# 3D periodic microscale structures on binary copper-nickel alloys via fs-DLIP: impact on E. coli-substrate interaction

Aisha Saddiqa Ahmed, Sebastian Wältermann, Pablo María Delfino, Daniel Wyn Müller, Jean-Nicolas Audinot, Jean-François Pierson, Frank Mücklich

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## To cite this version:

Aisha Saddiqa Ahmed, Sebastian Wältermann, Pablo María Delfino, Daniel Wyn Müller, Jean-Nicolas Audinot, et al.. 3D periodic microscale structures on binary copper-nickel alloys via fs-DLIP: impact on E. coli-substrate interaction. Materials & Design, 2025, 258, pp.114545. 10.1016/j.matdes.2025.114545. hal-05214286

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# 3D periodic microscale structures on binary copper-nickel alloys via fs-DLIP: impact on E. coli-substrate interaction

Aisha Saddiqa Ahmed<sup>a,b,*</sup>, Sebastian Wältermann<sup>a</sup>, Pablo María Delfino<sup>c,d</sup>, Daniel Wyn Müller<sup>a</sup>, Jean-Nicolas Audinot<sup>c</sup>, Jean-François Pierson<sup>b</sup>, Frank Mücklich<sup>a</sup>

## ARTICLE INFO

### Keywords:

Femtosecond

Direct laser interference patterning

Copper-nickel alloy

Antibacterial

Periodic three-dimensional structures

Line-like structures

Honeycomb structures

## ABSTRACT

In response to the rise of multidrug-resistant infections, researchers are increasingly focusing on modifying material surfaces through chemical or topographical changes to limit microbial spread. Copper and its alloys are particularly valued for their antimicrobial properties, playing a key role in combating pathogen transmission. To explore the potential of enhancing the antibacterial performance of copper-based alloys, this study focuses on surface modification of binary copper-nickel alloys. The goal is to create periodic microstructures at the scale of a single bacterial cell (*Escherichia coli*) and explore the bacteria-substrate interaction. Two copper-nickel alloy compositions with 10 and 30 wt-% nickel are selected to assess the impact of nickel on laser-material interaction. Additionally, two surface structures, lines and honeycomb, are fabricated to evaluate how structure type affects bacterial interaction. Results show that the line-like structures on the 10 wt-% nickel alloy enhance bacterial killing, while honeycomb structures show no improvement. Conversely, both laser-structured copper-nickel alloys with 30 wt-% nickel exhibit decreased bacterial killing, attributed to the reduced surface copper concentration (below 60 wt-%) as confirmed by chemical analyses. These findings highlight the importance of copper content and structural compatibility for effective contact-killing performance.

## 1. Introduction

Copper and its alloys are widely recognized for their ability to combat microorganisms, making them valuable materials in the fight against pathogen transmission [1,2]. With the growing threat of multidrug-resistant infections and the increasing prevalence of deadly microorganisms [3], research has expanded beyond conventional antibiotic development [4]. One promising approach is the modification of material surfaces via techniques that include chemical or topographical alterations to limit microbial spread through contact [5-7].

This study explores direct laser interference patterning (DLIP) [8], a surface modification technique that utilizes multiple laser beams to generate periodic micro- and nanoscale structures. DLIP has been widely employed to develop nature-inspired surface structures that enhance properties e.g., wettability, friction, electrical conductivity, and antibacterial activity [9-13]. When combined with ultrashort pulse laser processing, this technique enables precise modifications at the

microscale and nanoscale that can effectively inhibit bacterial adhesion and proliferation [5,14].

While copper itself exhibits strong antibacterial properties, it is normally alloyed to meet the properties required in practical applications [15]. Nickel is a commonly used alloying element in copper-based materials, particularly in high-humidity and underwater environments, where it improves strength and corrosion resistance [16,17]. Commercial Cu-Ni alloys typically contain multiple alloying elements; however, in laser-based surface modifications, it is important to isolate and understand the interaction between the laser and the material while minimizing other variables. For that purpose, only binary copper-nickel systems are investigated in this study.

Recent studies suggest that a copper alloy must contain at least 60 wt-% copper to exhibit significant antibacterial properties [2,18]. Based on this criterion, two binary Cu-Ni alloys with 10 % and 30 % nickel by weight were selected for this investigation. The surfaces of these alloys are modified using ultrashort pulsed DLIP (USP-DLIP) to create

<footnote><sup>a</sup> Functional Materials, Saarland University, 66123 Saarbrücken, Germany</footnote>

<footnote><sup>b</sup> Université de Lorraine, CNRS, IJL F-54000 Nancy, France</footnote>

<footnote><sup>c</sup> Advanced Instrumentation for Nano-Analytics (AINA), Luxembourg Institute of Science and Technology (LIST), L-4422 Belvaux, Luxembourg</footnote>

<footnote><sup>d</sup> University of Luxembourg, 4365 Esch-sur-Alzette, Luxembourg</footnote>

<footnote>* Corresponding author at: Functional Materials, Saarland University, 66123 Saarbrücken, Germany.</footnote>

<footnote>E-mail address: aisha.ahmed@uni-saarland.de (A.S. Ahmed).</footnote>
microstructures tailored for bacterial interaction and increase the con-
tact surface area. Furthermore, two distinct surface structures are
studied: first, a line-like structure, previously investigated for its anti-
bacterial properties on copper and other copper-based alloys
[5,14,19,20]; and second, a honeycomb structure was designed to offer a
varied contact area for bacteria and isolate them within the valleys to
prevent bacterial growth. The antibacterial efficacy of both Cu-Ni alloys,
along with their respective surface structures, is assessed against
Escherichia coli (E. coli) to determine the role of modification in bacteria-
substrate interaction.

## 2. Materials and methods

2.1. Materials

Two compositions of copper-nickel alloys were tested in this study. Most commercially available copper-nickel alloys contain additional alloying elements beyond copper and nickel. The primary goal of this study is to examine the specific effect of the nickel element and its weight percentage on the properties of the alloy. To achieve this, binary alloys consisting solely of copper and nickel were fabricated by the Forschungsinstitut Edelmetalle + Metallchemie in Germany, eliminating the influence of other alloying elements. The alloys were cast and then cold-rolled to a thickness of one millimeter. Subsequently, the sheets were cut to dimensions of 10 × 25 mm² to prepare the samples. These samples were then metallographically prepared for the laser process to achieve a mirror finish on the surfaces. For this purpose, the surfaces were first ground and then polished in three steps, as outlined in Ahmed et al. [19].

## 2.2. Direct laser interference patterning

A Ti:sapphire Spitfire laser system from Spectra Physics, featuring a femtosecond pulse duration, is used to modify surfaces at the microscale. Direct laser interference patterning is employed to create periodic structures at the scale of bacteria for this study. This technique involves two coherent laser beams that intersect through a lens and are then focused onto the sample surface, generating an intensity profile that leads to the formation of the desired 3D structures. An 800 nm centered wavelength and an ultrashort pulse duration of 150 fs (full width at half maximum) were employed. The optical configuration was chosen according to Ahmed et al. [19]. It comprises of five main components: a beam diameter aperture, a wave plate, a diffractive optical element, a mask, and a focusing lens system with a 100 mm focal length. In this study, two distinct structures are created. A line-like structure is produced using two-beam interference, while a honeycomb structure is generated using three-beam interference, both with a pulse fluence ($F_{pulse}$) of 0.65 J/cm² and an accumulated number of 18–19 pulses ($n_{acc}$).

Finally, the samples are immersed in 10 % citric acid to remove any process-related oxides. They are then stored at room temperature under ambient conditions for a minimum of 3 weeks to ensure stable wetting properties [5]. Subsequent examinations and tests are conducted after this 3-week period. For this study, samples that have undergone laser structuring are referred to as “as-processed,” while those that have undergone both laser structuring and etching are referred to as “etched.

## 2.3. Surface characterization

The surface topography was examined using confocal laser scanning microscopy (CLSM) with the LEXT OLS4100 3D Measuring Laser Microscope (Olympus) and scanning electron microscopy (SEM) in secondary contrast mode (Helios NanoLab 600™, FEI/Thermo Fisher Company). SEM images were captured at an acceleration voltage of 5 kV, with a current of 1.4nA, and the sample surface was tilted at a 45° angle.

Surface elemental analysis was conducted using the FEI/Thermo

Fisher Helios G4 PFIB CXe system, along with EDAX APEX software from Ametek Materials Analysis Division. Since the surface modification penetrated only a few micrometers, an acceleration voltage of 5 kV and a current of 5.5nA were used to limit the analysis to the outermost layer.

Phase analysis is performed using a high-resolution grazing inci-
dence X-ray diffractometer (GI-XRD), equipped with a Cu Kα source
(wavelength 1.5418 Å) and operated at a 1° grazing angle, utilizing the
PANalytical X'Pert PRO-MPD system. Additionally, Raman spectroscopy
(Horiba Jobin, Lab RAM HR with a 532 nm laser) is employed to char-
acterize oxides.

Surface chemical analysis was performed using a secondary ion mass spectrometer (SIMS) integrated to a Zeiss Orion NanoFab Helium Ion Microscope (HIM).[21] First, secondary electron (SE) images were acquired with 2048x2048 pixels, 25 keV He⁺, 4 pA, dwell time of 10 µs/ pixel, 4 lines average. Subsequently, SIMS images were acquired with 512x512 pixels, 25 keV Ne⁺, 5 pA, dwell time of 2 ms/pixel leading to a total of 8 min per image. Representative Regions Of Interest (ROIs) were selected for line-like and honeycomb structures with and without bacteria. Successive in situ SE and SIMS acquisitions provide morphological and chemical correlation from the same ROI.

The SIMS analysis was performed in dynamic mode, which operates under continuous ion bombardment. This configuration provides both a high spatial resolution (down to sub-15 nm) and an excellent detection limit for elemental analysis, making it particularly suitable for resolving nanoscale chemical distributions on structured surfaces and for detecting copper uptake at the bacterial interface as it is shown in section 3.3.2.

## 2.4. Contact killing and wet plating method

Contact killing followed by wet plating is used to determine the antibacterial activity on the surfaces. *E. coli* is grown aerobically overnight in Lysogeny broth (LB), and the bacterial suspension is prepared in a phosphate-buffered saline (PBS) according to Ahmed *et al.* [14]. Droplets with a volume of 40 µL each are placed on sample surfaces for 30 min, 60 min and 90 min. After the respective contact time, 5 µL of each droplet is retrieved, further diluted, and placed on LB-agar plates. After 24 h of incubation at 37 °C, the number of colony forming units (CFU) is counted to enumerate the surviving CFU/mL. The experiment was repeated three times for each material and contact time to ensure the results were reproducible.

## 3. Results and discussion

3.1. Topographical modifications

The surfaces were initially examined using secondary electron mi-
croscopy to assess the topographical changes. Fig. 1 a and b demonstrate
that the desired periodicity of 3 µm for the line-like structures, with a
depth of approx. 1 ± 0.2 µm, is achieved. As expected, the line-like
structures (Fig. 1 a and b) show wide valleys and narrow peaks
[14,19,22]. The periodicity aligns with literature values for the struc-
ture, which show an increased killing rate of E. coli on structured copper
and brass [5,14].

The three-beam interference generates an intensity profile that forms honeycomb-like point structures. The interference minima of the three beams create a region with hexagonal peaks range, while the interference maxima form a region with point valleys, resulting in a hexagonal pattern. Previous studies [23,24] demonstrate the fabrication of similar point structures using DLIP, though with different pulse durations, wavelengths, periodicities and materials. According to the findings of Mulko et al.,[23] the crater/valley width increases with higher fluence, and the shape of this ablated regions transitions from circular to hexagonal. In this study, the structures have a periodicity of 3.5 µm and a depth of 1 ± 0.2 µm, as shown in Fig. 1 c and d. With these dimensions, the valley width is approximately in the range of individual E. coli cells,
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** SEM-images of 3D-microscale periodic structures on CuNi10 and CuNi30. As-processed surfaces: a) and b) represent the line-like structures. c) and d) represent the honeycomb structures on both alloys in their as-processed state, respectively. Etched surfaces: e) and f) correspond to the line-like structures, and g) and h) to the honeycomb structures.</figcaption></figure>

which is ideal for contact with bacteria and similar to the line-like structure observed in this study and previous ones [5,14].

Overlapping beams create an intensity profile with maxima and minima, forming either a line-like (two-beam) or honeycomb (three-beam) pattern in the absorbed energy. Regardless of whether it is a two-beam or three-beam setup, the laser-material interaction remains similar for both structure types, as it is dependent on the pulse duration. In the case of femtosecond-pulsed DLIP, the pulse duration is shorter than the electron-lattice relaxation time (1–10 picoseconds) [25]. As a result, the high energy absorption leads to cold material ablation, primarily through phase explosion, which involves the creation of vapor and plasma phases that expand into the atmosphere, along with spallation [26,27]. This process is accompanied by a minimal heat-affected zone, resulting in little to no melting of the material [28–30]. Ultimately, it results in the formation of precise, periodic structures [31]. Due to the ultrashort pulse duration, the process is followed by rapid cooling of the molten matter and the redeposition of ablated material [32,33]. Additionally, since the phase explosion and evaporation occur outward from the interference maxima, the redeposition of ablated material predominantly happens on the peak regions of the structures, contributing to the roughness at the sub-micron scale [19].

In addition, another topographical feature at sub-µm is the formation of laser-induced periodic surface structures (LIPSS) [34,35]. The formation of LIPSS has been observed in previous studies using femtosecond laser processing on various materials and is reported to be dependent on several laser parameters, including wavelength, polarization, and scanning velocity [26,36,37]. Both alloys exhibit similar topographical features including LIPSS formation. In the case of line-like structures, the LIPSS are parallel to the lines, while in the honeycomb structure, they align with the vertical axis of the SEM images shown in Fig. 1 c and d.

Another notable difference is that the alloy with higher nickel content displays a small amount of flake-like structures that are associated with oxide formation [22]. The formation of these flake-like structures is directly related to the laser fluence [38]. Since both alloys have different chemical compositions, their laser-material interactions vary, which directly influences the chemical modification [39]. As a result, the threshold laser fluence required to form these flake-like structures may

differ depending on the alloy composition. Given that the pulse fluence used for both alloys are identical, it is highly likely that this is the reason for the variation in the concentration of these flake-like structures. Moreover, after etching these flake-like structures are removed from the surface and both alloys depict the same topography (see Fig. 1 e-h).

## 3.2. Chemical modifications

### 3.2.1. Elemental analysis

Chemical analysis was performed using multiple methods, beginning with SEM-EDS for elemental analysis of both peak (black spectra) and valley (red spectra) regions of as-processed samples. The results are displayed in Fig. 2 and summarized in Table 1 for both alloy compositions.

In the laser-processed CuNi10 samples (Fig. 2 a and c), both regions, peak and valley, show the presence of the same elements. One noticeable difference is the intensities of the spectra, likely caused by height differences in the measured area, which directly affects the information depth. The signal is in particular weak within the valley regions of the honeycomb structures, which are surrounded on all sides by the peak regions. This is probably due to the obstruction of both the irradiated beam and the emitted beam from reaching the detector. In the case of the line-like structures, the lines were aligned with the detector, allowing it to receive a comparatively stronger signal from the valleys. However, when comparing the weight percentages of the valleys for both structures, the compositions are similar.

Overall, compared to the bulk chemical composition, there is a slight increase in oxygen content and a significant increase in nickel content, approximately 7–8 wt-%, are noted. Oxygen content is slightly higher on the peaks of the lines, consistent with previous research on line-like structures [22]. Notably, the highest increase in nickel content, around 11 wt-%, is observed in the valleys of the honeycomb structures (see Table 1). It is important to note that, although the wt-% of oxygen listed in Table 1 is quite low, the measured increase in at-% is somewhat higher, ranging between approximately 4–7 at-%. This indicates that the surface contains a higher concentration of oxygen atoms, even though its overall mass contribution to the material is relatively small, given oxygen's lower atomic weight compared to the metals in the alloy.
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** EDX-spectra of laser-structured CuNi10 and CuNi30: a) & b) line-like structures c) & d) honeycomb structure. Each sample was analysed on the peak (black spectra) and in the valley (red spectra). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

Oxygen might be forming a thin oxide layer or could be adsorbed onto the surface.

CuNi30 samples in Fig. 2 b and d also exhibit the presence of the same elements. Compared to the bulk chemical composition, the peaks (black spectra) of both structured surfaces show a slight increase in oxygen content and an increase in nickel content ranging from approx. 4–5 wt-%. However, significant compositional differences are evident between peaks and valleys. The valleys (red spectra in Fig. 2 b) of line-like structures exhibit a slight decrease in nickel and oxygen content, aligning closely with the known bulk composition of polished samples in terms of copper and nickel wt-%. The difference in oxygen content between peaks and valleys correlates with the results observed in CuNi10 lines.

Furthermore, the valleys of honeycomb structures for CuNi30 demonstrate a slight increase in oxygen content and a significant increase in nickel content, up to approx. 12 wt-%. This led to a decrease in

copper content to approx. 54 wt-%. These findings suggest the presence of an oxide phase with high nickel content. According to the Ellingham diagram [40], the energy released during the formation of nickel oxide is more negative than that of copper, suggesting that the formation of a nickel-rich oxide phase is thermodynamically favored.

### 3.2.2. Phase analysis

Subsequently, phase analysis was performed on both polished and as-processed samples using GI-XRD. The resulting diffractograms are shown in Fig. 3 and Fig. 4, highlighting the prominent peaks: (111), (200), (220), (311), and (222) which appear in all samples. Fig. 3 a shows the diffractogram of polished CuNi10 with peaks at 43.65°, 50.67°, 74.59°, 90.45°, and 95.78°. In comparison, the peaks of CuNi30 are observed at 43.91°, 50.93°, 75.11°, 91.23°, and 96.56° (see Fig. 4 a). Since the atomic radii of nickel is lower than that of copper, the increase of the nickel content induces a shift in the peak positions towards higher 2θ values. These results are consistent with literature values [41–43] for pure copper, pure nickel, and binary copper-nickel alloys, confirming the correlation with known diffractograms.

Additionally, it is visible that for CuNi10 polished, the (111) and (200) peaks are more intense. According to the literature [44], both face-centered-cubic copper and nickel exhibit similar intensity ratios in between the peaks with (111) being the dominant one. While for CuNi30 polished, the (220) and (311) peaks are relatively more pronounced. This difference in relative intensities depends on various factors that influence the crystalline structure and texture of the sample, i.e. the agents and conditions used during sample fabrication, as well as the preparation of the sample surface [45–48].

The diffractograms of both CuNi10 as-processed samples, lines (Fig. 3 b) and honeycomb (Fig. 3 d) structures, are identical. However, as this is compared to the polished surface, a new low-intensity peak is visible at 36.63°. This can be attributed to the presence of copper or nickel oxide. According to literature [49–51], the 2θ-value of (111) peak for cuprous oxide is at 36.42° and for nickel oxide at around 37.25°. However, there are no further peaks visible for any oxides and the most dominant peak in the case of nickel oxide is (200) at 43.28° (Ref.: PDF # 00-044-1159). Furthermore, a study [52] shows the presence of (003) peak at 36.79° for nickel oxide with nickel vacancies (Ref. ICCD 04-011-2340). This suggests the presence of copper oxide, likely with defects, and the shift to a higher 2θ value compared to the literature value indicates that this may be related to the presence of nickel content. Another notable distinction is approx. a 0.1° shift observed in the (111) peak towards a lower value, and in the (200) peak towards a higher value. Additionally, there is a noticeable alteration in the relative intensities. Figures b and d illustrate that the (220) peak exhibits relatively high intensity, suggesting a preferred orientation of the CuNi10 surfaces after structuring.

The structured samples are also etched to remove any laser-process related oxide, as it has been shown in previous studies to enhance the killing of E. coli in the case of laser-structured copper alloys [14,19]. Fig. 3 c and e present the diffractograms of both etched laser-structured CuNi10 samples. These diffractograms are similar to their respective as-processed diffractograms with one exception, which is no visible sign of the oxide peak. This suggests the removal of oxides on laser-structured CuNi10 samples via etching.

<caption>**Table 1**</caption>

Weight percentages (wt-%) of copper and nickel in CuNi10 and CuNi30 samples after structuring, as determined by EDX analysis.

<table><thead><tr><th rowspan="3">wt-%</th><th colspan="4">CuNi10</th><th colspan="4">CuNi30</th></tr><tr><th colspan="2">lines</th><th colspan="2">honeycomb</th><th colspan="2">lines</th><th colspan="2">honeycomb</th></tr><tr><th>peak</th><th>valley</th><th>peak</th><th>valley</th><th>peak</th><th>valley</th><th>peak</th><th>valley</th></tr></thead><tbody><tr><td>Cu</td><td>79.5</td><td>80.5</td><td>78.9</td><td>74.7</td><td>60.7</td><td>68.4</td><td>62.4</td><td>53.9</td></tr><tr><td>Ni</td><td>17.8</td><td>17.4</td><td>16.8</td><td>21.0</td><td>36.3</td><td>29.2</td><td>34.9</td><td>41.8</td></tr><tr><td>O</td><td>1.91</td><td>1.28</td><td>1.86</td><td>1.54</td><td>1.76</td><td>1.34</td><td>1.74</td><td>2.57</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>Fig. 3. X-ray diffractogram of CuNi10 a) polished, b) lines as-processed, c) lines etched, d) honeycomb as-processed, and e) honeycomb etched.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 4. X-ray diffractogram of CuNi30 a) polished, b) lines as-processed, c) lines etched, d) honeycomb as-processed, and e) honeycomb etched.</figcaption></figure>

Fig. 4 displays the diffractograms of CuNi30 samples. First, as-processed samples are compared to the polished CuNi30. The diffractogram for the lines as-processed (Fig. 4 b) appears to be the same as that of the polished surface (Fig. 4 a). However, in the case of honeycomb as-processed (Fig. 4 d), a significant difference in the relative intensities is observed. The diffractogram shows that the (111), (200), and (220) peaks have similar intensities, suggesting a uniform distribution of these orientations. Furthermore, there are no new additional peaks related to oxides visible.

After etching laser-structured CuNi30, the diffractograms (Fig. 4 c and e) are similar in their peak positions and intensities, showing no change in chemical composition. However, a notable difference is observed in the relative intensity ratios of the peaks between the lines etched sample (Fig. 4 c) and the as-processed sample (Fig. 4 b). This suggests that the surface layer removed through etching exposes a layer with a different preferred orientation, which shows a uniform distribution of the (111), (200), and (220) peaks.

Overall, GI-XRD results indicate the formation of oxide phases solely on laser-structured CuNi10. However, compared with EDX results, an increased oxygen content is also observed on the laser-structured CuNi30 surface, particularly prominent in the valleys of honeycomb structures. A previous study [14] demonstrates similar effects on laser-structured brass surfaces, where the oxide phase was non-detectable via GI-XRD.

### 3.2.3. Oxide analysis

Next, Raman spectroscopy is utilized to identify the type of oxide present on both alloys. Since the GI-XRD results for laser-structured lines and honeycomb for CuNi10 as well as CuNi30 are identical, this section focuses exclusively on analyzing the line-like structures of each alloy. The resulting spectra are presented in Fig. 5, with the recorded band values summarized in Table 2.

A common feature across all spectra is the presence of bands in the 74–106 cm⁻¹ range. According to the literature [53], these bands can be attributed to the silent modes of Cu₂O, and the appearance of these bands is likely the result of defects in the oxide phase. Furthermore, the spectrum of as-processed samples in Fig. 5 a and b show a band at 219 cm⁻¹ and 240 cm⁻¹, respectively. This band cannot be assigned to any oxides relevant to this study. However, previous studies [19] have identified a similar band in laser-structured copper alloys. Since copper is the common factor in these studies, it suggests that this band may be related to a vibration of the bond involving copper.

The Raman spectrum of the CuNi10 lines in the as-processed state (black spectrum in Fig. 5 a) reveals a low-intensity band at 291 cm⁻¹, which can be attributed to the Ag mode of cupric oxide [48]. A

<figure><img src="image_8.png" /><figcaption>Fig. 5. Results of Raman spectroscopy on line-like structures of a) CuNi10 and b) CuNi30, each in as-processed and etched states.</figcaption></figure>
<table><caption>**Table 2**

Results of Raman spectroscopy on CuNi10 and CuNi30 in as-processed and etched conditions. As a comparison, the literature-values of copper oxide and nickel oxide.</caption><thead><tr><th rowspan="3">symmetry</th><th rowspan="3">activity</th><th rowspan="3">oxide</th><th rowspan="3">Raman shift ω<sub>e</sub> [cm<sup>-1</sup>]</th><th colspan="4">Literature</th></tr><tr><th colspan="2">CuNi10 lines</th><th colspan="2">CuNi30 lines</th></tr><tr><th>as- processed</th><th>etched</th><th>as- processed</th><th>etched</th></tr></thead><tbody><tr><td>E<sub>u</sub></td><td>silent</td><td>Cu<sub>2</sub>O</td><td>72–110 [53]</td><td>75<br>106<br>219</td><td>74<br>103<br>225</td><td>74<br>104<br>240</td><td>74<br>105<br>243</td></tr><tr><td>A<sub>g</sub></td><td>Raman</td><td>CuO</td><td>297 [48]</td><td>291</td><td>—</td><td>290</td><td>—</td></tr><tr><td>1P (TO)</td><td>Raman</td><td>NiO</td><td>400–440 [55],<br>548 [57]</td><td>554</td><td>551</td><td>539</td><td>523</td></tr><tr><td>1P (1LO)</td><td>Raman</td><td>NiO</td><td>498–570 [54–56],</td><td></td><td></td><td></td><td></td></tr><tr><td>1P (1LO)</td><td>Raman</td><td>NiO</td><td>656 [57]</td><td>635</td><td>633</td><td>—</td><td>—</td></tr><tr><td>B<sub>g</sub></td><td>Raman</td><td>CuO</td><td>609–636 [48,58]</td><td></td><td></td><td></td><td></td></tr></tbody></table>

prominent band appears at 554 cm⁻¹, accompanied by a low-intensity shoulder band at 635 cm⁻¹. Based on the literature [54–57], the prominent band can be attributed to the one-phonon (1P) modes of nickel oxide, specifically the transverse optical (TO) and longitudinal optical (1 LO) modes. Nickel oxide's Raman shift typically falls within the 400–600 cm⁻¹ range, with the TO mode varying between 400–548 cm⁻¹ and the 1 LO mode between 498–656 cm⁻¹. The precise position and intensity of these bands are influenced by several factors. For instance, doping can cause a shift toward higher Raman values. The greater the doping concentration, the more pronounced the shift [56]. One study on nanosized nickel oxide in powder form reported that structural defects significantly enhance the prominence of the 1P bands [54]. The shoulder band at 635 cm⁻¹ can be assigned to the B₉ mode of cupric oxide [48,58].

After etching the CuNi10 lines (pink spectrum in Fig. 5 a), the 219 cm⁻¹ band shifts to a higher value without a significant change in intensity. Conversely, the band corresponding to the A₉ mode (CuO) is no longer visible. Additionally, the 1P (NiO) and B₉ (CuO) bands shift to lower values, accompanied by a significant reduction in intensity. These observations suggest that most of the nickel oxide and cupric oxide are removed after etching.

Next, the CuNi30 lines are examined. The Raman spectrum of the as-processed surface (black spectrum), shown in Fig. 5 b, the A₉ (CuO) band is similar to the one observed in the CuNi10 lines in the as-processed state. Furthermore, a shoulder band is present at 539 cm⁻¹. However, the Raman shift value for the shoulder band is lower as compared to CuNi10 lines as-processed, indicating that it is not the B₉ (CuO) band, but rather another 1P (NiO) band. This suggests that the cupric oxide content is lower on CuNi30 in comparison to CuNi10. This difference is likely due to variations in the alloy composition, which affects the oxide phases formed during laser processing.

After etching (blue spectrum in Fig. 5 b), the CuNi30 lines exhibit a Raman shift trend similar to that of CuNi10. However, the intensity of the 1P (NiO) band is only reduced by half, indicating that the amount of nickel oxide remaining after etching is higher for CuNi30 than for CuNi10. On the other hand, the cupric oxide band is no longer visible, indicating its complete removal through etching.

The findings from chemical analyses suggest the formation of oxide phases with defects, consistent with results observed in previous studies on USP laser-structured copper-based alloys [14,19]. The results suggest that USP-DLIP processing leads to the formation of higher amounts of copper oxide on CuNi10, whereas higher nickel oxide content on CuNi30. According to the results of Raman spectroscopy, the cupric oxide content is higher on CuNi10 in comparison to CuNi30. This can be attributed to the significantly higher copper content in the bulk composition of CuNi10, which likely causes a concurrence between the formation of copper and nickel oxides. Although nickel oxide formation is more likely, this concurrence is likely to result in a higher formation of copper oxide content on CuNi10. Furthermore, etching leads to the removal of nearly all oxide phases from CuNi10, whereas in CuNi30, the

oxide phases are only partially removed, with comparatively higher nickel oxide content.

SEM images primarily show oxide-related flake-like structures on CuNi30, while EDX analysis indicates an increase in oxygen across all processed samples. XRD shows oxides only on CuNi10, contrasting with Raman spectroscopy, which detects oxides on both alloys. These differences highlight the complexities of oxide phase analysis at the nanoscale. After etching, the oxide on CuNi30 is only partially removed, likely due to surface modifications at the nanoscale. Previous studies suggest that the type of oxide formed depends on bulk composition, which can introduce analytical challenges, particularly in unstable, oxygen-rich regions [14,19].

Nevertheless, the combination of methods used in this study, supported by insights from prior research, provides a reliable and comprehensive understanding of oxide formation and surface modifications, despite the inherent challenges of nanoscale analysis.

For further experiments and characterization in this study, samples that are both laser-structured and etched will be used. Previous studies [14,19] have shown that etched samples are more effective at killing bacteria than as-processed samples, particularly in structured copper alloys like brass. This is primarily attributed to the removal of the oxide film during etching, which promotes increased copper corrosion on the surface, thereby improving antibacterial effectiveness. Importantly, this approach will further mitigate the influence of oxide formation due to laser treatment as far as possible, focusing instead on investigating the impact of surface topography on bacterial adhesion and killing.

### 3.3. E. coli on DLIP-structures

#### 3.3.1. Contact killing

The antibacterial activity on structured surfaces was evaluated using the contact killing method. The results, presented in Fig. 6 as the number of surviving colony-forming units (CFU/mL) over a 90 min contact period, show the following trends: the reference steel (black graph) and pure copper (purple graph) samples exhibit the expected killing rates—no killing for steel and significant killing for copper [5,19,59].

Next, the polished alloy samples (■ solid line graph: pink for CuNi10 and blue for CuNi30) are compared with copper. It is observed that the killing behavior is similar to copper after 30 min of contact. As the contact time increases, the killing rates of copper (purple graph) and CuNi30 (■ blue graph) converge. In contrast, CuNi10 (■ pink graph) shows a significant increase in killing efficiency, nearly an order of magnitude higher, after 90 min. Previous literature [14,19] indicate a decrease in the killing rate due to reduced copper content in polished copper alloys. However, this trend does not apply to copper-nickel alloys in this study, likely due to the presence of nickel. Nickel is known for its higher corrosion resistance in humid and underwater conditions [17]. This would typically suggest that copper-nickel alloys should exhibit higher corrosion rates, but the results in this study show a different trend. Based on the results from both polished copper-nickel surfaces
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Results of contact killing experiment, showing surviving CFU/mL over a 90 min period on laser-structured CuNi-alloys. Comparison of CuNi10 (pink graph) and CuNi30 (blue graph), showing polished samples (■ solid line graph), line-like structure (▲dash line graph), and honeycomb structures (● dot line graph). Also included are steel (black graph) and copper (purple graph) as references. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

and the known properties of nickel, it is likely that copper experiences more corrosion in the presence of nickel when in contact with the bacterial solution, leading to a higher killing rate on these surfaces. This discrepancy may be related to the presence of bacteria, as one previous study [60] suggests that even pure copper experiences varied corrosion in the presence of E. coli. However, further investigation is needed to explore this phenomenon in future studies.

The antibacterial performance of the structured samples was assessed. Both the line-like (▲ blue graph) and honeycomb (● blue graph) CuNi30 structures show a further decrease in killing efficiency compared to the polished sample (■ blue graph). In comparison, the honeycomb-like structure of CuNi10 (● pink graph) demonstrates a killing rate similar to that of polished CuNi10 (■ pink graph). Similarly, the line-like structure of CuNi10 (▲ pink graph) exhibits comparable behavior during the first 60 min of contact. However, after 90 min, a significant increase in killing efficiency is observed in the line-like structure (▲ pink graph). Note that the error bar for the CuNi10 lines at 90 min is not visible due to its value of the error being zero.

Overall, the results suggest that enhanced bacteria-substrate contact is achieved through a combination of optimal composition, structure, and increased contact area. CuNi10 line-like structures exhibited the best killing rate which is most probably due to a higher copper content and likely due to better compatibility between the structure and the shape of the bacteria.

The long-rod-shaped E. coli bacteria are more likely to align and spread along the grooves of the linear structures, leading to a larger and more continuous contact area compared to the honeycomb pattern or the polished surface. This improved alignment may facilitate stronger mechanical interactions or membrane stress, contributing to enhanced antibacterial activity.

Although the honeycomb theoretically offers greater total surface area, its complex and curved geometry may limit effective contact with bacteria due to mismatches in scale and orientation. These findings underscore that total surface area alone is not the key determinant of antibacterial performance—rather, the spatial compatibility between surface features and bacterial dimensions is critical.

While precise quantification of the actual bacteria-surface contact area was beyond the scope of this work, the qualitative assessment based

on structural parameters and bacterial size provides a solid basis for interpreting the observed trends. Future studies involving detailed contact area modeling and high-resolution morphological analyses will build on these findings by further explaining the underlying mechanisms.

Furthermore, the increased efficiency compared to pure copper is likely related to the larger contact area, and since the copper content in CuNi10 is relatively high, this effect may be further amplified in conjunction with the factors mentioned above.

Laser-structured CuNi30, regardless of the structure dimensions, showed reduced antibacterial activity, likely due to its copper content falling below 60 wt-%, as confirmed by EDX results. This aligns with prior studies on smooth copper-based surfaces, which suggest that copper content should not be lower than 60 wt-% in copper alloys to have significant antimicrobial properties [2,18]. The results of this study suggest that when working with laser-structuring, the copper content in the alloy should be higher, as it may decrease on the surface after the process, leading to a reduction in antibacterial efficacy. Specifically, for binary copper-nickel alloy system used in laser processing, the critical copper content threshold for the bulk composition appears to lie between 70 and 90 wt-%, with 70 wt-% already showing decreased antibacterial efficacy.

Nonetheless, the current results offer clear and compelling insights into how laser-induced microstructures and choice of material composition can be effectively enhance antibacterial properties, particularly in the case of copper-nickel alloys.

### 3.3.2. *Chemical analysis on structured surfaces with E. coli*

HIM-SIMS provides high-resolution chemical information of the surface through SIMS. Ni and Cu maps for etched laser-structured CuNi10 and CuNi30, with bacteria, are captured at different magnifications (field of view: FoV 10 (10 x 10 µm²)/15(15 x 15 µm²)/20 (20 x 20 µm²)). However, this section only shows FoV 10. Additionally, SE images are captured alongside the SIMS images to correlate Ni and Cu distribution with topographical features. The results of CuNi10 and CuNi30 are displayed in Fig. 7. In all cases, the elemental Ni and Cu maps replicate the pattern showing a clear distinction between topographical peaks and valleys. Secondary ion signal intensity is multifactorial dependent (it is mainly a function of the chemical composition of the sample, ion species, energy, current, and angle of incidence of primary ion beam), hence SIMS is usually restricted to qualitative analysis. To mitigate the topographic effects of the elemental SI signal intensity, Ni and Cu maps were normalized with the total ion count (TIC).

The results in Fig. 7 indicate a distinctively higher intensity of Cu and Ni in the valley compared to the topographical peaks. This difference is likely visible due to higher amounts of oxide on the peaks than in the valleys, as observed in previous studies as well [14,19,22]. Another significant finding is that Cu is present on the bacterial surface, whereas Ni is not. This is indicative of the copper-ion release from metallic phases which is effectively incorporated into the bacteria wall. The chemical information is estimated to correspond to a depth of no more than 13 nm of the bacteria's surface. This estimation is calculated by measuring the dimensions change in length and width of individual bacteria from SE (He⁺) images taken before and after the SIMS acquisitions.

The increased E. coli killing on CuNi10 lines, along with the higher copper content and copper oxide formation compared to CuNi30, suggests that copper is responsible for the enhanced antibacterial effect. Previous research indicates that copper ions kill bacteria by disrupting the cell membrane and degrading intracellular components [7,61,62]. In contrast, no studies indicate that nickel possesses antibacterial properties. Furthermore, the reduced E. coli killing effect observed in laser-structured CuNi30 samples with higher nickel content supports this. These findings are consistent with one another.
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** High-Resolution Secondary Ion Mass Spectrometry (HR-SIMS) analysis of etched laser-structured samples with bacteria: CuNi10 a) lines, b) honeycomb, CuNi30 c) lines, and d) honeycomb. The images were recorded with a field of view (FoV) of 10 x 10 µm². The first row displays the respective SEM images. The second and third rows present SIM images of the elemental map of nickel (Ni) and copper (Cu), respectively. Warmer colours indicate higher concentrations, and cooler colours represent lower concentrations.</figcaption></figure>

## 4. Conclusions

This study investigates the fabrication of two distinct periodic microscale structures, lines and honeycomb, on binary copper-nickel alloys with 10 wt-% and 30 wt-% nickel content. The focus is on the influence of bulk composition on laser structuring using ultrashort pulse durations, and its impact on the interaction between *Escherichia coli* (*E. coli*) and the structured surfaces.

The results show that topographical modifications are accompanied by the formation of copper- and nickel-rich oxide phases. Chemical analyses reveal that CuNi10 has a higher copper oxide content, whereas CuNi30 has a higher nickel oxide content. After chemical etching, most oxide is removed from the CuNi10 surface, while nearly half of the nickel oxide remains on CuNi30.

In the antibacterial tests, CuNi10 etched lines exhibited significantly faster bacterial killing compared to CuNi30, despite similar decreases in copper content. CuNi30's copper content decreased near or below the 60 wt-% threshold, which is the lower limit for copper-based alloys to be considered antibacterial, explaining the decreased antibacterial efficiency. These results underscore the importance of copper content in laser-structured alloys for effective bacterial killing.

Furthermore, line-like structures outperformed the honeycomb structures in terms of antibacterial performance. While the honeycomb structure was designed to prevent bacterial agglomeration by trapping bacteria in the valleys, it was not effective in promoting bacterial killing. This highlights the importance of ensuring dimension compatibility between surface structures and bacterial size, while also increasing the contact area. Future studies involving spherical bacteria may help clarify the suitability of such designs for different bacterial types.

In conclusion, this study highlights the critical role of copper content and laser structuring in enhancing antibacterial performance. It suggests

that the critical lower value of the copper content range for improved antibacterial activity in bulk composition through laser structuring lies between 70–90 wt-%. Future research should focus on determining the optimal copper content for bacterial killing in laser-structured copper-nickel alloys, exploring surface treatments like annealing to restore antibacterial effectiveness in CuNi30, and investigating the impact of bacterial presence on the corrosion behavior of these alloys.

### CRediT authorship contribution statement

**Aisha Saddiqa Ahmed:** Conceptualization, Investigation, Validation, Visualization, Writing – original draft. **Sebastian Wältermann:** Investigation, Validation, Writing – review & editing. **Pablo María Delfino:** Investigation, Validation, Writing – review & editing. **Daniel Wyn Müller:** Writing – review & editing. **Jean-Nicolas Audinot:** Resources, Supervision, Writing – review & editing. **Jean-François Pier son:** Investigation, Supervision, Resources, Validation, Writing – review & editing. **Frank Mücklich:** Funding acquisition, Resources, Supervision, Writing – review & editing.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgement

The Author would like to thank to the group of Prof. Rolf Müller from the Helmholtz Centre of Infection Research in Saarbrücken for providing the *E. coli*. The authors acknowledge financial support in the project
“MatInnovat” supported by the Saarland State Ministry of Economics with resources from the European Fund for Regional Development (EFRE) and funding by the German Research Foundation for the Femtosecond Pulsed Laser System (DFG, INST 256/562 FUGG). This work made use of the resources of the Correlative Microscopy and Tomography (CoMiTo) core facility at Saarland University. The authors would like to thank the technical platforms “Optic and Lasers” at Institut Jean Lamour (IJL, Nancy, France) for access to Raman facility.

## Funding

A.A. has been funded by the German Aerospace Center - Space Administration (DLR) within the project “Investigation of antimicrobial metal surfaces under space conditions - An effective strategy to prevent microbial biofilm formation” (project number 50WB1930). This work was funded by the German Research Foundation (DFG) within the project “Controlled bacterial interaction to increase the antimicrobial efficiency of copper surfaces” (project number 415956642). P.M.D thanks the funding to the Luxembourg National Research Fund (FNR) and the Agence Nationale de la Recherche (ANR) through the grants INTER/ANR/21/16227644 and ANR-21-CE05-0036-01.

## Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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