coatings

Article Kinetically Deposited Copper Antimicrobial Surfaces

Victor Champagne 1, Kristin Sundberg 2 and Dennis Helfritch 3,*

1 Army Research Laboratory, Aberdeen Proving Ground, MD 21005-5201, USA; [email protected] 2 Worcester Polytechnic Institute, Worcester, MA 01609, USA; [email protected] 3 Survice Engineering, Aberdeen, MD 21017, USA * Correspondence: [email protected]; Tel.: 443-890-7813



Received: 13 February 2019; Accepted: 11 April 2019; Published: 17 April 2019


Abstract: Bacterial and viral contamination of contact surfaces increases the risk of infection. A great deal of work has been done on the capabilities of copper and its alloys to protect against a variety of microorganisms endangering public health, particularly in healthcare and food processing applications. This work has conclusively shown the effectiveness of copper for touch surface disinfection; however, the optimum microstructural characteristics of the copper surface have not been established. The sterilization effectiveness of three kinetically sprayed copper surfaces and two copper feedstocks were examined. The surfaces were inoculated with methicillin-resistant Staphylococcus aureus (MRSA) and influenza A virus. After a two-hour exposure to the surfaces, the surviving microorganisms were assayed, and the results contrasted. These tests showed substantial antimicrobial differences between the coatings generated by the spray techniques and those obtained by different feedstock powders. The significance of the copper spray application was demonstrated, and the application-dependent mechanism for antimicrobial effectiveness was explained.

Keywords: antimicrobial; surface; copper; powder; spray

1. Introduction Microbial contamination of surfaces directly increases infections in hospitals and food processing facilities [1]. The microbial contamination of hospital surfaces, including patient rooms, nurse stations, and kitchens is well documented [2–5]. It is known that airborne influenza nuclei can infect environmental surfaces, leading to human infection with hand-to-surface contact [6–8]. In addition to the required cleaning practices, self-disinfecting surfaces would clearly inhibit the spread of infectious diseases. Laboratory studies have demonstrated copper’s ability to kill more than 99.9% of the following disease-causing bacteria within 2 h of contact time: Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli O157:H7, Pseudomonas aeruginosa, vancomycin-resistant Enterococcus faecalis (VRE), and methicillin-resistant S. aureus (MRSA). Increasing the copper content of alloys improves their antimicrobial effectiveness [9]. Rapid disinfection prevents the generation of protective biofilms [10]. The interaction of copper ions with cellular membranes resulting in a flow of ions in the intracellular matrix is hypothesized to be the microbial-killing mechanism [11]. TEM images show cytoplasm leakage from S. aureus and E. coli cell membranes on copper surfaces. These data show copper ion presence in the membranes of the microbes and indicate that the primary killing mechanism is membrane destruction by copper ions [12]. Viruses, as well as bacteria, have been shown to be killed by contact with copper. The Center for Disease Control and Prevention describes influenza transmission mechanisms as the airborne spread of infectious droplets onto individuals or surfaces [13]. Influenza A is a major cause of infection and

Coatings 2019, 9, 257; doi:10.3390/coatings9040257 www.mdpi.com/journal/coatings Coatings 2019, 9, 257 2 of 9 mortality of young children and the elderly [14]. Between 20 and 30 million people died in the influenza pandemic of 1918. During the 2012–2013 influenza season, the deaths attributed to pneumonia and influenza in the United States peaked at 9.9%, and exceeded the epidemic threshold for 13 consecutive weeks [15]. The Copper Development Association lists more than 400 copper alloys that are registered with the U.S. EPA as antimicrobial [16], and all surfaces have a minimum copper content of 60%. These registered materials can be utilized for the control and inactivation of microbes on frequently touched surfaces in hospitals [17].

2. Materials and Methods Surfaces that make contact with people and foods should be composed of copper or copper alloys, which can be achieved with solid copper or through a copper surface coating. Copper coatings are favored over solid structural copper because of cost considerations. Several metal spray methods can be used for depositing a copper surfaces, and an optimum deposition method should be identified.

2.1. Antibacterial Surface Coating Three metal spray techniques were evaluated regarding the antibacterial activity of the copper surfaces produced by each. The commercial metal spray techniques evaluated were cold spray (high speed, kinetically deposited solid-state powder particles), plasma spray, and wire arc spray (moderate speed, kinetically deposited molten particles). The copper powder used for the antibacterial tests was ACuPowder 500A (mean diameter 16.9 µm, ACuPowder, Union, NJ, USA) for cold spray. The plasma arc used Praxair Cu-159 ( 31/+5 µm, Praxair, Danbury, CT, USA), and the wire arc spray used copper − wire as feedstock. Approximately 0.3 mm-thick coatings were sprayed onto stainless steel coupons. Table1 gives the estimated particle impact conditions for the three processes, as well as the porosity and oxide ranges of the resulting deposits. Ranges for particle velocities and temperatures result from the range of particle diameters present in the powders. Porosity and oxide ranges are typical for the three spray methods.

Table 1. Particle impact and deposition characteristics.

Spray/Property Temperature ◦C Velocity m/s Porosity % Oxides % Plasma Arc 1500–2500 100–400 ~5 ~2 Wire Arc 1500–2500 50–100 ~10 ~15 Cold Spray 150–400 500–1000 <1 <1

Figure1 shows cross sections of the deposits generated by the three spray methods. Di fferences in microstructures can be expected on the basis of the large differences experienced by impacting particles for the three methods. These differences in microstructure suggest that a variance in microbial destruction may also occur. Particle melting can be seen for the high-temperature plasma and wire arc processes. Large voids can be seen in the wire arc process cross-section. Coatings 2019, 9, 257 3 of 9

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Figure 1. Deposited copper cross sections. Plasma (a), wire arc (b), cold spray (c). Stainless steel FigureFigure 1. 1. DepositedDepositedDeposited copper copper copper cross cross cross sections. sections. Plasma Plasma ( (aa),), w wirewireire a arcarcrc ( (bb),), coldccoldold spraysspraypray ( (c (cc).)). . StStainless Stainlessainless steel steel substrates can be seen at the bottom. substratessubstratessubstrates can can be be seen seen at at the the bottom. bottom. 2.2. Antiviral Surface Coating 2.2.2.2. AntiviralAntiviral SurfaceSurface CoatingCoating Antiviral effectiveness was compared for cold-sprayed conventional copper powder feedstock AntiviralAntiviral effectivenesseffectiveness waswas comparedcompared forfor coldcold--sprayedsprayed conventionalconventional coppercopper powderpowder feedstockfeedstock and nanometer-particle-diameter feedstock powder. The nano copper powder was composed of andand nanometer nanometer--particleparticle--diameterdiameter feedstock feedstock powder. powder. The The nano nano copper copper powder powder wawass composed composed of of micron-sized agglomerates (mean 25 µm) of nanoscale particles. The conventional (Praxair Cu-159) micronmicron--sizedsized agglomeratesagglomerates (mean(mean 2525 µm)µm) ofof nanoscalenanoscale particles.particles. TheThe conventionalconventional (Praxair(Praxair CuCu--159)159) and nano (Eltron Research and Development Inc., Boulder, CO, USA) copper powder feedstocks used andand nano nano (Eltron (Eltron Research Research and and Development Development Inc. Inc.,,, Boulder, Boulder, CO, CO, USA USA)) copper copper powder powder feedstocks feedstocks us useded for cold sprays are shown in Figure2. forfor coldcold sprayssprays areare shownshown inin FigureFigure 2.2.

Figure 2. Conventional (a) and nanoscale (b) copper powders. FigureFigure 2. 2. Conventional Conventional ( (aaa))) and andand nanoscale nanoscalenanoscale ( ((bb))) copper coppercopper powders. powders.powders.

CrossCross sections sections of ofof the thethe deposits deposits generated generated by by cold cold spray spray for for antiviral antiviral teststests areare shownshown inin FigureFigure 3.33.. TheThe deposits deposits produced produced byby bothboth powderspowderspowders formedformedformed densedense coatings.coatingscoatings... T TheThehe nano nano-structurednano--structuredstructured deposit deposit wa waswass nonnon-uniform,non--uniform,uniform, exhibiting exhibiting microstructural microstructuralmicrostructural differences. didifferences.fferences. This This may may be be representative representativerepresentative of ofof the the non non-uniformnon--uniformuniform naturenature of of of the the the nano nano nano feedstock. fee feedstock.dstock. The The The variance variancevariance inmicrostructures ininin microstructure microstructure microstructure impliesss impliesimpliesimplies a diff erence aa differencedifference in biological ininin biological biological biological activity activitybetweenactivity betweenbetween conventional conventionalconventional and nano andand copper nanonano surfaces. coppercopper surfaces.surfaces.

Figure 3. Cross-sectional views of the conventional powder (a) and the nanoscale powder (b) cold- FigureFigure 3. 3. CrossCrossCross-sectional--sectionalsectional viewsviews views ofof theofthe the conventionalconventional conventional powderpowder powder ((aa)) andand (a) the andthe nanoscalenanoscale the nanoscale powderpowder powder ((bb)) coldcold (b-)- sprayed deposits. sprayedcold-sprayedsprayed deposits.deposits. deposits.

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2.3.2.3. Microbiological Microbiological Procedure Procedure TheThe copper copper-coated-coated coupons coupons generated generated by by the the thermal thermal spray spray devices devices in in Section Section 2.1 2.1.,, as as well well as as structuralstructural copper copper coupons coupons,, were were inoculated inoculated with with MRSA. MRSA. The The copper copper-coated-coated coupons coupons produced produced by by coldcold sp sprayray for for the the conventional conventional and and nanoscale nanoscale feedstock feedstock powders powders were were inoculated inoculated with with influenza influenza A virus.A virus. Stainless Stainless-steel-steel coupons coupons were were used used as control as control substrates substrates for both for bothbacterial bacterial and viral and viraltests. tests.The inoculatedThe inoculated coupons coupons were were then then kept kept at room at room temperatur temperaturee for for a a given given time time period, period, after after which which survivorssurvivors were were re re-suspended-suspended and and cultured. cultured. The The EPA EPA Protocol Protocol,, “Test “Test Method Method for for Efficacy Efficacy of of Copper Copper AlloyAlloy Surfaces Surfaces as as a Sanitizer” a Sanitizer” was was followed followed for the for bacteria the bacteria tests [18 ]. tests The [18] EPA.T antimicrobialhe EPA antimicrobial procedure procedurewas used with was someused changeswith some for changes the influenza for the A influenza tests as there A tests is no as EPA there copper is no surfaceEPA copper procedure surface for procedurevirus. The reduction for virus. of The inoculated reduction microbes of inoculated was normalized microbes by was the normalizedresults of the by control the results exposure of tothe a controlstainless-steel exposu surface.re to a Thestainless bacterial-steel measurements surface. The bacterialwere taken measurements after 2 h of continuous were taken exposure after 2 to h the of continuoussurfaces. The exposure viral measurements to the surfaces. were The taken viral after measurements several intermediate were taken exposures after several up to intermediate 2 h. The full exposuresdetails of theup proceduresto 2 h. The full are details given in of reference the procedures [19,20]. are given in reference [19,20].

3.3. Results Results FigureFigure 44 showsshows thethe percentpercent ofof survivingsurviving S.S. aureus after 2 h of surface exposure. The The results results for for coldcold-sprayed-sprayed and and structural structural copper copper were were below below the the measurement measurement thresholds thresholds and and are are thus thus reported reported asas “less “less than”. than”. A A three three order order of of magnitude magnitude difference difference in disinfection efficiency efficiency between the the plasma plasma andand wire wire arc arc methods and and the the cold spray method was observed observed.. Differentiating Differentiating between between the the results results forfor the the cold cold-sprayed-sprayed surface surface and and the the structural structural copper copper surface surface wa wass not not possible, possible, because because of of the the measurementmeasurement limit. limit.

FigureFigure 4. 4. PercentPercent methicillin-resistant methicillin-resistantStaphylococcus Staphylococcus aureus aureus(MRSA) (MRSA surviving) surviving after a two-hour after a two exposure-hour exposureto copper to surfaces. copper surfaces.

TheThe results results for for viral viral killing killing are are shown shown in in Figure Figure 55.. As opposed to expectations, thethe result forfor thethe conventionalconventional copper copper cold cold-sprayed-sprayed surface surface was was very very similar similar to to that that of of the nano copper cold cold-sprayed-sprayed surface,surface, so so it it is is not shown. The The results results obtained obtained for for the the inactivation inactivation of of i influenzanfluenza A A on on structural ((wrought)wrought) copper copper (C1100) (C1100) [21] [21] were added to the graph graph for for comparison. comparison. The The difference difference in in procedure procedure betweenbetween this this current current work and that of Noyce et al al.. is is that that this this work work utilized utilized the the TCID TCID5050 AssayAssay Protocol, Protocol, whilewhile Noyce, Noyce, et et al al.. use usedd a fluorescent a fluorescent dye dye that that illuminate illuminatedd viral viral particles particles that thatwere were still infectious still infectious and countand counteded virus virusparticles particles recovered recovered from fromthe coupons the coupons under under a microscope. a microscope. Measurements Measurements were taken were afttakener 10, after 30, 10,45, 30,60, 45,90, 60,and 90, 120 and min 120 exposures min exposures for the forcurrent the current work, while work, measurements while measurements were made were aftermade 60 after and 60 360 and min 360 minexposure exposuress for wrought for wrought copper. copper. In Inboth both cases, cases, the the number number of of survivors survivors on on stainlessstainless-steel-steel controls controls were were orders orders of of magnitude magnitude higher higher than than on on th thee copper copper surfaces. surfaces. For For clarity, clarity, the the endend points points of of the the cold cold-sprayed-sprayed copper copper data data and and the the wrought wrought copper copper we werere 0.13% 0.13% at at 120 120 min min and and 0.03% 0.03% atat 360 360 min min,, respectively. respectively.

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FigureFigure 5. 5.Percent Percent of of influenza influenza A A survivors survivors as as a a function function of of exposure exposure time time to to copper copper surfaces. surfaces.

4.4. Discussion Discussion TheThe antimicrobial antimicrobial eff ectiveness effectiveness obtained obtained with sprayed with sprayed copper depositioncopper deposition methods in comparison methods in withcomparison that achieved with with that copper achieved feedstocks with copper needed feedstocks an examination neededto an determine examination the etoffects determine of powder the feedstockeffects of andpowd depositioner feedstock process and deposition on the copper proce surface.ss on the In copper order to surface. relate the In order nature to of relate the deposit the nature to itsof antimicrobial the deposit to e ffitsectiveness, antimicrobial we neededeffectiveness, to know we the need mechanismed to know by the which mechanism contact killingby which occurred. contact killingRecent occur studiesred. show that significant numbers of copper ions, Cu(II), are taken up by E. coli over a 90 minRecent period, studies when anshow aqueous that significant suspension numbers (a standing of copper drop) isions, used Cu(II), to apply are cells taken to up copper by E. coupons. coli over Whena 90 min cells period, are plated when on copperan aqueous using minimumsuspension liquid (a standing and a drying drop) time is used of 5 s,to theapply buildup cells ofto coppercopper ionscoupons by the. When cells was cells even are plated greater, on quickly copper reachingusing minimum a high concentration. liquid and a drying The copper time of ion 5 s,level the buildup in the cellsof copper remained ions high by the throughout cells was the even disinfection greater, quickly phase, suggestingreaching a thathigh the concentrat cells becameion. The overwhelmed copper ion bylevel intracellular in the cells copper remain [22ed]. high throughout the disinfection phase, suggesting that the cells became overwhelmedCopper ions by added intracellular in solution copper to influenza [22]. A virus resulted in morphological abnormalities [23]. StudiesCopper show ions an increased added in sensitivitysolution to ofinfluenza covered A viruses virus result to coppered in morphological ions as compared abnormalities to uncovered [23]. viruses,Studies indicating show an i thatncreased the lipid sensitivity membrane of covered of the virus viruses becomes to copper overwhelmed ions as compared by ionic copper, to uncovered which isviruses, similar toindicating the bacterial that disinfection the lipid membrane mechanism of [24 the]. virus becomes overwhelmed by ionic copper, whichThus, is similar ionic copper to the b isacterial likely responsibledisinfection for mechanism microbial [24] killing;. hence copper layers that maximize ionic copperThus, ionic diffusion copper to theis likely surface responsible are the best for antimicrobials.microbial killing; Ionic hence diff usioncopper in layers metals that is enhanced maximize byionic grain copper dislocations. diffusion Known to the surface as “pipe are di theffusion”, best antimicrobials. significant ionic Ion diicff diffusionusion proceedsthrough in metals is enhanced these dislocations.by grain dislocations. The relationship Known between as “pipe dislocation diffusion”, density significant and ionic didiffusionffusivity proceeds is giventhrough by [25]: these dislocations. The relationship between dislocation" density and!# ionic diffusivity is given by [25]: 2 Dd Deff = D0 1 +2 πa 퐷ρdd 1 (1) 퐷eff = 퐷0 [1 + π푎 ρd ( ⁄ D −−1)] (1) 퐷00

where Deff is the effective ionic diffusivity; D0 is the lattice diffusivity; Dd is the pipe diffusivity; ρd is where Deff is the effective ionic diffusivity; D0 is the lattice diffusivity; Dd is the pipe diffusivity; ρd is thethe dislocation dislocation density; densitya; ais is the the average average dislocation dislocation radius. radius. InIn addition addition toto dislocations,dislocations, increasedincreased graingrain boundaries caused by by impact impact breakup breakup result result in in a asimilar similar augmentation augmentation of of didiffusion.ffusion. The The generation of of ultra-fine-grained ultra-fine-grained structures structures (including(including dislocations,dislocations, dislocation dislocation cells, cells and, and twinning) twinning caused) caused by the by high the velocity high velocity impact of impact cold spray of cold particles spray hasparticles been extensivelyhas been extensively documented documente [26–28].d [26 Figure–28]6. Figureshows 6 the shows extreme the extreme plastic plastic deformation deformation of a copperof a copper particle particle after impactafter impact by cold by spraycold spray [29]. A[29] jet. A of jet plasticized of plasticized material material is typically is typically ejected ejected at the at particle–substratethe particle–substrate interface. interface.

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Figure 6. Example of cold-sprayed copper particle impact.

Gubicza et al. [30] measured the change in dislocation density by means of X-ray peak profile analysis of an initially Figure annealedFigure 6. 6.Example Example copper of of specimen cold-sprayedcold-sprayed, which copper copper wa particles particle subsequently impact. impact. deformed by equal channel angular pressing. Their results are shown in Figure 7. The imposition of strain to the Gubicza et al. [30] measured the change in dislocation density by means of X-ray peak profile specimen,Gubicza similar et al. to [30 what] measuredFigure would 6. Examplehappen the change ofto cold cold in-sprayed dislocation-sprayed copper particles, densityparticle impact.can by increase means of the X-ray displacement peak profile analysis of an initially annealed copper specimen, which was subsequently deformed by equal analysisdensity ofby an a factor initially of annealedsix. copper specimen, which was subsequently deformed by equal channel channel angular pressing. Their results are shown in Figure 7. The imposition of strain to the angularLuo pressing.Gubicza and Li [31]et Their al. show [30] results edmeasure how are thed shown the dislocation change in Figure in density dislocation7. The of imposition cold density-sprayed by of means straincBN/NiCrAl of to X the-ray specimen,wa peaks decreased profile similar analysisspecimen, of similar an initially to what annealed would copperhappen specimento cold-sprayed, which particles,was subsequently can increase deformed the displacement by equal tofrom what its would original happen cold-sprayed to cold-sprayed condition particles,as the coating can increasewas annealed. the displacement Figure 8 shows density a decrease by a factorof channeldensity by angular a factor pressing. of six. Their results are shown in Figure 7. The imposition of strain to the ofseveral six. orders of magnitude as the annealing temperature was increased from 750 to 900 °C. specimen,Luo and similar Li [31] to showwhated would how thehappen dislocation to cold density-sprayed of coldparticles,-sprayed can cBN/NiCrAl increase the wa displacements decreased densityfrom its by original a factor cold of six.-sprayed condition as the coating was annealed. Figure 8 shows a decrease of severalLuo orders and Li of [31] magnitude showed howas the the annealing dislocation temperature density of wacolds increased-sprayed cBN/NiCrAl from 750 to 900was °C. decreased from its original cold-sprayed condition as the coating was annealed. Figure 8 shows a decrease of several orders of magnitude as the annealing temperature was increased from 750 to 900 °C.

Figure 7. Copper dislocation density as a function of strain. Figure 7. Copper dislocation density as a function of strain.

Luo and Li [31] showed how the dislocation density of cold-sprayed cBN/NiCrAl was decreased Figure 7. Copper dislocation density as a function of strain. from its original cold-sprayed condition as the coating was annealed. Figure 8 shows a decrease of several orders of magnitudeFigure as the7. Copper annealing dislocation temperature density as a was function increased of strain from. 750 to 900 ◦C.

Figure 8. The displacement density of cold-sprayed cBN/NiCrAl as it is annealed. Figure 8. The displacement density of cold-sprayed cBN/NiCrAl as it is annealed.

FigureFigure 8. The 8. The displacement displacement density density of cold cold-sprayed-sprayed cBN/NiCrAl cBN/NiCrAl as it as is it annealed. is annealed.

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Thus, the process of cold-spraying copper can improve copper antibacterial activity through the generation of dislocations. The known effect of work hardening by cold spray is also a result of dislocation density growth. The results showed that bacterial inactivation by cold-sprayed copper surfaces was more complete than viral inactivation. Hardier viral defenses may be one explanation. Roughness of the copper surface can contribute to improved contact-killing levels of influenza A virus by cold-sprayed copper, as opposed to wrought or structural copper, as shown in Figure5. Bacteria such as MRSA range in size from 0.5 to 1.0 µm in diameter, while viruses such as influenza A are on an 80–120 nm scale [32,33]. The size difference may have affected the ability of viruses to be fully affected by the extended surface area of cold-sprayed surfaces. While not explicitly considered in this effort, the effects of copper surface oxidation on antimicrobial effectiveness has been studied by Hans et al. [34]. They found that the predominant oxide formed by atmospheric oxygen, Cu2O, did not affect copper ion transport or antimicrobial effectiveness; however, CuO, formed by wet oxidation, could decrease the effectiveness.

5. Conclusions The significant anti-microbiological differences between copper coatings originating as powders or as wrought material and the differences between powder application techniques demonstrate the importance of the deposition structure. The cold spray method showed greater antimicrobial effectiveness relative to the other two thermal spray methods, due to the much greater impact velocity of the sprayed particles which resulted in high dislocation density and high ionic copper diffusivity. Cold spray is a commercial process, used for a variety of applications requiring metal coatings. The cold spray process can thus apply effective anti-microbial copper coatings onto touch surfaces and will not damage heat-sensitive substrates. For example, a hospital tray and its entire metal support structure was coated with cold-sprayed pure copper at the Army Research Cold Spray Center.

Author Contributions: Conceptualization, V.C. and D.H.; Methodology, K.S. and D.H.; Formal Analysis, V.C.; Investigation, D.H. and K.S.; Writing—Original Draft Preparation, D.H.; Writing—Review and Editing, V.C.; Supervision, V.C.; Project Administration, V.C. Funding: This research received no external funding. Acknowledgments: The sanitizer testing conducted against MRSA and influenza A virus and reported in this paper was carried out under contract by ATS Labs at Eagan MN. The work was performed by Amy Jeske, Becky Lien, and Shanen Conway. Conflicts of Interest: The authors declare no conflict of interest.

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