The Effect of Cu Additions on the Antibacterial Properties of Metallic Glassy Ni₅₀TM₅₀ (TM; Ti, Zr) Binary Systems

Abstract

Antibacterial agents derived from classic organic compounds have been frequently employed for a number of years as a protective layer for biofilms. On the other hand, these agents often comprise dangerous components that, due to their interaction with toxic compounds, may be damaging to human beings. This hazard may be caused by the agents’ proximity to the toxic substances. Over the course of the past three decades, a variety of approaches, such as the utilization of a broad spectrum of metallic and oxide materials, have been the subject of research in order to develop a diverse selection of antibacterial coating layers that are acceptable. One of these approaches is the use of silver nanoparticles. It has been established that the cold spray technique, a solid-state method compatible with nanopowders, has shown higher performance and is the most effective strategy for coating materials. This has been proven via testing. It is possible to produce one-of-a-kind material coatings in ways that are not even remotely imaginable with any other thermal coating method, which is the primary reason for its prominence in contemporary production. The capacity to do so is what provides it with an advantage over its rivals in the market. This current study was conducted, in part, to investigate the effects of Cu-alloying elements on the antibacterial behavior of metallic glassy alloys on Ni₅₀TM₅₀ (TM; Ti, Zr) and Cu₅₀TM₄₀Ni₁₀ (TM; Ti, Zr) systems prepared by the mechanical disordering technique, in conjunction with the cold spray method. These alloys were created by combining the mechanical disordering technique with the cold spray method. The arc melting process was employed to generate master alloys consisting of Ni₅₀Ti₅₀, Ni₅₀Zr₅₀, Cu₅₀Ti₄₀Ni₁₀, and Cu₅₀Zr₄₀Ni₁₀ for the purpose of this investigation. The master alloys were then used as feedstock materials for the creation of metallic glassy powders. Following the pulverization of the alloys of each system into a powdered form, the mixtures were charged through a high-energy ball milling operation for a duration of 50 h. Using the cold spray technique, the as-milled powders, which were metallic glasses, were applied singly in order to coat SUS304 sheets. The method was employed for this purpose. After the addition of Cu to the two binary Ni₅₀TM₅₀ (TM; Ti, Zr) alloys, the antibacterial properties of their corresponding metallic glassy phases were found to be significantly enhanced. This was shown by the fact that they were successful in preventing the development of biofilm by E. coli in contrast to the other systems that were evaluated.

1. Introduction

Most of the technologically significant alloys are, in some respect or another, metastable [1]. This indicates that they deviate from a state of equilibrium in their compositions because they do not have enough free energy of the lowest attainable value. In order to develop new alloys that have enhanced characteristics, it is often necessary to increase the amount of displacement from equilibrium, in either the final product or in a stage of the manufacturing process that comes in between. Metallic glasses (MGs) are a type of metastable alloy, which came into existence during the last sixty years as a result of the use of the novel preparation procedures employed for obtaining significant deviations from equilibrium [2]. The rapid solidification of metals or vapors, the atomic disordering of crystal lattices, solid-state reactions between metallic thin films, and high-energy ball milling are some of the techniques that have been utilized to conduct huge departures from equilibrium [3]. The absence of the long-range atomic order, which is characteristic of crystals, is the feature that serves as the distinguishing characteristic of this newly discovered group of MGs [4]. Therefore, even though MGs have such a distinctive short-range order structure, it was possible to incorporate new properties into these new materials. Some of these new properties include a high mechanical ductility and yield strength, high magnetic permeability, low corrosive forces, unusual corrosion resistance, temperature-independent electrical conductivity, and a great number of other properties [1,4]. As a consequence, MGs have emerged as potentially advantageous candidates for high-tech applications across almost the whole spectrum of industrial, medical, athletic, and life science fields [5]. Recent researches on several families of MGs reveal that some of them have antibacterial properties that are superior to those of stainless steel, which is a metal that is often used in the manufacturing and preservation of medical and food sectors [6].

Surface engineering is concerned with the design and modification of the surfaces of bulk materials in order to obtain certain physical, chemical, and technical qualities that are not intrinsically included in the original bulk materials [4]. The development of mature biofilm on surfaces may result in a significant loss in a variety of different industrial sectors, including the food industry, water systems, and surroundings in health care [7].

Because biofilm formation is responsible for the widespread development of bacterial resistance to antibiotic treatments, it is necessary to fabricate antibiofilm-coated surfaces that are both effective and safe to apply [8,9]. The inadequate attention that has been given to the analyses and testing of the bioactive components that are present in the protective coating [10,11,12] presents a barrier for products that are now available on the market. This has been a source of difficulty for those products. Due to the delayed antibacterial activity and accompanying toxicity of silver compounds, researchers are under rising amounts of pressure to produce an alternative that is less dangerous [13,14]. It is still proving to be a challenging endeavor to develop a covering that is effective against microorganisms on a global scale and is appropriate for usage, both inside and outdoors. This is a result of the fact that there are connected risks to not only one’s health but also one’s safety.

The objective of discovering an antibacterial agent that is less hazardous to human health, and determining how to include it into a coating matrix that has a longer shelf life, is being pursued with a great deal of interest [15]. The most modern antimicrobial and antibiofilm materials have been developed with the goal of killing bacteria, either via direct contact or being in close vicinity after the active agent has been released. This goal may be accomplished by either blocking the initial bacterial attachment, which requires counteracting the creation of a protein coating on the surface, or by killing bacteria by disturbing the cell wall. Both of these strategies are viable options.

Austenitic stainless steel alloys (SUS316 and SUS304), which have a high chromium (Cr) content ranging between 12 and 20 wt% and are employed in the manufacturing of surgical instruments, make up the majority of the instruments that are used in the medical and food industries. It is generally agreed upon, that the addition of Cr metal to steel alloys as an alloying element has the potential to significantly improve the standard steel alloy’s resistance to corrosion. Despite their excellent resistance to corrosion, stainless steel alloys do not demonstrate significant antibacterial characteristics [15,16]. This is in contrast to the fact that they have great resistance to corrosion. As a consequence of this, it is feasible to foresee the development of infection and inflammation, both of which are mostly produced by the adherence and colonization of bacteria on the surfaces of stainless steel biomaterials. Significant difficulties could develop if there is a link between the pathways that bacteria use to adhere to surfaces and the pathways that they use to form biofilms. These difficulties could lead to a decline in health, which could have a variety of knock-on effects that could either directly or indirectly affect human health.

We investigated the feasibility of using a high-energy ball milling technique for the production of metallic glassy Cu₅₀Ti₄₀Ni₁₀ and Cu₅₀Zr₄₀Ni₁₀ ternary systems with the purpose of producing an antibiofilm/SUS304-surface protective coating. The present study is a component of a project (EA074C) that was funded by the Kuwait Foundation for the Advancement of Sciences (KFAS) under contract number: PR1915EC01. The present study was done to investigate the antibacterial qualities of a composite made of coated metallic glassy particles/SUS304 to be compared with the results obtained upon coating with pure metals Ti, Zr, Cu and Ni, as well as the binary Ni₅₀Ti₅₀ and Zr₅₀Ni₅₀ systems.

2. Materials and Methods

2.1. Preparations of the Starting Master Alloys

For the aim of the present study, multiple ingots of binary Ni₅₀TM₅₀ (TM; Ti and Zr) and ternary Cu₅₀TM₄₀Ni₁₀ (TM; Ti and Zr) systems were prepared using an arc melting process that was operated in a pure Ar gas environment. In these experiments, the starting bulk materials (above 99.95 wt.%) were first balanced in order to obtain the desired average composition of each system (

Table 1. Compositional analysis performed by scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) of as-arc-melt and high-energy ball milling materials.

Composition (at. %)
Nominal	As-Arc-Melt	As 50 h Milling
Ni50Ti50	Ni49Ti51	Ni49.3Ti50.7
Ni50Zr50	Ni48.7Zr51.3	Ni48.6Zr51.4
Cu50Ti40Ni10	Cu49.3Ti40.9Ni9.8	Cu49.2Ti40.8Ni10
Cu50Zr40Ni10	Cu49.1Zr41.8Ni9.1	Cu49.2Zr40.7Ni10.1

). Thereafter, they were bathed in acetone and ethanol for ten minutes each and then dried at a temperature of 200 °C in an oven for 30 min. The reactant bulk materials were placed into a copper open-hearth cooled with water (Figure 1a).

The melting procedure started by melting pure (99.99 wt.%) Zr-getter with a current and voltage of 250 A and 20 V, respectively, using a W-metal electrode (Figure 1b). Then, the reactant bulk materials were melted to give pre-alloyed ingots. In order to assure that the final product would be homogeneous throughout, the ingots were turned over at regular intervals and re-melted five times. Figure 1c displays the outer surface appearance of three different typical alloys of Cu₅₀Zr₄₀Ni₁₀.

2.2. Preparations of Metallic Glassy Powders

The as-prepared binary and ternary master alloy buttons were simply crushed separately into small pieces with a 20-ton cold press. The as-snipped bulk pieces were disintegrated into large particles of ~150 μm in diameter (Figure 2a) using a Vibratory Disc Mill RS 200, provided by Retsch GmbH, Retsch-Allee 1-5, 42781 Haan, Germany. About 50 g of the disintegrated particles for each system were individually charged with 50 Cr–steel balls (12 mm in diameter) into a Cr–steel vial inside a glove box (UNILAB Pro Glove Box Workstation, mBRAUN, M. Braun Inertgas-Systeme GmbH, Dieselstraße 31, 85748 Garching, Germany) filled with helium gas. The ball-to-powder weight ratio was 10 to 1. The mechanical disordering (MD) process was started by mounting the vials on a planetary-type high-energy ball mill PM4, provided by Retsch GmbH, Retsch-Allee 1-5, 42781 Haan, Germany, for 50 h (Figure 2b).

Because the milling stock and the balls come off the inner wall of the vial (milling vial), the ball milling media in this type of mill have very high energy. This is because the effective centrifugal force may reach up to 20 times the gravitational acceleration [3]. The milling charge is subject to the effects of the centrifugal forces, which are brought about by the rotation of the supporting disc and the vial’s independent spinning (balls and powders). As a result of the supporting disc and the vial revolving in different directions, the centrifugal forces alternate between being synchronized and opposed to one another. Because of this, the milling media and charged powders alternately roll on the inner wall of the vial, and then they are lifted and hurled out across the bowl at a rapid speed (Figure 2b,c).

2.3. Fabrication of Metallic Glassy Powders Coated/SUS304 Composites by Cold Spray Process

It was realized that the as-prepared powders, which were noncrystalline (amorphous) powders, would crystallize into a stable (crystalline) phase when heated beyond their crystallization temperature. This was based on the fact that the powders were noncrystalline (amorphous) powders. As a result of this, the objective of this project work is to explore the impact that metallic glassy alloy powders have on the formation of biofilms; it is essential that the glassy phase be maintained throughout the spraying process.

Figure 3 shows the powders being loaded into the cold spray feeder, which is then exposed to a high-pressure argon gas flow (Figure 3a) as it travels via a pipeline attached to a supersonic jet before being sprayed onto the surface of a stainless steel substrate (Figure 3b). This procedure was carried out a total of five times on either side of the sheet. As a direct result of this, the sheets made of SUS304 were subjected to a cold spraying procedure so that both sides could be covered, as the substrate metal, stainless steel (SUS304) sheets, were utilized. These sheets were first cleaned with acetone and ethanol, and then dried in an oven at 150 °C for 1 h.

At room temperature, the surface of the substrate was treated with alumina blasting prior to the commencement of the coating method. This treatment took place before commencing the coating process. The cold spray technique, in contrast to the thermal spray combustion-based approaches, was carried out at a low temperature (in the range of 100 to 900 °C), which was significantly lower than the melting points of the feedstock powders. This is because the feedstock powders are atomized rather than powdered. In this study, the process of cold spraying, which was accomplished by utilizing a supersonic jet at a very high velocity (1200 m/s), was carried out at a temperature of 600 °C for the elemental Ti, Zr, Ni, and Cu powders. On the other hand, it was carried out at 340 °C and 300 °C, respectively, for the metallic glassy Ni₅₀Ti₅₀ and Ni₅₀Zr₅₀ systems. During this time, the coating process was completed at temperatures of 540 °C for the ternary Cu₅₀Ni₅₀Ti₅₀ metallic glassy system and 630 °C for the Cu₅₀Ni₅₀Zr₅₀ metallic glassy system.

2.4. Materials Characterizations

2.4.1. Structural Characterizations

Using equipment from Rigaku-SmartLab (Rigaku, Kawasaki, Japan), 9kW x-ray diffraction (XRD) was used to evaluate the general structural changes that occurred as a result of ball milling the master alloys. Using the CuKα radiation with a wavelength (λ) of 0.15418 nm and an operating voltage of 45 kV, 200 mA, all the samples were evaluated at a speed of 2 degrees/min through a continuous 2θ/θ scan mode. The detector utilized was a high-speed 1D X-ray detector, called D/teX Ultra 1D mode (D/teX), with a Ni filter. Using a step size of 0.02/2θ and a duration of 1 s/step, the diffraction patterns were acquired across a range of 20 degrees to 80 degrees. The XRD was produced as a consequence of the constructive and destructive interference brought on by the scattering of X-rays by atoms arranged in a regular array. Diffraction lines appeared at angles that were consistent with Bragg’s approach.

Using the JEOL 2000F model (JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan) microscopes with a resolution of 0.17 nm and operating at a voltage of 200 kV, a field-emission high-resolution transmission electron microscope (FE-HRTEM) that was equipped with energy-dispersive X-ray spectroscopy (EDS) was used to examine the powder samples of as-synthesized materials. After dissolving the sample powders in ethanol, a few drops of the resulting suspension were placed on a copper microgrid and allowed to dry in a vacuum.

Following the above, the microgrid was mounted onto the TEM transfer rod prior to being moved into the vacuum sample chamber of the TEM. In addition to using EDS to conduct elemental analyses on the materials, micrographs were collected for the bright-field image (BFI), the dark-field image (DFI), and the selected-area electron diffraction patterns (SADPs).

2.4.2. Morphological Characterizations and Elemental Analysis

In order to explore the morphological characterizations of the samples as well as their elemental compositions, field-emission scanning electron microscopy with energy-dispersive spectroscopy (FE-SEM/EDS) was used on a JEOL (JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan) JSM-7800F instrument that was run at an accelerated power of 15 kV. Afterwards, the powder samples were positioned on a piece of double-sided sticky carbon tape before being positioned on a copper sample holder. The samples avoided the possibility of any charge occurring in the micrograph, and they maintained the consistency of the powder. In order to conduct the analysis, the samples were placed within the chamber of the FE-SEM. The methods of TEM/EDS and SEM/EDS were used in order to ascertain the levels of the metallic alloying elements that were present in the powders after they had been ball milled.

2.4.3. Thermal Stabilities

The Shimadzu Thermal Analysis System/TA-60WS (SHIMADZU CORPORATION, 1, Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604-8511, Japan) with a differential scanning calorimeter (DSC) was used to investigate the thermal stability of the as-ball milled powders. This was accomplished at a heating rate of 40 °C/min, and the results were indexed by the transition glass temperature (T_g) and crystallization temperature (T_x).

2.5. Bacterial Strain and Biofilm Growth Conditions

The test organism was Escherichia coli (ATCC 25922). Triplicate coupons (22-mm²) for the sterile monocoated systems (Cu, Zr, Ni), binary systems (ZrNi, CuZr), and ternary systems (CuZrNi) were positioned vertically in 50 mL conical tubes containing 6 mL of pre-warmed BHI (Brain Heart Infusion). An amount of 100 μL of 0.5 McFarland standard suspension (equivalent to 1.5 × 10⁸ CFU mL−1) of a 24 h culture of planktonic cells was added to each tube. Bacterial inoculum preparations were conducted as follows: an overnight bacterial culture was centrifuged (8000× g, 10 min) to form a cell pellet, which was then washed with deionized water before being resuspended in BHI with an optical density of 10⁸ CFU/mL. The tubes were then incubated on a shaker to allow biofilm to form. At each time point (24, 48, and 72 h), triplicate-coated coupons were removed and rinsed with phosphate buffer solution (PBS) to eliminate the non-adherent bacterial cells. After that, the coated coupons were placed in a new tube with 6 mL of BHI and vortexed for 1 min at the maximum speed. After vortexing, the suspension was serially diluted in PBS and plated on nutrient agar (NA) for a viable count.

3. Results and Discussion

3.1. Elemental Metal Coated/SUS 304 Substrate

It is widely known that the growth and development of a biofilm is a complicated process that involves the adherence of microorganisms, such as bacteria, viruses, and fungi, as well as the creation of extracellular polymeric substances (EPS) and the dissemination of microorganisms into the surrounding environment [17]. Furthermore, it is well-recognized that once a mature biofilm has developed, it is exceedingly difficult to remove [18]. As a consequence of this, there is a widespread consensus that the development of inhibitory techniques to prevent the adhesion of microbes is an essential first step. As a consequence of this, there is a rising interest in studying both current and new antibiofilm surface materials for their potential use in clinical and culinary contexts [19].

In this study, the biofilm formation on pure powder elemental alloys (Cu, Zr, Ni, Ti), binary (Ti₅₀Ni₅₀, Zr₅₀Ni₅₀), and ternary (Cu₅₀Ti₄₀Ni₁₀, Cu₅₀Zr₄₀Ni₁₀) systems individually cold sprayed onto a SUS 304 substrate, as well as the viability of cells released from coated and noncoated coupons, were investigated after various times (24, 48 and 72 h) to determine the inhibitory effect of the metallic glass coating/SUS304 on the biofilm’s formation. The Gram-negative E. coli, ATCC 25922, was used as the model bacteria. The colony-forming unit (CFU)/mL was used to quantify the inhibitory effects of coated surfaces. The mean colony counts for both types of coupons are displayed.

Figure 4a,b show the inhibitory effects of the mono elemental metals after 24, 48 and 72 h. Despite the fact that the antibacterial activity of copper ions is dose-dependent [19], only the pure Cu 100% nano-coating demonstrated significant inhibition of biofilm formation by E. coli at all testing times in comparison to the SUS304-noncoated coupons (10⁶ CFU/mL after 72 h exposure), and showed no viable bacterial cells after 72 h of incubation (Figure 4a). Moreover, in comparison to SUS304, the Ni-coated coupons showed no significant inhibitory effect against the tested organisms for all exposure times. Similar to the Ni-coated coupons, the Ti-coated coupons showed no inhibitory effect against the biofilm formation by E. coli (10⁶ CFU/mL viable count); and only after 72 h, the formed biofilm seems to be reduced by one log (10⁵). On the other hand, Zr alone showed a poor inhibitory effect (it reduced the viable count by one log) against the E. coli biofilm formation after 72 h.

3.2. Metallic Glassy Ni₅₀TM₅₀ (TM; Ti, and Zr) Coated/SUS 304 Substrate

Since the elemental metallic coating did not demonstrate significant antibacterial behavior (Figure 4), we studied the effect of the metallic glassy coating of the Ni₅₀Ti₅₀ and Ni₅₀Zr₅₀ systems on improving the antibiofilm characteristics of the SUS304 substrate upon coating with the binary metallic glassy powders.

Figure 5a,b show the X-ray diffraction (XRD) patterns of the buttons produced from as-arc-melt Ni₅₀Ti₅₀ and Ni₅₀Zr₅₀, respectively. The Bragg peaks, shown in Figure 5a, revealed a monoclinic structure (Hermann Mauguin: P2₁/m 11), which was associated with an equiatomic NiTi system. On the other hand, the XRD patterns shown in Figure 5b displayed a typical orthorhombic crystal structure (Hermann Mauguin: Cmcm 63), which was a good match for an equiatomic NiZr system. In order to obtain the amorphous phases of the NiTi and NiZr systems, the disintegrating powders of arc-melt alloys were high-energy ball milled for a period of 50 h. This milling step caused significant lattice defects, which led to the formation of amorphous phases. In the mechanical alloying (MA) process, the starting materials were elemental powders of the diffusion couples [20], but in the mechanical disordering (MD) process, the starting materials have been already homogeneous alloys [21]. This is in contrast to the mechanical alloying (MA) process, in which the starting materials are elemental powders.

Figure 6 displays, in a condensed form, a depiction of the free energy associated with a binary AB system. Point 1 in the figure corresponds to the free energy of the initial AB alloy, which illustrates the most stable phase. This phase is shown as being the most stable. This stable phase has a propensity to transform into a disordered (metastable) phase when severe lattice defects are generated by the process of high-energy ball milling. It is possible for such flaws to raise the free energy from point 1 to point 2 (shown in Figure 6), which causes a change in phase from a long-range order to a short-range order and the production of an amorphous phase. Increasing the MD duration also results in a greater frequency of collisions between the ball and powder balls (Figure 6).

The XRD patterns of the Ni₅₀Ti₅₀ and Ni₅₀Zr₅₀ powders obtained after 50 h of high-energy ball milling are displayed in Figure 5c and Figure 5d, respectively. The samples for both systems revealed diffuse halo patterns, with the absence of any Bragg lines related to the starting intermetallic phases (Figure 5c,d). This indicates the formation of amorphous phases.

Differential scanning calorimetry (DSC) was used to examine the thermal stability of the Ni₅₀Ti₅₀ and Ni₅₀Zr₅₀ powders after high-energy ball milling for 50 h of MD time (final product). The DSC thermograms of the final product for the Ni₅₀Ti₅₀ and Ni₅₀Zr₅₀ powders are presented in Figure 7a and Figure 7b, respectively. The samples of both systems revealed two opposite thermal events in a temperature range between 150 °C to 550 °C, as displayed in Figure 7. The first events, which were endothermic, refer to the glass transition temperature (T_g), whereas the second ones took place at higher temperatures due to the crystallization of the formed metallic glassy phases. The T_g of the Ni₅₀Ti₅₀ and Ni₅₀Zr₅₀ powders were reordered to be 295 °C and 277 °C, as shown in Figure 7a and Figure 7b, respectively. On the other hand, the onset of the crystallization temperatures (T_x) of the two sharp exothermic peaks related to the metallic glassy Ni₅₀Ti₅₀ and Ni₅₀Zr₅₀ powders was determined and found to be 366 °C and 315 °C, as shown, respectively, in Figure 7a,b. Meanwhile, in the super-cooled liquid regions, ΔT_x (ΔT_x = T_x−T_g), the metallic glassy Ni₅₀Ti₅₀ and Ni₅₀Zr₅₀ powders were calculated from the DSC traces displayed in Figure 7 and found to be 71 °C and 38 °C, respectively.

In an attempt to investigate the local structure of the metallic glassy powders that were formed following the cold spray coating on the SUS304 substrate, a field-emission high-resolution transmission electron microscope (FE-HRTEM) was used. Figure 8a shows the FE-HRTEM picture, and Figure 8b shows the matching nanobeam diffraction pattern (NBDP) for the Ni₅₀Ti₅₀ powders after the coating process was carried out at 340 °C. Figure 8c and Figure 8d, respectively, illustrate the FE-HRTEM and NBDP results for the coating powders after cold spraying at 630 °C. The powders of both the Ni₅₀Ti₅₀ and the Ni₅₀Zr₅₀ systems exhibit a maze-like fine structure, despite the lack of crystalline phases, as illustrated in Figure 8a and Figure 8c, respectively. These morphological properties are beyond the nano-level. This suggests that the amorphous phase (shown in Figure 7) maintained its short-range order structure, even when the cold spray procedure was carried out at a temperature that was much lower than the T_x. This is suggested by the fact that the spot-free diffuse NBDPs for the Ni₅₀Ti₅₀ and Ni₅₀Zr₅₀ powders, respectively, are shown in Figure 8b and Figure 8d.

The SUS304 coupons were coated with the Ti₅₀Ni₅₀ and Zr₅₀Ni₅₀ alloys, as demonstrated in Figure 9. The binary system showed an improved inhibitory effect of coating against the biofilm formation by E. coli by a minimum of two logs in the case of the coupons coated with Ti₅₀Ni₅₀ after 24 and 48 h of exposure with 10⁴ and 10³ viable counts, respectively. This was in comparison to the SUS304-noncoated coupons, which showed more viable counts at all the exposure times (10⁶ viable counts). The coupons coated with Zr₅₀Ni₅₀ showed a less inhibitory effect in comparison to the SUS304 control and Ti₅₀NI₅₀, with a viable count of 10⁵ at all times of exposure.

3.3. Metallic Glassy Cu₅₀TM₄₀Ni₁₀ (TM; Ti, and Zr) Coated/SUS 304 Substrate

We used metallic copper as a superior antibiofilm agent for alloying evenly with Ni₁₀TM₄₀ (TM; Ti, and Zr) in order to increase the thermal stability and antibacterial properties of the fabricated glassy powders. This may have allowed the coating powders to be more resistant to bacteria. The XRD patterns of the powders obtained after high-energy ball milling of the arc-melted Cu₅₀Ti₄₀Ni₁₀ and Cu₅₀Zr₄₀Ni₁₀ buttons for 50 h of MD are shown in Figure 10a and Figure 10b, respectively. The two samples revealed diffuse halo patterns, implying the formation of amorphous phases, as presented in Figure 10a,b.

The morphological characteristics of the Cu₅₀Ti₄₀Ni₁₀ and Cu₅₀Zr₄₀Ni₁₀ amorphous powders obtained after 50 h of MD time are presented in Figure 11a and Figure 11b, respectively. After this final stage of milling, the powders consisted of aggregated particles and possessed nearly spherical-like morphologies with an apparent particle size ranging between 0.35 μm and 2.1 μm, as shown in Figure 11.

In order to explore the local structure beyond the nanoscale of the as-fabricated ternary system, the FE-HRTEM approach was used. Figure 12a and Figure 12c, respectively, illustrate the atomic-resolution TEM images of the Cu₅₀Ti₄₀Ni₁₀ and Cu₅₀Zr₄₀Ni₁₀ amorphous powders. Both of these powders were prepared using the TEM technique. The photos indicated a conventional short-range order structure with a morphology resembling a labyrinth. Because neither the crystalline phase, nor nanoclusters’ organized structure could be identified at this high TEM resolution, the creation of a single amorphous phase was hypothesized to have taken place (Figure 12). In addition, the NBDP of the Cu₅₀Ti₄₀Ni₁₀ and Cu₅₀Zr₄₀Ni₁₀ systems both exhibited an amorphous structure, which can be recognized by their smooth halo-diffuse patterns, which are shown, respectively, in Figure 12b,d.

The glass forming ability and crystallization characteristics of the final powder products, indexed by T_g and T_x, were examined by the DSC technique (Figure 13). As expected, adding Cu as a major alloying element has led to improving the thermal stability of both the Cu₅₀Ti₄₀Ni₁₀ and Cu₅₀Zr₄₀Ni₁₀ amorphous powders.

Comparing the T_g and T_x values (510 °C, 572 °C) for Cu₅₀Ti₄₀Ni₁₀ with binary Ni₅₀Ti₅₀ (71 °C, 366 °C), it demonstrated the significant role of Cu in enhancing the thermal stability of the obtained metallic glassy phase (Figure 13a). Likewise, the Cu₅₀Ti₄₀Ni₁₀ system and Cu₅₀Zr₄₀Ni₁₀ system revealed high T_g (562 °C) and T_x (641 °C) values (Figure 13b).

Figure 14a,b displays the bright-field image (BFI) and the corresponding atomic-resolution TEM images of the Cu₅₀Ti₄₀Ni₁₀ powders after cold spraying on the SUS304 substrate. During the cold spray procedure, which was repeated five times at 540 °C for 1800 s, a significant volume fraction of nanocrystalline spherical grains were obtained, as indicated by the nano-contrast presented in Figure 14a. The HRTEM image of a selected spheroid particle indicates the existence of elemental Cu metal, as shown in Figure 14b. This suggests that a considerable volume fraction of Cu metal was released from the amorphous Cu₅₀Ti₄₀Ni₁₀ alloys upon the cold spray process.

In order to assess the inhibitory effect of Cu in a metallic alloy coating, we explored the ternary systems preparations, including the addition of Cu (Cu₅₀Ti₄₀Ni₁₀, Cu₅₀Zr₄₀Ni₁₀). Cu₅₀Ti₄₀Ni₁₀ (Figure 15) demonstrates a significant inhibitory effect against biofilm formation by E. coli by a minimum of four log reductions (10²) in comparison to the noncoated SUS304 control (10⁶). Interestingly, less concentration of Cu in the ternary systems (50%) showed a similar inhibitory effect against biofilm formation, as shown in Figure 4. Moreover, Cu₅₀Zr₄₀Ni₁₀ showed a significant inhibitory effect against biofilm formation after 72 h of exposure in comparison to the SUS304 control coupons.

The antibacterial mechanism of action of copper surfaces has been widely investigated [22,23]. The processes of contact death may vary depending on the microorganism and its state in planktonic or matured biofilms. The discharge of copper ions from metallic surfaces is primarily responsible for the antibacterial activities of copper surfaces; however, direct contact between the microbes and surfaces may also be required. Moreover, when bacteria come into contact with copper, several processes are proposed: the cell envelope (outer and inner membrane) destruction, oxidative damage mediated by the generation of reactive oxygen species (ROS), enzymatic inhibition, and nucleic acid degradation. Furthermore, various metallic glass materials have been developed, including antibacterial stainless steels 304-Cu, 420-Cu, and 317L-Cu, as well as Ti-6Al-4V-xCu (x = 1, 3, 5) alloy, by adding Cu to the preparations, showing very effective antimicrobial properties due to the continuous release of Cu ions into the environment [24]. Many studies demonstrate the inhibitory effect of Ti against biofilm formation. For example, the development of Titania nanotubes (TiO2NTs) containing Ag had an effective inhibitory effect against Porphyromonas gingivalis (ATCC 33277) and Actinobacillus actinomycetemcomitans (ATCC 29523) [24]. Our results, along with others [24,25], showed that Ti could inhibit biofilm formation in comparison to the noncoated SUS304. However, in order to improve the performance of Ti, known antibacterial materials were added to the alloy, such as Cu [25]. In this study, we evaluated the effects of various material alloys separately: Cu, Ti, Ni, and Zr, which demonstrated a poor inhibitory effect against biofilm formation; binary binding materials, excluding Cu (Ti₅₀Ni₅₀, Zr₅₀Ni₅₀), which showed an improved inhibitory effect against the biofilm formation by E.coli; and ternary elemental alloys, including Cu (Cu₅₀Ti₄₀Ni₁₀ and Cu₅₀Zr₄₀Ni₁₀), which showed an excellent inhibitory effect.

Our current findings support and imply that the Cu₅₀Ti₄₀Ni₁₀- and Cu₅₀Zr₄₀Ni₁₀-coated materials will be extremely successful at preventing biofilm formation. Furthermore, when Ni was coupled with Cu and Zr, the antibacterial action of Ni improved, suggesting a synergistic effect.

4. Conclusions

The mechanical disordering technique, using high-energy ball milling, was utilized for the preparation of the metallic glassy Ni₅₀Ti₅₀, Ni₅₀Zr₅₀, Cu₅₀Ti₄₀Ni₁₀, and Cu₅₀Zr₄₀Ni₁₀ powders. The prepared alloy powders, obtained after 50 h of ball milling, were used to produce metallic glassy coats/SUS304-antibacterial protective coatings, and the Cu₅₀Ti₄₀Ni₁₀ powders, after cold spraying on the SUS304 substrate. The results have indicated that during the cold spray procedure of Cu₅₀Ti₄₀Ni₁₀ powders at 540 °C for 1800 s, significant volume fractions of nanocrystalline spherical Cu grains were obtained. This suggests that a considerable volume fraction of Cu metal was released from the amorphous Cu₅₀Ti₄₀Ni₁₀ alloys upon the cold spray process. Compared to the other systems tested, the ternary systems (CuTiNi and CuZrNi) were the most effective coating metals for inhibiting E. coli bacterial cell attachment, according to the findings of this study. Despite the fact that the CuTiNi and CuZrNi metallic alloys had the best antibiofilm inhibitory effect against E. coli, they should not be considered the only solution to biofilm formation on surfaces. Bacterial strains have been shown to develop metal resistance pathways similar to those developed in response to antibiotic treatments. As a result, research into how to eliminate biofilm resistance to metal should continue.