Saeed Behzadinasab1, Myra D Williams2, Mohsen Hosseini1, Leo L M Poon3,4,5, Alex W H Chin3,4, Joseph O Falkinham2, William A Ducker1. 1. Department of Chemical Engineering and Center for Soft Matter and Biological Physics, Virginia Tech, Blacksburg, Virginia 24061, United States. 2. Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061, United States. 3. School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China. 4. Centre for Immunity and Infection, Hong Kong Science Park, Hong Kong, China. 5. HKU-Pasteur Research Pole, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
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 a 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 min and inactivates 99.98% of SARS-CoV-2 virus in 1 h. PDA/Cu2O kills 99.94% of P. aeruginosa and 96.82% of MRSA within 10 min and inactivates 99.88% of SARS-CoV-2 in 1 h.
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 a 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 min and inactivates 99.98% of SARS-CoV-2 virus in 1 h. PDA/Cu2O kills 99.94% of P. aeruginosa and 96.82% of MRSA within 10 min and inactivates 99.88% of SARS-CoV-2 in 1 h.
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, a gram-negative bacterium, is another
causative agent of HAIs and has also been labeled 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,
for example, 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 four 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 modeling
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 seven 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 h (on a dead human skin model[22]) or up to 4 days (on dead pig skin[23]) 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,[30,31] and 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 transparency,[46] 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 a variety of shapes.
The coatings are thus easy to prepare and also consist of inexpensive
and relatively safe materials.These coatings have an 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 min. 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 min. The PDA/Cu and PDA/Cu2O coatings inactivate 99.98 and 99.88%, respectively, of SARS-CoV-2
virus within 1 h, compared to uncoated glass.
Results
and Discussion
Antimicrobial Coatings
are Transparent
The antimicrobial coatings are transparent
and allow for different
colors to be clearly seen when the coatings on glass are held in front
of a computer screen. The visible spectra data (Figure ) confirm 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 a longer wavelength
as expected,[46] which counteracts the absorption
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,C)
photographs of PDA/Cu and PDA/Cu2O coatings, respectively,
held in front of a computer monitor. Both coatings are transparent
and allow for all colors to be seen in transmission. (B,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 PDACu). (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 PDACu). The spectra of the two PDAs (i.e.,
PDACu and PDACu) are similar. To
ensure that coatings formed on one side only for the optical measurements,
the PDACu 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.
(A,C)
photographs of PDA/Cu and PDA/Cu2O coatings, respectively,
held in front of a computer monitor. Both coatings are transparent
and allow for all colors to be seen in transmission. (B,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 PDACu). (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 PDACu). The spectra of the two PDAs (i.e.,
PDACu and PDACu) are similar. To
ensure that coatings formed on one side only for the optical measurements,
the PDACu 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.
Other Characterization of the Coatings
For antimicrobial activity, the active ingredients, that is, 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 A), 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 ) and
additional small-scale roughness is visible (see Figure S3E,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 S3E,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 the 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 and Figure S4.
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 and 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 the PDA/Cu2O coating, we incorporated a heat treatment step at 80 °C for
30 min. 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 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 C,D). XPS data show 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° (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°.
Inactivation
of Microbes
Both Transparent Coatings
Kill Bacteria
in 10 Min
Figure shows very low survival of P. aeruginosa and MRSA on each of the transparent coatings, even after only 10
min. Survival is a comparison between the input titer and the titer
at a later time and is calculated from
Figure 3
Time course of survival of P. aeruginosa (PA) and methicillin-resistant S. aureus (MRSA) on transparent PDA/Cu (left) and
PDA/Cu2O (right)
coatings. Survival was calculated with eq , so zero represents the CFU in the initial
droplet. For the coatings, each symbol represents the average of three
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 the glass control, within 10 min, 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 three-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.
Time course of survival of P. aeruginosa (PA) and methicillin-resistant S. aureus (MRSA) on transparent PDA/Cu (left) and
PDA/Cu2O (right)
coatings. Survival was calculated with eq , so zero represents the CFU in the initial
droplet. For the coatings, each symbol represents the average of three
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 the glass control, within 10 min, 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 three-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 shows that survival
is very high on uncoated glass. Our performance metric is “reduction”,
which is a comparison of the titer on the coated and uncoated samplesThe % reduction by PDA/Cu
is 99.99% (4-logs) for P. aeruginosa and 99.18% (>2-logs) for MRSA within
only 10 min (Figure ). The reduction on PDA/Cu2O is 99.94% (>3-logs) for P. aeruginosa and 96.82% (∼2-logs) for MRSA
in 10 min. This compares favorably to the typical United States Environmental
Protection Agency (EPA) standards of 99.9% (3-logs) in 1 h 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, Figures S7
and S8), confirming that copper and Cu2O are the active
ingredients in the two coatings.
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 h, respectively compared
to a glass control (see Figure ). 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 three 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 were 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 h. 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.
Time course of SARS-CoV-2 titer for transparent
PDA/Cu (left) and
PDA/Cu2O (right) coatings. Each marker shows the average
of three 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 were 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 h. 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 1 h.
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, and so forth, 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 h,[20] while here, ∼99.9% of it is inactivated within 1 h on our
coatings. Note that in the work on copper,[20] 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.
Mechanism of Action
The mechanism
of action of copper or Cu2O against microbes is one or
combination of three 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 ions[57] where the composition
of copper species depends on the presence of other ions.[58] These dissolved species are typically cationic
and can interact with anionic biomolecules such as DNA,[59] RNA, some lipids, parts of proteins, and so
forth. 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 (O2–•), hydroxyl radicals
(•OH), and hydrogen peroxide (H2O2),
which can oxidize biological materials.[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]
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, that is, 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 performed within about 4–5 min 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.
Other Aspects of the Coatings
The
polymerization of dopamine is achieved in water, which is an environmental-friendly
and non-toxic solvent. Likewise, PDA is non-toxic: PDA 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 for
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 that was wetted with
70% ethanol, 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.
Conclusions
Two methods are described for rapidly preparing
transparent antimicrobial
coatings based on PDA using inexpensive and relatively safe materials.
These coatings are very effective at both 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 min compared
to a glass control, as well as 99.98% reduction of SARS-CoV-2 virus
infectivity in 1 h. 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 min, as
well as 99.88% of SARS-CoV-2 virus in 1 h 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.
Experimental Section
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%) and dimethylamineborane (DMAB, 98%). Cu2O particles (Chem Copp HP III UltraFine Type -5; mean size = 5.4
μm) 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.
Fabrication of the Surface Coatings
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 × 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 h. 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, PDA-coated glass-slides were immersed in the solution.
The reaction was stopped 30 min after the solution color changed to
dark green. The samples were washed with DI water and dried with high-pressure
nitrogen.
Fabrication of PDA/Cu2O Coating
A 0.2% w/w suspension of Cu2O particles in tris solution
(10 mm) was sonicated for 1 h. 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 ×
15 mm pieces of cleaned glass slides, to give a Cu2O-loading
of 1.27 × 10–4 g/cm2. Two to 3 min
after deposition, the samples were heat-treated at 80 °C in an
oven for 30 min to dry. Subsequently, they were forcefully blown with
high pressure nitrogen gas. These samples were coated on one side
of the glass only.
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.
Antibacterial Assay
Microbial Strains
The microbial
strains employed in this study were P. 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 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 TSA (BD, Sparks, MD) and incubated
at 37 °C for 48 h 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 (5000g 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 s. Those suspensions were centrifuged (5000g for 20 min), the supernatant was discarded, and the washed
cells suspended in 5 mL of sterile PBS by vortexing for 60 s. 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
h 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 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 s 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, that is, each surface coating
at each time. Note that the bacterial suspension contacts only one
side of the glass.
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.
Calculation of Microbe
Survival and Reduction
Microbe survival and reduction are
defined in eqs –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.
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.
Data Availability Statement
The raw data that support the
findings of this study are available
in the Supporting Information file.
Authors: Ostap Lishchynskyi; Yana Shymborska; Yurij Stetsyshyn; Joanna Raczkowska; Andre G Skirtach; Taras Peretiatko; Andrzej Budkowski Journal: Chem Eng J Date: 2022-05-18 Impact factor: 16.744