Characterization and antibacterial activity of co-sputtered Cu-doped TiO2 coatings deposited on Ti6Al4V alloy

Copper has gained increased interest because of its important role in metabolism and antimicrobial activity. In this study, composite target material of Ti and Cu was used to deposit TiO2 coatings doped with copper on Ti6Al4V alloy. The aim was to examine the influence of the applied bias during the deposition of Cu-doped TiO2 coating by sputtering in a glow-discharge in a pure O2 atmosphere for a deposition time of 240 min. Different substrate values, selected from 0 to -150 V, were used in the process. The increase in bias voltage from -50 to - 150 V decreased the thickness of the oxide coatings and improved their adhesion to the substrate while increasing the Cu2O phase at the expense of a CuO phase decrease. Simultaneously, the increase in bias voltage decreased Cu content from about 32 wt% for the -50 V biased down to around 11 wt% for the -150 V biased specimens. The antimicrobial efficacy against E. coli estimated by direct contact experiments on the top of the uncoated (control) and coated Ti6Al4V alloy revealed about 94% inhibition for the -50V biased down to around 37% for the -150 V biased coatings as opposed to the control.


Introduction
Because of their low density, high strength, good corrosion resistance, and biocompatibility, titanium alloys are widely used for orthopedic implants [1][2][3].Nonetheless, they are bioinert materials that cannot trigger osseointegration with the surrounding bones [4].Moreover, titanium alloys do not possess antibacterial properties and offer no bactericidal protection [5].To lessen the likelihood of implantassociated infections and increase the bioactivity of titanium implants, it is important to develop effective antibacterial coatings via the incorporation of antibacterial agents.Incorporating copper (Cu) into biomedical coatings can enhance both osteogenic and antibacterial activity to mitigate implantassociated infections because Cu is an essential trace element that participates in enzyme-based processes for bone metabolism, enhances neovascularization, and promotes early skin wound healing [6].It also demonstrates very good bactericidal properties against many pathogens [7].According to earlier studies, adherent TiO2-based coatings containing Cu in the form of Cu2O [8] or CuO [9] can be produced efficiently using micro-arc oxidation.So far as we know, only a small number of studies using co-sputtering of Ti and Cu in an oxygen atmosphere [10] or subsequent annealing in air [11] were previously conducted to improve the performance of biomedical devices.None of these studies have looked at substrate biasing, which can alter the microstructure and properties of the coatings because it regulates the adatom mobility during film growth [12].Therefore, in this work, co-sputtered Ti and Cu oxide coatings deposited at varying bias voltage were prepared by glow-discharge sputtering and their structure, composition, adhesion and antibacterial activity against E. coli were studied systematically.

Materials and methods
Ti6Al4V (Gr 5) alloy with a composition of 6.23 wt% Al, 4.18 wt% V, 0.12 wt% Fe, 0.17 wt% O, 0.014 wt% H and Ti -the rest, was used for the experiment.Samples with a dimension of 14×14×2 mm were laser cut and subjected to dual acid etching.The specimens were etched using pure HF and HCl (PanReac AppliChem) acids for exposure times of 3 min (at RT) and 2 hours (at 60 °C), respectively.Then, the samples were washed with dH2O and ultrasonically cleaned in 96% ethanol for 5 min.
For the glow discharge deposition, a П-shaped sputtering system in a cubic vacuum chamber with water-cooled walls was used.The sputtering chamber was evacuated to a base pressure of 3×10 -1 Pa.A composite Ti-Cu (Ti:Cu = 179:1) cathode was used at a target voltage of 1200 V (3 A current).To improve the adhesion of the oxide film, the samples were bombarded by a glow discharge for 15 min.in a pure argon atmosphere at a working pressure of 6×10 0 Pa.Then, the deposition of the oxide coating took place in a pure O2 atmosphere at a working pressure of 8×10 0 Pa for a deposition time of 240 min.During the process, the substrate bias was varied from no bias (0V, grounded), -50, -100, and -150 V at a constant distance from the cathode of 55 mm.Polished Ti6Al4V samples for adhesion evaluation and pure Ti foils for cross-section observations were also coated during the processes.
The implant surface morphology and composition were examined with a scanning electron microscope (SEM, LYRA I XMU, Tescan) equipped with an energy dispersive spectrometer (EDS, Quantax 200, Bruker).The phase composition was analyzed by X-ray diffraction (XRD, URD-6 Seiferd&Co, Germany) within 2θ range of 20-80° in a step of 0.1°, Ni-filtered CuKα radiation (λ = 0.154178 nm) and a symmetrical Bragg-Brentano mode.The scanning rate was 6 s per step.CSEMscratch tester equipped with a standard Rockwell-C indenter and an optical microscope was used to characterize optically the adhesion of the coating up to a load of 50 N.
The antibacterial properties of the coatings were revealed by the plate counting method.The Escherichia coli (E.coli) K12 AB1157 (F-thr-1 leu-6 proA2 his-4 argE3 thi-1 lacY1 galK2 ara-14 xyl-5 mtl-1 tsx-33 rspL31 supE44) strain was purchased from the National Bank for Industrial Microorganisms and Cell Cultures (Sofia, Bulgaria).A single colony of E.coli was inoculated into 5 ml of sterile LB medium containing 1% protein hydrolysate, 0.5% yeast extract, and 0.5% NaCl adjusted to pH 7.4.Cells were cultivated overnight at 37°C.The next day, 100 μl of these cells were taken in 10 ml of new sterile LB medium.Cells were cultured at 37°C to 0.6 OD (optical density).Then 1 ml of cells were centrifuged for 5 min at 2500 rpm.The supernatant was discarded and the bacterial pellet was dissolved in 1 ml of sterile PBS.The 100 μL of bacterial suspension was dropped on the coated and etched Ti6Al4V alloy and was incubated at 37 °C for 24 hours.After incubation, aliquots of 10 μl were taken and diluted 100,000 times.A 100µl from the dilution was seeded on LB agar plates.After 24 h incubation at 37 ºC, the bacterial colonies were pictured and counted.
The inhibition percentage was calculated by the following formula: R = (B -A) / B ×100, % where A and B are the colony numbers on the test and control samples, respectively.

Results and discussions
The surface morphology of the as-deposited Ti-Cu oxide films characterized by SEM is shown in figure 1. Figure 2 shows that, for the same set of parameters, the deposition rate dropped as the substrate bias voltage changed from -50 to -150 V due to increased ion bombardment.The layer thickness increases when the bias is low (-50 V) because the neutral groups and positive ions in the plasma migrate to the surface, speeding up the reaction kinetics and adsorption.Due to the competing effects of substrate biasing and thickness, even at -100 V bias, the grain size was much greater (figure 1).Larger crystalline grains in thinner films are typically the result of increased adatom surface mobility brought on by an increase in the kinetic energy of the particles affecting the coating.Moreover, densification of microstructure can also lead to a slight decrease in film thickness.A higher substrate bias (-150 V) may result in higher energetic impingement of positive ions triggering ion-bombardment-indicated defects and higher re-nucleation rates causing the grain size to decrease.
The coating composition is significantly impacted by the bias voltage (table 1).The phenomenon known as "self-sputtering," which etches the coated surface, will intensify with an increase in bias voltage [13].As shown in table 1, a reduction in Cu content is observed as the bias voltage increases.At the same time, the films deposited at high substrate bias voltage indicate a higher incorporation of Ti in the coatings.It is assumed that at zero biasing sputtering of the deposited film does not take place since the ions have energy below the sputtering threshold.With an increase of substrate biasing, during the reverse sputtering Cu may undergo higher self-sputtering due to its higher sputtering yield than Ti [14].The XRD patterns of the samples are presented in figure 3. The patterns are typical for materials characterized by polycrystalline structure.All diffraction maxima are indexed, and no amorphous-like halo can be observed at the lower Bragg angles, meaning that the coatings are crystalline, and no amorphous-like structure is formed in all considered cases.The results indicate that as the bias voltage increased the diffraction peaks of the coatings became stronger and sharper implying that the lattice imperfections, namely vacancies, dislocations, and stacking faults of the coatings decreased.The diffractograms of the biased samples indicate the existence of both monoclinic CuO and cubic Cu2O phases with an increasing intensity ratio of Cu2O diffraction peaks when applying higher bias implying the predominant formation of Cu2O.Peaks corresponding to both anatase and rutile TiO2 are seen in all samples.The more distinct peaks of anatase TiO2 when decreasing the monoclinic CuO phase with higher bias can be attributed to the similarities in tetragonal anatase and monoclinic CuO lattice constants.Accordingly, it is believed that the bias voltage and, therefore, the level of ion bombardment, is a significant factor affecting the phase composition of the Ti-Cu oxide coatings.
The scratch tracks of the coatings (figure 4) showed relatively higher critical loads (indicated in red color in figure 4) of the biased samples than those deposited without bias.The adhesion of the deposited coatings is improved by raising the bias voltage.The primary cause is that when bias is applied, the substrate is bombarded with more particles with more energy, which encourages the creation and  The Cu-doped Ti oxide coatings demonstrated effective antibacterial activity against important colonizers of implants such as E. coli compared to Ti6Al4V alloy (table 2).In contrast to the large number of colonies on Ti6Al4V confirming that the alloy has no antibacterial properties, only several bacterial colonies were observed on the coated at 0, -50 and -100 V substrate bias voltage samples.Despite the lower content of Cu in a -100 V biased sample than that in the non-biased, the former demonstrated higher contact-killing activity due to the higher antimicrobial effect of Cu2O than CuO [15].It is thought that Cu 1+ ions cause higher cytotoxicity against bacteria than Cu 2+ since they can migrate across the cell membrane to the cytoplasm and bind to sulfhydryl groups [16].However, this inhibitory effect was concentration-dependent and decreased for the -150 V biased coatings with the lowest Cu content.Cu 2+ ions were also found to be quite active [17] which was confirmed by the high antibacterial rate (84%) of the non-biased specimen with predominant CuO phases present in the multicomponent oxide coating.

Conclusions
In this study, we have demonstrated that the bias voltage applied during the glow-discharge deposition of Ti-Cu oxide coatings is a crucial factor that influences the chemical, phase composition, microstructure and adhesion properties of the deposited films and provides a way to change the surface antimicrobial activity.Compared to other groups, -50 V biased coatings showed the best bactericidal activity due to their chemical and phase composition.However, Cu release from the coatings should be below the cytotoxic level and evaluation of the interaction with osteoblast cells needs to be addressed in future studies.

Figure 3 .
Figure 3. XRD pattern of the non-biased and biased sample coatings.

Figure 4 .
Figure 4. Typical scratch morphologies of the Ti-Cu oxide coatings deposited at (a) 0 V, (b) -50 V, (c) -100 V and (d) -150 V bias voltage.From left to right the normal load increase from 0 to 50 N.

Table 2 .
Antibacterial activity of control and coated samples against E. coli.Results are shown as average ± SD of 3 independent experiments.CFU -colony-forming unit; R -reduction (inhibition %).