Bakır alaşımları esaslı anti-bakteriyel yüzey kaplamalarının üretimi ve karakterizasyonu
Anti-bacterial efficacy of wire arc sprayed copper alloy coatings against various pathogens
Yazar: SAMİ ARDA KOCAMAN
Danışman: PROF. DR. ÖZGÜL KELEŞ
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Metalurji ve Malzeme Mühendisliği Ana Bilim Dalı / Metalurji ve Malzeme Mühendisliği Bilim Dalı
Derece: Doktora, 2022
Konu: Klinik Bakteriyoloji ve Enfeksiyon Hastalıkları = Clinical Microbiology and Infectious Diseases ; Metalurji Mühendisliği = Metallurgical Engineering
Dizin: Antibakteriyel ajanlar = Anti-bacterial agents ; Antibakteriyel aktivite = Antibacterial activity ; Bakır kaplama = Copper plating ; Gram negatif bakteriler = Gram negative bacteria ; Gram pozitif bakteriler = Gram positive bacteria ; Metal kaplama = Metal coating ; Yüzey kaplama = Surface coating ; Yüzey kaplama malzemeleri = Surface coating materials ; Yüzey kaplama yöntemleri = Surface coating methods ; İlaçlara direnç-bakteriyel = Drug resistance-bacterial

Özet:
Günümüz dünyasında artan nüfus ve etkileşim sebebi ile patojenlerin bulaşma riskine karşı mücadelede, antibakteriyel malzemelere daha çok ihtiyaç duyulmaktadır. Medeniyet tarihi boyunca insanlığın yakından aşina olduğu bir metal olan bakırın, antibakteriyel etkisinin bilinirliği de eskidir. Bakırın antibakteriyel özelliği bilinir olmasına rağmen, özgül ağırlığının yüksekliği nedeni ile üretilen parçaların ağır olmaları, sertliğinin düşük ve karakterinin sünek olması sebebi ile işlenme esnasında zorluklar yaşanması ve deforme olmaya yatkınlığı dolayısı ile yapısal bütünlüğünün korunmasında problem olması, bakırın, gündelik objeler içerisinde kullanımını yok denecek seviyede kısıtlamaktadır.
Bu çalışmanın hipotezi, bakır alaşımlarının, farklı yüzeylere uygulanabilir bir kaplama yöntemi olarak seçilen ısıl püskürtme kullanılarak kaplandığında anti-bakteriyel özelliklerinin kütlesel forma göre korunduğu ve bu sayede uygulanabilirliğinin arttırıldığıdır. Diğer bir deyişle, modern dünyada uygulanabilirliği geçerli bakır alaşımlarının kaplama olarak tatbik edildiğinde karakteristik antibakteriyel özelliklerinin korunarak ve hatta kitlesel formda taşıdığı tüm dezavantajlardan sıyrılarak iyileştirdiği, çalışmanın çıkış noktasını oluşturmaktadır.
Çalışmanın, literatürde gerçekleştirilmiş az sayıda mevcut olan benzer çalışmalara göre önemli farklarını oluşturan özgün yönleri ise, geniş alaşım ve bakteri yelpazesinde inceleme yapılmış olmasıdır. Önemle vurgulanması gereken bir diğer unsur da literatürde, bakteriyel testlerdeki indirgenme süreleri uzun zaman aralıklarında incelenir iken, mevcut çalışmada kısa zaman adımları alınarak indirgenme gözlenmiştir. Ayrıca, literatürdeki çalışmalarda, taze kolonilerin statik şartlarda indirgenmesini incelenmiş, bu çalışmada ise, her kontrol sayımı esnasında sürekli taze koloni ekimi ile dinamik şartları temsil eden, sürekli indirgenme etkisini gözlem altına almıştır.
Bakır ve alaşımlarının (Cu-Al, Cu-Sn, Cu-Zn, NiCuZn) kaplanması, termal sprey yöntemi olan ark püskürtme ile sağlanmıştır. Kaplama parametrelerinin iyileştirilmesi için sonlu elemanlar analizi yöntemine dayalı benzetim yapılmış ve doğrulanması da püskürtme ölçüm sistemi ile sağlanmıştır. Parametrelerin kontrollü ele alınması sayesinde, gündelik hayattaki tüm yüzeyler istenilen alaşımlar ile, kaplama sürekliliğine ve altlık ile arasında mukavim bağa sahip olarak kaplanabilmiştir. Kaplamaların karakterizasyonunda yüzey pürüzlülüğü için optik profilometre, yapısal analizi için XRD, morfolojik analiz için SEM kullanılmıştır. Antibakteriyel etkinlik testleri için standart kültür bakterileri ile klinik izole bakteriler, 15 dakika, 1 saat ve 2 saat zaman adımlarında tekrarlı ekimler sonrasında koloni indirgenme sayıları ölçülmüştür. Yüzey pürüzlülüğünün etkisi saf bakır için incelenmiştir. Alaşımlara karşı bakteri dirençleri tek yüzey tipi için, paslanmaz çeliğe karşı kıyaslamalı olarak değerlendirilmiştir.
Bakırın antibakteriyel etkinliği, E. Coli, S. Aureus, PsA başta olmak üzere VRE, MRSA süper bakterilerine karşı da yüksek verimlilik ile (>%99,9) elde edilmiştir. Yüksek yüzey pürüzlülüğüne paralel olarak özellikle tel püskürtme sürecinin doğası gereği yüksek soğuma gradyanları ve yüksek kinetik enerji ile yığma sayesinde, kaplamaların yüksek iç gerilmelere sahip olması, bakterilerin hücre duvarlarının delinmesinde etkili bakır iyonlarının deşarjını olumlu yönde arttırmaktadır. Söz konusu etki mekanizması sayesinde, kısa sürelerde dahi agresif indirgenme davranışı elde edilmiş ve etkinliğin sürekliliğe sahip olduğu da gözlenmiştir.
Elde edilen kaplamalar, gerçek hayatta ihtiyaç duyulan ahşap, polimer, seramik yüzeylere başarı ile tatbik edilerek, diğer doktora çalışmalarından farklı şekilde teknolojik hazırlık seviyesi açısından incelendiğinde, THS7 seviyesinde ürün üretimi de gerçekleştirilmiştir.

Abstract:
Copper is a well-known antimicrobial metal in human civilization that among its various properties such as high thermal, electrical conductivity, and ductility, recently, it has been catching interest due to its biocidal effect. Although exact mechanisms on how copper exerts its biocidal effect are not fully understood but theories assert that copper ions bind to thiol groups near cell walls resulting in respiratory enzyme inhibition which would result in cell membrane wall tearing off, causing depletion of cytoplasm in cell and oxidation of its nuclei. Therefore, copper-based alloys that do not require any supportive agent for activation and that are continuously active independently of external conditions are taken into consideration for anti-microbial applications on touch surfaces.
Pilot installations have been made in medical facilities around the world to investigate biocidal effectivity of copper. Many different types of touch surface applications were realized on these installations including door handles, operating plates, air ventilation ducts, reception tables, etc. When pathogen reduction is concerned, gathered results were overwhelming. However, the feasibility of these applications was an issue due to bulk form of copper which is expensive, heavy, and not competitive by means of structural integrity.  Therefore, applying copper as a coating would make it feasible and would unlock its potential in antimicrobial properties to be carried into real-life applications.
Various technologies are available for coating copper alloys such as electroless coating, electroplating, physical or chemical vapor deposition, cold spray, and thermal spray. Due to its low cost, flexibility and most importantly having no requirement in pre or post processing prior to use, a thermal spraying process known as wire arc spraying is chosen in this study. All thermal spray processes run on same principles that heat load and kinetic energy are transferred to the particles generated from coating material in consideration, but they differ in applicability by the level of output particle speed and velocity. In High Velocity Oxy-Fuel coating process, high speed of the particles damage soft substrate materials. In Plasma Spray coating process, high thermal and radiative load to target surface does affect heat sensitive materials such as thermoplastics, wood, and similar. Cold Spray deposition has good compatibility for wide range of substrate materials but due to its high operation cost related to type and quantity of process gases involved limits its applicability. Therefore, due to its low operation costs while providing a high adhesion with low oxygen and low porosity levels, wire arc spraying stands out sharp among other thermal spray methods. Since wire arc transfers low amount of heat on target, it enables copper alloys to be deposited virtually on any material such as ceramics, polymers and even on wood with no damage. Since many different types of material surfaces are available in hospital environment such as stainless steel, aluminum, wood, ceramics, plastics, etc., it is inevitable to seek for the compatibility of the coating process on these materials which is satisfied by occupying wire arc.
In developing the application for this study, first, a series of coating simulations are made utilizing a finite element method (FEM) package (Comsol Multiphysics) to evaluate the effect of process parameters on particle temperature and speed upon arrival on targets to manage accumulation of heat load that might damage the target while at the same time to optimize splat formation that would directly influence final coating properties. Process is monitored using Accura Spray G3C measurement system to validate particle simulation results. Parallel to particle simulations, results are plugged into droplet simulations and spreading of a droplet while solidifying has been investigated. Regarding optimum parameters gathered via simulations, coatings are realized that are carried to a series of antibacterial activity tests against standard pathogens (E. Coli, Staph. Aureus, PsA) as well as against hard to kill super-bugs such as VRE, MRSA which have not yet been investigated for wire arc thermal sprayed copper alloy coatings. This study differs from available literature in a sense of having tests over a wide range portfolio of bacterium, having fresh colony insertion at measurement intervals to reflect dynamic conditions and by having small time intervals such as 15 minutes, 1 hour and 2 hours.
Computational flow field simulations are based on high Mach number flow approach by occupying quarter symmetry of 3D geometry that includes all barrel, nozzle, and free discharge domains. Non-transient k-ɛ turbulence model is built on air properties which are obtained by an interpolation of the data from the literature and one-way coupled particle tracing is studied based on acquired flow field results including phase transfer within particles. Only primary breakup is assumed to be available, and particles are considered as perfect spheres with homogeneous temperature profile along their radius which is also proved to be valid. Measured data do fit on calculations for particle temperature and velocity by an error margin of %10. Regarding achieved results, FEM based particle trajectory and particle temperature simulations during travel until the target helped optimizing spraying distance which is set so to get the highest number of liquid fractions maximizing deposition rate with the lowest temperature of particles minimizing damage to substrate. Having a balance in process parameters are uttermost importance to droplet spreading phenomena when droplets reach target and start accumulating on top of each other. Therefore, droplet simulations based on phase-field method including phase change helped study the optimum parameter range of particles to achieve ideal coating formation scenarios. Sensitive surfaces such as wood and thermoplastics therefore are made possible to be coated with proposed coating parameters as well thanks to insight gained by the aid simulation results.
Coatings are applied to coupons with a diameter of 25.4 mm, made from 316L sheets of 2 mm thickness. Twin wire-arc system Sparc 400 from GTV is used with a converging diverging nozzle which is fed by 1.6 mm diameter wire stocks. Coupons are positioned on a specially designed sample holder which is held on 4 axis robotic platform. Prior to coating, substrates are cleaned with alcohol and sand blasted with 36 grit aluminum oxide particles. For comparison of surface roughness on antimicrobial efficiency, group of samples are blasted with 16 grit aluminum oxide particles resulting in three times higher roughness on as sprayed profiles. The plain stainless-steel substrate that is in comparison in antibacterial tests was also sand blasted to overcome any error in the bacterial efficacy investigation.
After completion of coating production, all specimens have been analyzed for thickness, porosity, roughness, and oxygen content. Coating thickness and coating morphology is determined via coating surface and cross-section by scanning electron microscopy (SEM) from JEOL 5410. Surface roughness of as sprayed coatings are measured by 3D Optical Profilometer from Veeco. Oxygen content analysis for the coating are determined by XRD from Philips (PW3710 System) with a 2θ range of 30–140o steps of 0.02o (with CuKα at 40 kV and 30 mA).
Antibacterial tests are done with respect to a modified procedure based on Environmental Protection Agency Test Method for the Continuous Reduction of Bacterial Contamination on Copper Alloy Surfaces (EPA-800R09004). All tests were carried to reflect dynamic conditions by adding fresh colonies at observation intervals onto previous ones to see reduction effect in continuous pattern. Carrier samples were cleaned with alcohol, rinsed with deionized water and air dried. Five tests were carried per sample per organism per time. S. Aureus and E. Coli are taken from standard stock cultures while PsA, VRE and MRSA were clinically isolated. To produce fresh cultures from root cultures, bacteria were held at 37°C for 24 hours. The fresh cultures were then put into a suspension of 0.85% saline (0.5 Mc Farland) and later were diluted by 1/10,000 ratio followed by inoculation to Mueller Hinton agar plates using sterile calibrated pipettes by 100μl of each. Following inoculation, each specimen was held at 37°C for separate set of time intervals (15 min, 1 h, 2 h). Inoculums were taken and harvested on Mueller Hinton agars and incubated for 48 hours at 37°C after exposure. Colony counting was done for after the incubation where number of colony forming units (CFU) was gathered from the growth of viable bacteria 37°C after 24h. Tests were repeated five times for each sample for statistical reliability.
Tests results show that control sample of 316L stainless-steel, has a very weak antibacterial rate comparatively to copper alloys. Copper alloy coatings continuously reduced bacterial count, even re-inoculation was done at set time intervals. One may have expected that re-inoculation on top of dead colonies would promote bacterial growth positively, since dead colonies would act as a feeding site for fresh colonies. However, the high effectivity of copper did continuously kill any upcoming fresh colonies and was faster in killing bacteria then it could have multiplied as well. E. Coli, S. Aureus and PsA completely vanished from the surface within 15 minutes. Superbugs such as VRE and MRSA were also completely removed from the surface successfully in 2 hours. VRE had shown a resistance at initial 15 minutes but was effectively eliminated at further intervals and MRSA survived until 1 hour but as well was eliminated successfully in 2 hours.
Copper has a reduction mechanism based on ion discharge which is complex and involves many external parameters including environmental temperature, relative humidity, surrounding medium which were kept constant during the tests. Therefore, reduction rate is related to copper ion availability on the surface which is the dominant mechanism in killing bacteria. When copper surfaces get in contact with water, or even with organism's fluid, ion discharge rises. The copper ions attack the bacteria by rupturing their cell membrane and by binding with their enzymes. This phenomenon is universal to all bacterium types and by being non-selective it works on any living organism. It is proposed that unique semi-porous, rough texture of wire-arc sprayed the coating would have a profound influence in the reduction of bacterial count however pure copper did kill every bacterium in test as good as it did with its polished surface in comparison, showing that surface roughness has no measurable influence on effectivity. However, it should be stated that the porous structure of wire-arc coatings with micro-cracks and micro-holes does host sites for oxygen release and copper solute transport. Additionally, high energy grain boundaries and internal stresses formed due to rapid cooling upon impact in the wire-arc spray process, help ions to discharge more. Therefore, wire arc sprayed copper coating showed tremendous reduction ability. An important aspect for copper itself to note is that the killing rate of copper is high enough to overcome horizontal gene transmission. 
Gram positive bacteria such as S. Aureus has a thick but a loose cell that makes diffusion easier. However, gram negative bacteria such as E. Coli has a thinner but multi layered outer membrane over its cell that makes diffusion slightly slower which enables this type of bacterium resist to chemical agents more. However, no difference was observed in biocidal performance when the coating is exposed to both gram negative or positive bacteria. The antibacterial efficacy slowed down for superbugs which are known to have thicker cell membranes, possibly causing a harder ion diffusion through the cell membrane. Characteristics of the wire arc copper alloy coatings, that are rough surfaces, having pores and internal stress in its structure have made possible to effectively eliminate all bacterium types in short time.
After achieving desired results, real-life examples have been created by acquiring real-life objects and coating related alloys on their surfaces. Having post surface treatments over coated active copper alloys on ceramic, thermoplastic, wood surfaces proved that this technology is ready for field use and installations, which sets the technology readiness level (TRL) to 7 for this project.