CoV-2 virus. Using a thin layer of copper deposited on a PDA layer, we achieved a Reduction of >99.99% of P. aeruginosa and 99.18% of MRSA within 10 minutes compared to a glass control, as well as 99.98% Reduction of SARS-CoV-2 virus infectivity in 1 hr. Using a sparse layer of Cu₂O bound to, but protruding from PDA, we obtained a Reduction of 99.94% of P. aeruginosa and 96.82% of MRSA within 10 minutes, as well as 99.88% of SARS-CoV-2 virus in 1 hr. compared to a glass control. The activity against SARS-CoV-2 virus is similar to the activity obtained from much greater solids loading in previous work. The coatings are thus highly effective against important pathogens and should find applications on common-use objects to reduce the spread of microbial diseases. A transparent coating is an essential feature for a coating on touch screens, and also extremely desirable for maintaining the visual appeal of an object while achieving a strong antimicrobial effect.

4. EXPERIMENTAL SECTION

4.1. Materials

Glass slides (25 × 75 × 1 mm), 200 Proof ethanol, 70% ethanol, nitric acid (70%, ACS grade), and sodium hydroxide (beads, ACS grade) were purchased from VWR. The following were purchased from Fischer Scientific: dopamine hydrochloride (99%), dimethylamineborane (DMAB, 98%). Cu₂O particles (Chem Copp HP III UltraFine Type −5; mean size = 5.4μm) were purchased from American Chemet Corporation. The following were obtained from Sigma Aldrich: copper(II) chloride (97%), boric acid (ACS reagent), tris(hydroxymethyl)aminomethane (Tris, ACS reagent), and ethylenediaminetetraacetic acid (EDTA, >99%). Tryptic soy broth (TSB) and Tryptic Soy Agar (TSA) were purchased from BD, Sparks, MD. Cell suspensions were diluted in either phosphate-buffered saline (PBS) or DE Broth (BD, Sparks, MD). Water was purified by Milli-Q Reference system. Glass slides were serially cleaned with DI water, 70% ethanol, DI water, nitric acid (6 M), and DI water, and subsequently dried with high pressure nitrogen.

4.2. Fabrication of the surface coatings

4.2.1. Fabrication of PDA/Cu coating:

A two-step procedure was used in which the first step was coating preparation of a thin coating of PDA.^{47, 63} Pieces of cleaned glass slides (12 x 12 mm) were immersed in tris solution (10 mM in water) at 60°C and stirred. Dopamine hydrochloride was added to achieve a final concentration of 5 g dopamine /L and then stirring at 60°C continued for 4 hours. After cooling to room temperature, the glass slides were washed with purified water and dried with nitrogen gas. PDA makes conformal coatings on solids, so we assume that the coating forms on both sides of the glass,⁴⁷ but we always tested the coating on the side that was not touching the container. Copper was deposited onto a preformed layer of PDA, using a method from the literature,^{47, 51–54} but with a thinner layer than in previous work. The copper was electrolessly deposited using an aqueous solution containing EDTA (50 mM), CuCl₂ (50 mM), and boric acid (100 mM) with the pH adjusted to 7 using NaOH solution.^{47, 51–54} The solution was placed on a heater at 37°C under stirring. DMAB was added to achieve a final concentration of 100 mM and subsequently, polydopamine-coated glass-slides were immersed in the solution. The reaction was stopped 30 minutes after the solution color changed to dark green. The samples were washed with DI water and dried with high-pressure nitrogen.

4.2.2. Fabrication of PDA/Cu₂O coating:

A 0.2% w/w suspension of Cu₂O particles in tris solution (10 mM) was sonicated for 1 hr. Dopamine hydrochloride was added to achieve a concentration of 0.05 g dopamine/L. Next, 140 μL of Cu₂O and dopamine suspension was drop cast on 15 x 15 mm pieces of cleaned glass slides, to give a Cu₂O-loading of 1.27 x 10⁻⁴ g/cm². Two to three minutes after deposition, the samples were heat-treated at 80°C in an oven for 30 minutes to dry. Subsequently they were forcefully blown with high pressure nitrogen gas. These samples were coated on one side of glass only.

4.3. Characterization

SEM (JEOL IT500) was used to characterize the morphology of the coatings (the samples were coated with 5 nm of Pt/Pd prior to SEM measurements). XPS (PHI VersaProbe III with a monochromatic Al Kα source of 1486.6 eV) was utilized for measuring of chemical composition of the samples. UV-Vis transmittance was measured using an Agilent model 8453 UV-VIS spectrometer. Water contact angles were measured using First Ten Angstroms FTA125.

4.4. Antibacterial Assay

Microbial Strains.

The microbial strains employed in this study were Pseudomonas aeruginosa strain (DSM-9644) and methicillin-resistant S. aureus (MRSA) strain (MA43300, obtained from the Danville Community Hospital, VA).

Growth of Microbial Strains.

Each bacterial strain was grown in 5 mL of Tryptic Soy Broth (TSB, BD, Sparks, MD) to mid-exponential phase at 37° C with aeration (60 rpm). Following growth, the purity and identity of the cells in the cultures were verified by streaking bacterial cultures on Tryptic Soy Agar (TSA, BD, Sparks, MD) and incubated at 37° C for 48 hr and examining colonies for species-specific traits (e.g., pigmentation and surface texture).

Preparation of Microbial Strains for Testing.

Grown cells were collected by centrifugation (5,000 g for 20 min), the supernatant medium discarded, and the cells were resuspended in 5 mL of sterile phosphate-buffered saline (PBS) by vortexing for 60 sec. Those suspensions were centrifuged (5,000 g for 20 min), the supernatant was discarded, and the washed cells suspended in 5 mL of sterile PBS by vortexing for 60 sec. The number of colony-forming units (CFU)/mL of each washed suspension was measured by spreading 0.10 mL (in duplicate) of serial dilutions in PBS on TSA plates.

Measurement of Cell Number.

The cell number of PBS-suspensions of bacteria were measured as colony-forming units (CFU)/mL of suspension. A 10-fold dilution series was prepared for each suspension in either PBS or DE Broth, 0.1 mL of each dilutions was spread on TSA in triplicate, and colonies were counted after 48 hr. incubation at 37° C. If zero colonies were present for the least dilution, then to enable a log transformation of all data, we designated the zero as one. This is the detection limit, which is an upper bound.

Measurement of Surface-Killing.

For each microbial strain, a 5 μL droplet of bacterial cells in PBS was placed on each coated or uncoated glass sample. Immediately and after 10 min and 20 min (or longer where noted), each sample was transferred to a separate sterile 50 mL centrifuge tube containing 5 mL of PBS or DE broth, vortexed at the highest setting for 10 sec and sonicated for 1 min in a Branson Model 12 Ultrasonic Cleaner (Shelton, CT) to detach cells from the solid, and then the CFU/mL of the resulting bacterial suspension measured by removing a sample of the suspension without coming in contact with the glass sample and preparing a dilution series. The process was repeated at each time point, and three different samples were used for each condition, i.e., each surface coating at each time. Note that the bacterial suspension contacts only one side of the glass.

4.5. SARS-CoV-2 assay

The viral assay methods were described previously.^{25, 27} In summary, the stock virus (BetaCoV/Hong Kong/VM20001061/2020, isolated from a confirmed COVID-19 patient in Hong Kong) was prepared in Vero-E6 cells cultured in Dulbecco’s Modified Eagle Medium with 2% fetal bovine serum and 1% v/v penicillin-streptomycin at 37°C with 5% CO₂. Prior to tests with the virus, all of the surfaces were sterilized with 70% ethanol and air-dried. A 5 μL SARS-CoV-2 droplet at 7.8 log₁₀ unit TCID₅₀/ml was placed on the test solid (temp=22–23°C and 60–70% relative humidity) and after a prescribed time, the solid was soaked in 300 μL of viral transport medium (Earle’s balanced salt solution including 0.5%(w/v) bovine serum albumin and 0.1%(w/v) glucose, pH 7.4) to elute the virus. Subsequently, the eluted virus was titrated by 50% tissue culture infective dose (TCID₅₀) assay in Vero E6 cells.^64–65 In brief, confluent Vero E6 cells on 96-well plates were infected with serially diluted virus in quadruplicates. The infected cells were incubated at 37°C with 5% CO₂. On day 5 post-infection, the cells were examined for a cytopathic effect. The TCID₅₀/ml is the dilution that caused a cytopathic effect in 50% of treated Vero E6 cell cultures. Three independent samples were tested at each condition.

4.6. Calculation of Microbe Survival and Reduction

Microbe survival and Reduction are defined in equations 1–4. We use the word survival for simplicity but acknowledge that the CFU assay measures those cells that can reproduce to form a colony and that the TCID₅₀ assay does not measure numbers of virions.

4.7. Statistical Analysis

The statistical analysis used N-way ANOVA in MATLAB. The error bars were calculated for each figure with Post Hoc multiple comparison using Dunn-Sidák’s approach. We chose a significance level of 0.05. Before statistical analysis, each microbial titer was log₁₀ transformed.

Supplementary Material

am-2021-155057 SI

Supporting Information includes additional methods, characterizations, results, and tables of raw data, as follows:

Figure S1. Schematic of abrasion tests

Figure S2. Particle Size Distribution of Cu₂O particles

Figure S3. Additional SEM images of PDA (prior to copper deposition) and PDA/Cu

Figure S4. Additional SEM images of coatings

Figure S5. XPS results

Figure S6. XPS results of Ar⁺-etched coatings

Figure S7. Bacteria assay for PDA sample

Figure S8. SARS-CoV-2 titer for PDA sample

Figure S9. Characterization of PDA/Cu₂O coating on glass that was prepared by spray-coating

Tables S1-S7. Data tables

Data Availability Statement

The raw data that support the findings of this study are available in the Supporting Information file.