# Contribution to the understanding of antibacterial properties of CuTiZn thin film: from crystalline alloy to metallic glass

D. Boivin, P. Birnal, Pascal Brault, F. Brulé-Morabito, A. Caillard, E. Bourhis, P. Andreazza, B. Aspe, T. Vaubois, E. Menou, et al.

► To cite this version:

D. Boivin, P. Birnal, Pascal Brault, F. Brulé-Morabito, A. Caillard, et al. Contribution to the understanding of antibacterial properties of CuTiZn thin film: from crystalline alloy to metallic glass. Surface and Coatings Technology, 2025, 499, pp.131879. 10.1016/j.surfcoat.2025.131879. hal-04930839

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Contribution to the understanding of antibacterial properties of CuTiZn thin film: from crystalline alloy to metallic glass

D. Boivin a, P. Birnal a, P. Brault a, F. Brulé-Morabito d, A. Caillard a, E. Bourhis c, P.

Andreazza c, B. Aspe a, T. Vaubois b, E. Menou b, M. Cavarroc-Weimer b, A.L. Thomann a*

a GREMI université d'Orléans/CNRS, 14 rue d'Issoudun, BP6744, 45067 Orléans Cedex2, France

b SAFRAN Tech, rue des jeunes bois, Châteaufort, CS 80112, 78772 Magny-les-Hameaux, France

c ICMN université d'Orléans/CNRS, 1b rue de la Férollerie, Orléans Cedex 2, France

d ART ARNmessager US55-INSERM / LI²RSO / Université d'Orléans, CHU d'Orléans, 14 avenue de l'Hôpital, 45100 Orléans La Source, France

a Université de Nantes, CNRS—Institute of Materials of Nantes Jean Rouxel, IMN,F-44000 Nantes, France

**Abstract**

In this work, thin films of CuZn and CuTiZn alloys were synthesized by magnetron sputter deposition. Their antibacterial activity on the proliferation of *Escherichia coli* was investigated after incubation during 24h at 37.0°C by measuring the bacteria solution absorbance at 600nm. Among tests usually employed in laboratories to evaluate the antibacterial properties of coatings, this one specially characterizes the action of chemical elements dissolved in the solution, i.e. the so-called release killing mechanism. The obtained results are discussed depending on the film chemical composition (in depth and at the surface), microstructure and crystallinity. A good antibacterial activity was found for pure Zn and CuZn films, whereas a rapid degradation was observed as soon as Ti is added, which coincides with the formation of a dense amorphous phase. At close chemical compositions, formation of this stable metallic glass phase seems to be detrimental to the ion release mechanism. This work highlighted that the microstructural and crystalline properties (more or less porous polycrystalline film / dense, defect free metallic glass) significantly influence the antibacterial properties of such alloy thin films.

**Keywords:** Sputtered alloy thin films; antibacterial activity of metallic thin films; bacteria degradation by release killing; metallic glass thin film, polycrystalline thin films
# 1. Introduction

The pandemic COVID-19 crisis highlighted the need to limit the survival and the spread of pathogens through surfaces. Most gram-positive bacteria, such as Enterococcus spp., *Staphylococcus aureus*, or *Streptococcus pyogenes*, survive for months on dry surfaces. This is also the case of gram-negative species, such as Acinetobacter spp., *Escherichia coli*, Klebsiella spp., *Pseudomonas aeruginosa*, *Serratia marcescens*, or Shigella spp.. A few others, such as *Bordetella pertussis*, *Haemophilus influenzae*, *Proteus vulgaris*, or *Vibrio cholerae*, however, persist only for days [1]. Most viruses can persist on surfaces for at least a few days. Respiratory viruses (*coronavirus*, *coxsackievirus*, *influenza virus*, *SARS* or *rhinovirus*) can persist on surfaces for a 3 to 5 days. For intestinal viruses (*astrovirus*, *HAV*, *poliovirus* and *rotavirus*) this delay reaches 2 months, whereas viruses transmitted by blood (HIV, HBV) can survive about a week. Herpes viruses (CMV, HSV) can persist on surfaces for a few hours to 7 days [1]. J.M. Gwaltney published a 10-year-study in 2002 showing that surface contamination can be considered as one of the significant factors in transmitting viruses, e.g. rhinovirus [2].

Cleaning contaminated surfaces with biocides is the most widely used method despite some limitations: cleaning is efficient for a short given time before reinfection, parts can be accidentally skipped; the cleaning product can affect the surface and are generally not environmentally friendly. In public places, the pathogens are easily reloaded on cleaned surfaces because of continuous aerosol and frequent touching [3-5]. This observation calls for a response in terms of “antimicrobial” surfaces. A promising solution to achieve cost-effective, eco-friendly, antimicrobial surfaces is to develop metallic coatings. Metals have been used as antimicrobial agents for ages before the introduction of organic antibiotics in the 20th mid-century [6-7]. The most used metals are transition metals from the d-block, such as Cu, Ag, Zn, Ti, Ni, Cr, and the metalloids from the 13-16 periodic group [7]. The rise of antibiotic resistance, and the decrease in the use of antibiotics led to a renewed interest for metals as antimicrobial agents [8]. Antimicrobial properties of metals are associated with the delivery of ions and/or the presence of nanoparticles. Changes in the surface chemistry, texture, and structure of the metallic coatings can influence the long-term antimicrobial efficiency [9].

In an attempt to understand the antibacterial activity, different processes have to be taken into account, related to the composition but also to the structure and morphology of the surface [10, 11]. This last parameter determines the physical interaction with bacteria. Depending on the pattern scale, rough surfaces may promote adhesion and even induce retention of bacteria, which could lead to the formation of a biofilm. Lutey *et al*. reported that *E. coli* and *S. aureus*
were less attached to nanostructured stainless steel surface than to mirror polished or micro-
spike-patterned ones [12]. It has been shown that sharp edge patterns with size lower than that
of the bacteria are able to damage their membrane and cause their death [13]. This mechanism
called “contact killing” is not completely understood but seems to be related to the size and to
the mechanical properties of the micro-tips present at the surface [13, 14]. On the contrary, very
smooth surfaces are expected to reduce adhesion and help preventing the biofilm formation
[11]. Usually, hydrophobic character is expected to be beneficial since it limits the interaction,
bacteria being always present in an aqueous medium. But this rule is far from being universal
and various behaviours have been highlighted depending on the bacteria themselves [12]. For
instance, Fadeeva et al. have proved that S. aureus cells were able to colonize a super-
hydrophobic titanium surface whereas no Pseudomonas aeruginosa cells were able to attach to
the surface [15]. In their work Lutey et al. highlighted that the growth of E. coli is reduced on
hydrophobic Laser Induced Periodic Surface Structures (LIPSS) as compared to hydrophilic
LIPSS, whereas S. aureus is able to colonize hydrophobic LIPSS surface but is not detected on
hydrophilic ones [12].

The chemical action on bacteria is related to the release of metallic ions from the surface. These
ions are capable of initiating reactions that cause oxidative damage to microorganisms through
proteins, nucleic acids, and lipids [16], leading to DNA perturbation [10]. The “release-killing”
process requires the liberation of ions in the solution. This supposes that the film is somehow
degraded by the solution and evolves during the interaction. It has been pointed out in several
works that the release of ions occurs when the film is sensitive to bio corrosion. Nkou Boualaa
et al. evidenced that the antibacterial activity of ZrCuAg TFMGs is promoted when the Ag
content increases, as could be expected. But this is associated to an enhanced sensitivity to
corrosion [17]. A compromise has to be found between the chemical killing activity and the
integrity of the film [18]. Regarding this chemical aspect, a last feature of the film has to be
taken into account: the porosity or more precisely the specific surface. The concentration of
released metallic ions is directly correlated with the area in contact with the solution.

Our aim is to design and manufacture antimicrobial metallic alloy coatings exhibiting specific
properties such as high hardness, good abrasion resistance etc. depending on the targeted
application. Among complex metallic alloys, the ability of Zr-based metallic glasses, containing
Cu or Ag, to fulfil these requirements has been investigated [10, 19-22]. Thin film metallic
glasses (TFMG) appear as good candidates since their homogeneous composition and
amorphous nature prevent the formation of any crystalline defects such as grain boundaries or
dislocations [20, 23]. Consequently, good corrosion resistance, mechanical properties etc. [18,
19, 24] can be expected and associated to antimicrobial activity if constituting elements are
judiciously chosen. Moreover, it has been shown that the high density and smooth surface of
the TFMG prevent bacterial adhesion and limit the formation of biofilms [10, 18, 21].

In this work, the relationship between the antimicrobial activity and the chemical composition,
structure and crystallinity of thin metallic alloy films synthesized by dc magnetron sputter
deposition is investigated. This well-known versatile technique allows for easy composition
tuning and exhibits a good glass forming ability due to deposition conditions ensuring very fast
cooling rates [18, 19, 25]. Our objective is to bring particular emphasis into the formation of
the amorphous phase and its influence on the properties. Recently some bacteria resistance has
been highlighted for silver [26], consequently copper was chosen in association to zinc to ensure
bactericidal and antiviral properties by "release killing" [27, 28]. Titanium is added to improve
the mechanical properties of the film, while ensuring good biocompatibility [29]. To our
knowledge, the antimicrobial behaviour of CuTiZn alloy family has never been studied. It was
decided to start with the binary alloy CuZn with compositions close to the equimolarity, and to
progressively add Ti into the films.

The microstructure of the films was investigated on cross-section views obtained by scanning
electron microscopy (SEM). The mean chemical composition was measured using energy
dispersion spectroscopy (EDS), while the surface chemistry was investigated by X ray
photoelectron spectroscopy (XPS). Rutherford Backscattering Spectroscopy (RBS) was used to
check the composition's homogeneity throughout the thickness. Grazing incidence X ray
diffraction (GIXRD) was carried out to characterize the crystalline properties of the films. In
an attempt to get a better understanding of the organisation at the atomic level, molecular
dynamic simulations of the film growth were conducted in conditions close to the experimental
ones. Film characteristics were correlated to the antibacterial activity measured on *Escherichia coli* using a protocol developed for films or surfaces, widely used in literature [19].

**2. Materials and methods**

2.1. *Coating preparation*

The experimental setup is composed of a UHV deposition chamber connected to an airlock.
Both parts are pumped separately by primary and turbo-molecular pumps. The residual pressure
in the deposition chamber reached 10⁻⁵ Pa. Argon flow was set to 10 sccm. The pressure was
measured using a capacitive gauge and set to 0.5 Pa during the deposition. Three balanced 4"
diameter circular magnetrons are inclined at 30° relative to the substrate surface plan on which
3 mm thick targets are fixed. Deposition experiments were performed at fixed DC powers in
the range 20 to 400 W. Thin films were deposited on single crystal silicon substrates (P-doped
Si (100)) cleaned in an ultrasonic bath, 10 minutes in isopropanol and 10 minutes in ethanol.
The substrates were pasted on the sample holder with carbon tape. The sample holder was
located 11.5 cm away from a plane passing through the centre of the three magnetrons. The
substrate rotation during deposition was set to 26 rpm. The substrate was neither cooled nor
heated during deposition.

## 2.2.Film characterization

The crystallographic structure of the coatings was analysed by X-ray diffraction (XRD) using
an AXS Bruker D8 Advance diffractometer with the Cu Kα radiation (λ= 0.15406 nm). GIXRD
measurements were performed between 20° and 80° at a grazing incidence of 1°, to limit the
contribution of the substrate and to enhance the scattering coming from coatings. Thickness and
morphology were determined by scanning electron microscopy (SEM) using a Zeiss Supra 40
FEG-SEM operating at 3 kV. In addition, energy dispersive X-ray spectroscopy (EDS) at the
accelerating voltage of 15 kV was carried out on (200×200) nm² areas with a detector Bruker
QUANTAX to estimate the mean composition of the coatings. RBS, performed at the CEMHTI
[30] laboratory on a Pelletron accelerator with 2 MeV alpha particles (with 166° scattering
angle), allowed in-depth composition homogeneity assessment. RBS analysis was performed
on thinner thin films (200-300 nm) than those used for EDS, in order to properly quantify Ti
content. Since Zn and Cu feature close atomic weights, the Cu/Zn ratio cannot be accurately
determine by this technique. Spectra simulations were carried out with the SIMNRA software
[31].

The chemical composition of the surface prior to bacteria exposure was determined by X-ray
Photoelectron Spectroscopy (XPS) using a Thermofisher Escalab Xi+. The performance of the
X-ray monochromatized source (Kα Al =1487 eV) and the detection setup allow to achieve a
photoelectron energy resolution of 0.1 eV. Position of the C1s spectrum at 285 eV was taken
as reference [32]. The spectra fits were done with the functionalities of the “Advantage”
software. Images at the micrometre scale were taken with a Keyence digital microscope (VHX-
5000).

## 2.3.Antimicrobial tests
Measuring the antibacterial properties according to the standards requires very specific laboratories with dedicated teams and tools as targeted bacteria are pathogenic. In literature, authors usually develop their own test in collaboration with biologists. Different kinds of bacteriological tests have been optimized for coatings. Many of them are based on the count of the number of colonies grown in a bacteria solution after interaction with the sample surface during several hours [14, 21, 33]. A comparison with a positive control (free growth) allows calculating the antibacterial rates that stand for the impact of the film on the bacteria proliferation. The way of counting may differ. For instance, after interaction, the bacteria solution is spread on Petri dishes at various dilutions and colonies are counted after a second culture step. Other teams, following the standardized JIS Z 2801:2000 test, deposit a droplet of bacteria solution directly on the thin films and cover with a sterilized PE film [20, 34]. After an incubation of about 24 hours, the bacteria were harvested, spread onto agar medium and counted or marked in solution using fluorescent reagents. After a 15 minutes incubation time in the dark at room temperature, a droplet of the solution was observed by confocal microscopy. Some protocols are based on the measurement of the “non contaminated” area around a sample that has been put on a seeded petri dish, the active surface being in contact with the bacteria. To evaluate the bacteria adhesion, after 3 hours of interaction with bacteria in humidity, P-T Chiang et al. attached the samples to a Mueller-Hinton agar plate and incubated at 37.0°C for 24, 48 and 96 hours [35]. The areas of microbe growth were recorded by serial subtraction photography. Izquierdo-Barba et al. compared the adhesion of several bacteria on Ti6Al4V and nano-Ti6Al4V after interaction during 90 minutes with a seeded solution. The samples were then washed to remove any unattached bacteria and stained before fluorescence was excited at different wavelengths in order separate living and dead bacteria [36].

Although these different antibacterial tests do not measure exactly the same antibacterial property, some focusing more on bacterial adhesion and others on chemical release, they all allow TFMG to be compared with each other and to evaluate activity compared to a control. Among them, we have chosen to measure the absorbance at 600 nm of a bacteria solution after 24 hours – 37°C interaction with the sample. This gives some insight into the antibacterial activity via chemical element release. For example, Lu et al. [19] report this kind of measurement. All the binary and ternary alloy coatings have been submitted to a non-pathogenic strain of E. coli (CIP54 117). In an attempt to determine the component driving the activity, thin films of pure Cu, Zn, Ti deposited by magnetron sputter deposition have also been tested. In order to achieve a concentration of colony-forming units (cfu) per millilitre of 10⁹,
100 µL of bacteria were inoculated in 5 mL of nutrient solution in a shaking incubator. A dilution down to $10^2$ cfu/mL is done and 500 µL of the new solution is spread over 7 wells each one containing one sample. One well is kept empty (without sample) as control. The liquid medium with the samples and the control were placed in a shaking incubator at 37.0°C during 3 hours and 30 minutes. 10 µL were taken out every thirty minutes and dispersed into a 1/6 area of a Petri dishes filled with a solid autoclaved nutrient medium (NaCl, Tryptone, yeast extract, PH=7±0.2). Examples of Petri dishes are presented in figure 1 showing the result for a binary and a ternary alloy thin film (B1 and T-Ti10) with the control.

<figure><img src="image_2.png" /><figcaption>Figure 1: E. coli bacteria colonies grown after interaction between the seeded solution and the samples during 30 min (0); 1 hour (1); 1 hour and 30 min (2); 2 hours (3); 2 hours and 30 min (4); 3 hours and 30 min (5); for the control, a binary and a ternary alloy, respectively.</figcaption></figure>

This first experiment gives an idea of the bacteria growth kinetic and a first glance of the coating activity. In the present case, it indicates that the impact of the coating on the bacteria proliferation is visible at the very beginning (after 30 minutes) and after 3 hours of incubation. Moreover, the ternary alloy seems less active than the binary one. For a better quantification, and to lie close to standard tests (see discussion above), the absorbance of the solution in interaction with the sample was measured after a total interaction time of 24 hours at 37°C, using an UV/Visible spectrophotometer adjusted on 600 ± 5 nm (VICTOR Nivo Multimode Microplate Reader). The recorded data is a measure of the absorbance defined as the ratio between the light intensity after passing through $1 \text{ cm}^3$ of the nutrient solution and the light intensity after passing through $1 \text{ cm}^3$ of the bacteria solution. Since bacteria absorb light at this wavelength, the lower the measured value, the lower the concentration of bacteria in solution, hence a higher impact of the coating on their growth. The absorbance is directly proportional to the bacteria density relative to the control.
### 3. Results

#### 3.1. Thin film mean composition

The composition of the films measured by EDS is given in Table 1. Two binary CuZn alloy films have been deposited, one Zn-rich (B1) and one Cu-rich (B2). Each element at. % (as determined by EDS) lies in the range of 40-60 %. The Ti content varies from 1 to 42 at. %. The ratio Cu/(Cu+Zn) is also calculated for ternary alloys. The chemical composition at the surface measured by XPS is given for four of the synthesized films and will be discussed below.

<table><caption>Table 1: Chemical composition of the films determined by EDS (mean value) and XPS (surface chemistry) and proportion of Cu as compared to Zn calculated from EDS data.</caption><thead><tr><th rowspan="2"></th><th colspan="3">At. % (EDS)</th><th colspan="3">At. % (XPS)</th><th rowspan="2">Cu/(Cu+Zn) x 100</th></tr><tr><th>Cu</th><th>Ti</th><th>Zn</th><th>Cu</th><th>Ti</th><th>Zn</th></tr></thead><tbody><tr><td>B1</td><td>41</td><td>0</td><td>59</td><td>3</td><td>0</td><td>97</td><td>41</td></tr><tr><td>B2</td><td>61</td><td>0</td><td>39</td><td>47</td><td>0</td><td>53</td><td>61</td></tr><tr><td>T-Ti3</td><td>41</td><td>3</td><td>56</td><td></td><td></td><td></td><td>42</td></tr><tr><td>T-Ti10</td><td>46</td><td>10</td><td>44</td><td>18</td><td>4</td><td>78</td><td>51</td></tr><tr><td>T-Ti24</td><td>38</td><td>24</td><td>38</td><td></td><td></td><td></td><td>50</td></tr><tr><td>T-Ti42</td><td>33</td><td>42</td><td>25</td><td>14</td><td>37</td><td>49</td><td>57</td></tr></tbody></table>

The Ti at. % measured by RBS is in agreement with EDS results: 3 %, 9 % and 20 % have been obtained for T-Ti3, Ti-Ti10 and T-Ti24, respectively. The indicates that the chemical composition is constant over the whole depth as illustrated by the RBS spectra shown figure 2 where perfect top-hat signals are visible for Ti and Cu+Zn (using the Cu/Zn ratio found by EDS).
<figure><img src="image_3.png" /></figure>

Figure 2: Experimental and simulated RBS spectra of the T-Ti10 film. Contributions of Si, Ti and Cu+Zn included in the simulation are reported. The simulated spectrum corresponds to one layer of 1300 10<sup>15</sup> at.cm<sup>-2</sup> with an in depth constant composition of 44 at. % of Cu, 9 at. % of Ti and 47 at. % of Zn.

### 3.2. Antimicrobial activity

Figure 3 presents the absorbance at 600 nm as function of the Ti at. % for binary and ternary alloy films. Values obtained for pure Cu, Ti and Zn films and for the silicon substrate are given for comparison

<figure><img src="image_4.png" /></figure>
Figure 3: absorbance at 600 nm of bacteria solutions after interaction with the samples at 37°C during about 24 hours as a function of the Ti at. % measured by EDS.

It is clearly seen on this graph that both Ti and Si have no antibacterial activity on *E. coli*, e.g. the measured absorbance is close to one. On the contrary, the pure Zn film shows a reduction of 80% of the bacteria in solution compared to the control. These results are in line with what was expected regarding the reported activity of these elements: Ti and Si have been proved un-active, whereas Zn is known to be antibacterial (see reference 28 for instance). Both CuZn alloy films exhibit the same activity as Zn, with an absorbance value between 0.1 and 0.2. This could appear as a surprise as one would have expected that adding Cu, a highly antimicrobial material, would have promoted the destruction of bacteria. However, in 2008 H. Kawakami *et al*. have studied the antimicrobial activity of many elements using the JIS Z 2801 protocol dedicated to surfaces. They have found similar activity of Cu and Zn against *E. coli*, Zn being even more active versus *Staphylococcus aureus* than Cu [34]. Nevertheless, the total inactivity of the pure Cu film obtained in our experiment is very surprising. This will be discussed in the following. A very important trend visible in figure 3 is a clear decrease of the activity when Ti is added to the binary alloy. Above 30 at. % of Ti, the absorbance reaches 1, meaning that no antibacterial activity is detected for the films in the present conditions of test.

Thus to try to depict the reason for the activity evolution plotted figure 3, it is necessary to go deeper into the film characterization since chemical composition alone does not drive the complex mechanisms of the bacteria/surface interaction.

### 3.3. Film characteristics

Our previous works showed that different phases are stabilized in the films depending on the Cu to Zn proportion; even if the composition range is narrow [37]. Both binary alloy films are polycrystalline. At low Ti content, a CuZn phase is detected determined by this Cu/(Cu+Zn) ratio, but with shifted and broadened peaks. When the percentage of Cu is close to 40%, (B1 and T-Ti3) the γ-Cu₅Zn₈ phase (BCC, Body-Centered Cubic) is formed, whereas at 60% (B2) the α-CuZn phase (FCC, Face-Centered Cubic) is obtained. In the case of T-Ti10 the Cu percentage is close to 50 (see table 1), and a mixture of both phases is suspected. The most important point, for the present study, is the loss of crystallinity when Ti at. % at increases. This is clearly visible from the GIXRD peak shape and intensity and from the very smooth and feature less morphology evidenced on SEM images. Note that on figure 4 diffraction patterns
are only given to show the degree of crystallinity, which is the relevant data for this study. A
complete interpretation of the diffraction patterns, along with the phase identification, are out
of the scope of this paper and can be found in reference [37]. All the pure element films are
polycrystalline exhibiting the low temperature phase: HCP (hexagonal close-packed) for Zn
and Ti and FCC for Cu (diffraction patterns not shown).

<figure><img src="image_5.png" /><figcaption>Figure 4: A) top view and cross section SEM images (× 50 000, 3kV) and GIXRD diffraction patterns of binary and ternary alloy thin films; B) top view and cross section SEM images (× 50 000, 3kV) of pure Cu, Ti, Zn films. Black scale at the bottom of the figure refers to all top view images. White line in cross section images corresponds to 400 nm too. NB: GIXRD patterns are only given to stand for the degree of crystallinity of the films.</figcaption></figure>
SEM images of the films are given in figure 4 where a large variety of film structures is visible. Pure Zn film appears highly porous formed of meandering columns and nanoparticles. Ti film is composed of sharp-edged and V-shaped columns; the structure is not homogeneous in the thickness. On the contrary, Cu film appears denser, made of a mixture of columns and what many authors called vein-like patterns, which are usually associated to a viscous behaviour [25, 38]. The trends are not surprising. Microstructures of magnetron sputtered films have been widely studied and correlated with the deposition conditions and with the element intrinsic properties. Many authors have investigated this aspect. The so-called structure zone model defines microstructure types depending on the energy deposited at the surface during the growth as compared to the ability of the adatom to become mobile, which is related to the fusion temperature [39-41].

Binary alloy films have very different microstructures: the Zn-rich one (B1) exhibit a columnar morphology, whereas the Cu-rich one (B2) presents a porous, vein-like, aspect. This is not completely different from the pure Cu film microstructure and is thus in line with the chemical composition (Cu-rich binary alloy) and the type of crystalline phase (FCC) present in both films. It is interesting to note that the Zn-rich binary alloy exhibits a microstructure very different from that of the pure Zn film, which can be related to the formation of the γ-Cu₅Zn₈ phase having a BCC symmetry (instead of HCP) and a much higher melting temperature than the α-Zn one [37]. The T-Ti3 film cross section shows irregular columns. An almost featureless cross section structure is observed for T-Ti10. Above this Ti content, the films appear very dense.

To go further, XPS analysis was conducted on multiple films, which confirmed the expected presence of various native oxides on the top surface (refer to Figure 5). The titanium spectrum displays four distinct peaks that correspond to TiO₂ (458.1 eV), Ti₂O₃ (456.8 eV), TiO (455.2 eV), and metallic Ti bonds (453 eV). In the zinc spectrum, two main components are observable: ZnO at 1022.2 eV and metallic Zn at 1021.7 eV. The analysis of the copper spectrum was more challenging due to the overlapping of the Cuº and Cu₂O components at the same binding energy (932.2 eV); however, copper is known for its high susceptibility to oxidation in ambient atmosphere, so the presence of Cu₂O and CuO (932.6 eV) can be suspected. The presence of the metallic peaks suggests that the native oxidized layer is very thin, with a thickness less than 10 nm. This is what is expected for most metals. A rough estimation of the oxide/metal ratio evaluated from XPS results shows that more than 90 % of the chemical bonds are Metal-O. Thus, even if metallic areas co-exist with oxide zones at the
top surface, they are very small. Several studies indicate that oxides of copper, zinc or titanium exhibit antibacterial and even antiviral properties [42, 43]. However, it is important to note that native oxides formed under humid air exposure are generally non-crystalline and may include hydroxyl groups. Nevertheless, results from other groups clearly show that magnetron sputtered TFMG can reduce the bacteria proliferation, and even causes their death, without any pre-treatment of the surface. It can be concluded that the presence of a thin native oxide layer does not usually hinder the antibacterial activity.

<figure><img src="image_6.png" /><figcaption>Figure 5: a) XPS Zn2p and Cu2p spectra of B2 and b) XPS Zn2p and Ti2p spectra of T-Ti42 films. In this last sample Cu2p signal is too low to be fitted.</figcaption></figure>
An important point is that the proportion of Zn is higher than the mean content measured by EDS or as predicted by RBS spectrum simulation (see table 1). This suggests a migration of this element at the surface. As mentioned above, the Cu/Zn ratio cannot be precisely determined by RBS. Nevertheless, simulations of the RBS spectra performed with the hypothesis of great variations of the Cu/Zn ratio (using values found by XPS) over more than 20-30 nm, failed. This result, correlated to XPS data, show that the existence of a Zn concentration gradient along the thickness is very unlikely and that the Zn enrichment only concerns the top surface. This superficial phenomenon has already been predicted by MD simulation of the film growth carried out considering the energy distribution function of each element in the corresponding experimental conditions. Complete description of the MD calculations and hypotheses can be found in references 44 and 45. Segregation of Zn at the film surface and grain boundaries is visible on the examples of snapshots obtained for T-Ti24 and T-Ti42 samples presented in figure 6.

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Figure 6: Results from MD simulations. Color-coding: brown Cu, light grey Ti, dark grey Zn, light brown Si substrate.</figcaption></figure>

Zn is known to have a particular behaviour during magnetron sputter deposition. The sputtering of a Zn target is difficult to control because of its low melting temperature, and its proportion inside the film does not always fit what is expected. During the growth, because of the energy transferred by all the energetic species, as mentioned above, the local temperature increases and adatom mobility is high. This could lead to Zn atom diffusion. The surface Zn enrichment is observed by XPS on binary and ternary alloys (Table 1).

## 4. Discussion

Antibacterial results have to be discussed in light of film characteristics. First, the fact that the pure Cu film does not show any antibacterial activity could question the test conditions themselves. However, this test has been duplicated several times and the results have been confirmed. Moreover, Cu sulphate solutions have shown activity related to the concentration,
confirming the validity of our test conditions. A bulk Cu piece of the same size than all the samples was also tested. In this case, a very low activity was found. A highly porous film composed of 2-5 nm in diameter Cu nanoparticles (with an expected high specific surface area) was prepared using a gas aggregation source. They have an absorbance value about 0.6. All the above results indicate that our protocol, which has often been used in literature for thin films, is very sensitive to the surface accessible to the bacteria solution. That means that the sample size has to be precisely calibrated to ensure accurate comparison (which is required in standard protocols) and, more important, that the specific surface should be taken into account in the interpretation of the results.

With that in mind, the very good activity of the highly porous Zn pure films can be understood, as compared to the dense copper film one, although Cu is known to be an antibacterial agent. Due to the similar antibacterial activity of these elements on E. coli [34], adding Cu to Zn, is expected not to significantly modify the activity of the film. This is what is observed on B1 and B2, which exhibit the same absorbance as pure Zn film (figure3). Even if these two films do not appear as porous as the pure Zn one on SEM images, voids exist in their structure (between columns on B1 and as pores in B2). This explains their sensitivity to bio-corrosion evidenced by the degradation of the film visible to the naked eye after the antibacterial test. Optical microscope images are presented figure 7 proving severe modification of B1 and T-Ti3 films at the micrometre scale. Considering that the bio-corrosion mechanism induces ion release, it can be speculated that for both samples Cu and Zn ions were present in the solution during interaction with the bacteria.

<figure><img src="image_9.png" /><figcaption>Figure 7: photo and optical microscope images of thin films taken after interaction with the bacteria solution during 24 hours at 37°C. For comparison the images of as-deposited T-Ti3</figcaption></figure>
and T-Ti42 are given. Whereas B1 and T-Ti films are clearly degraded, which highlights bio-corrosion phenomenon, T-Ti24 and T-Ti42 samples show very little or no modification after the test.

The sharp decrease of the antibacterial activity observed even at low at. % of Ti, in a narrow composition range, could be attributed to the modification of the crystallinity of the films and the formation of an amorphous phase. This lead to a reduction of the surface accessible to the bacterial solution and to a better resistance to bio-corrosion, in line with the general higher chemical stability, when an amorphous, crystalline defect free, phase is present [18, 24]. This last assumption is corroborated by the resistance of the film to the test observable on figure 7. Both features tend to limit the ability of metallic ions to be released.

The absence of activity of a Cu-TFMG may appear in contradiction with the literature. Interestingly, however, the effect of Cu itself is not always clearly demonstrated. In a combinatorial approach, Steyer *et al.* investigated the properties of ZrCuAg TFMG deposited by magnetron sputtering [20]. They found activity on *E. coli* for a Cu at. % between 30 and 65. In a similar study on ZrCuAlAg TFMG, Liu *et al.* evidenced a decrease of 80% of *E. coli* bacteria growth after 9 hours interaction with the films [19]. However, both works highlight a non-direct relationship between Cu content and the antibacterial activity. In Steyer *et al.* study the predominant role of silver is clearly shown. The same trend can be emphasized from Liu's work, in which surprisingly, the highest activity is obtained in the composition range where Zr and Al are the most abundant elements.

When Cu is the only antibacterial element in the alloy, its role has been studied and could appear questionable. Studying the properties of ZrCuNiAl (at. 30% of Cu) TFMG under the Japanese Industrial Standard JIS Z2801: 2000, Chu *et al.* correlated the antimicrobial efficiency with both the smooth surface state of the metallic glass and to the release of Cu ions in the solution [10]. Unfortunately, since they compared films of fixed composition with “rough” stainless steel, the respective role of each factor cannot be elucidated. Nkou Bouala *et al.* investigated the effect of silver addition in ZrCu alloy thin films [22]. They found that the binary alloy (Zr<sub>52</sub>Cu<sub>48</sub>) and low Ag containing ternary alloys have no efficiency on the proliferation of *E. coli* and *S. aureus*. This confirms that, even at high proportion of Cu in the film, the antibacterial activity may not be significant. All these observations suggest that the release of Cu ions in the solution depends on the chemical or thermodynamic stability of the alloy phase. The results obtained by Nkou Bouala *et al.* for instance confirm the chemical stability of the Cu-TFMG
phase. The other reason for a low activity of Cu alloy thin films may be the absence of Cu at the surface. Unfortunately, the analysis of the top surface chemical composition is rarely carried out.

The present work clearly shows that Zn, an antibacterial element, is present at the surface. Despite that no, or at least less, activity has been found as soon as the CuTiZn metallic glass is formed. This indicates that obtaining this amorphous phase may not be favourable to the “release killing” mechanism since the surface accessible to the solution is reduced and the chemical stability is enhanced. Comparing with other kind of TFMGs, we can deduced from this work that CuTiZn family has limited effect on *E. coli* bacteria. Thus coupling the action of Cu and Zn in presence of Ti, in an attempt to further the effect on microbes while keeping biocompatibility, does not seem to be a valuable solution.

### 5. Conclusion

This work demonstrates the capability of magnetron sputter deposition to synthesize CuZn and CuTiZn thin films with a good control of the Cu/Zn proportion in the 40 to 60 % range. XPS analysis shows that Zn (metal or oxide) is abundant at the surface, where Cu concentration is very low. This is confirmed by molecular dynamic simulations of the film growth. Ti addition greatly perturbs the crystallinity of the film, leading to the formation of a dense, stable amorphous phase from 30 at. %. This induces a sharp decrease of the antibacterial activity, despite the presence of Zn at the surface. As discussed in this paper, the dependence of the antibacterial activity to the morphology and microstructure of the films prevent from determining whether or not the ternary alloys become completely inactive. However, what can be drawn from the results is that coupling Cu and Zn in such kind of alloys may not be beneficial, since only Zn lies at the surface.

In the antibacterial test protocol we used, the activity is correlated with 1) the metallic ion release ability, which depends on the biocorrosion resistance of the film (crystallinity and composition) and on the specific surface (open porosity) and 2) the composition that determines the intrinsic activity (via the presence of active element). Our work has shown that comparing the antibacterial activity of thin films of very different features could be very tricky. Evidencing the intrinsic chemical action (release killing) requires to study films of similar nature. This is not always possible since the microstructure/crystallinity of thin film alloys depend not only on the deposition conditions, but on the chemistry itself. This is what has been showed in this study
when Ti is added to CuZn alloy. Fortunately, such issues do not rise when comparing thin films
in a limited composition range that means when the amorphous phase remains stable.

Acknowledgements

Region Centre - Val de Loire is acknowledged for supporting this work through ARD-MATEX grant # 2021 - 00145829. This work has received support under the CERTEM 2020 Program, with the financial support of the Regional Council Centre-Val de Loire (CERTEM 2020 201400096812) and co-funded by the European Union through the European Regional Development Fund (ERDF 2017-EX002247). We acknowledge the French EMIR&A network for provision of irradiation beam time and assistance in using the CEMHTI-Pelletron facility.
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