# Femtosecond-DLIP on tin-alloyed bronze: Effect of pulse fluence on surface topography for antibacterial application

Aisha Saddiqa Ahmed, Diego Sancio, Daniel Wyn Müller, Jean-François Pierson, Frank Mücklich

► To cite this version:

Aisha Saddiqa Ahmed, Diego Sancio, Daniel Wyn Müller, Jean-François Pierson, Frank Mücklich. Femtosecond-DLIP on tin-alloyed bronze: Effect of pulse fluence on surface topography for antibacterial application. Materials Today Advances, 2025, 27, pp.100596. 10.1016/j.mtadv.2025.100596. hal-05108412

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# Femtosecond-DLIP on tin-alloyed bronze: Effect of pulse fluence on surface topography for antibacterial application

Aisha Saddiqa Ahmed<sup>a,b,*</sup>, Diego Sancio<sup>a</sup>, Daniel Wyn Müller<sup>a</sup>, Jean-François Pierson<sup>b</sup>, Frank Mücklich<sup>a</sup>

## ARTICLE INFO

Keywords:

Bronze

Copper alloy

Direct laser interference patterning

Ultrashort pulse duration

Femtosecond

Surface modification

Antibacterial

## ABSTRACT

The rise of antibiotic resistance has prompted the exploration of other strategies, including the use of material surfaces, to prevent the spread of contagious microorganisms. Copper, renowned for its antimicrobial properties, holds potential in this area. However, its application is often limited from a materials perspective. Therefore, it is crucial to investigate copper alloys to assess their effectiveness in these promising new approaches. For that purpose, the surface of the bronze with 6 % tin content was modified using ultrashort-pulsed direct laser interference patterning (USP-DLIP) to produce 3 µm periodic line-like structures aimed at enhancing antibacterial properties. This study examines how pulse fluence impacts surface topography and subsequently, the killing of *Escherichia coli* (*E. coli*). The results show a variation in topography with varied peak-to-valley width ratios. Chemical analyses confirmed an increased copper content and the formation of tin- and copper-rich oxides. Subsequently, the flake-like oxide structures, formed due to laser treatment, were removed via etching with 5 % citric acid. The wettability tests showed increased hydrophobicity over 3 weeks, with etched surfaces exhibiting lower hydrophobicity than as-processed ones. Overall, the etched laser-processed surfaces demonstrated an *E. coli* killing rate of an order of magnitude higher than polished bronze and comparable to pure copper. This enhancement is attributed to the increased contact area and optimized topography achieved through USP-DLIP. This approach presents a promising strategy for antimicrobial surface design.

## 1. Introduction

To address the growing spread of pathogenic microorganisms, antimicrobial surfaces have become a major focus of research due to their potential to reduce microbial transmission [1,2]. Copper, long known for its antimicrobial properties, remains one of the most promising materials due to its ability to release metallic ions that disrupt bacterial membranes and degrade RNA/DNA upon contact [3-6]. Although copper's antibacterial performance can be significantly enhanced using laser-based surface treatments such as Direct Laser Interference Patterning (DLIP) [7], its limited mechanical strength constrains broader application. Alloying copper with elements like tin improves properties such as hardness, strength, corrosion resistance, and wear resistance. [8], making copper alloys—such as brass and bronze—viable candidates for antimicrobial infrastructure in public spaces like

hospitals, schools, and transport systems [9-11]. Bronze, in particular, is suitable for high-touch components (e.g., door handles, railings, bed rails) where its antibacterial properties can help mitigate pathogen spread.

Over recent decades, several strategies have been developed to improve the antibacterial performance of surfaces. One common approach involves the use of copper-based nanoparticles, which release ions and generate reactive oxygen species that damage bacterial membranes and DNA [12,13]. Surface coatings offer another route, combining protective and antimicrobial properties; copper coatings applied by cold gas spraying or onto textiles have shown effective bacterial inhibition [14,15]. In addition to nanoparticles and surface coatings, laser surface structuring has emerged as a powerful technique for enhancing antibacterial activity by modifying the physical and chemical characteristics of material surfaces [7,16-20]. These textures can reduce

Abbreviations: CFU, colony forming units; CLSM, confocal laser scanning microscopy; *E. coli*, *Escherichia coli*; fs, femtosecond; GI-XRD, grazing incidence X-ray diffractometer; LIPSS, laser induced periodic surface structures; SEM, scanning electron microscopy; USP-DLIP, ultrashort-pulsed direct laser interference patterning.

E-mail address: aisha.ahmed@uni-saarland.de (A.S. Ahmed).

<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>* Corresponding author. Functional Materials, Saarland University, 66123, Saarbrücken, Germany.</footnote>
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** SEM images of laser treated bronze surfaces with an accumulated fluence of 10 J/cm². Each sample varies in their single pulse fluence *F*<sub>pulse</sub>: (a) 0.16 J/cm² (b) 0.31 J/cm² (c) 0.47 J/cm² (d) 0.62 J/cm² (e) 0.78 J/cm² (f) 0.94 J/cm² (g) 1.09 J/cm². Additionally, SEM images of (h) 0.16 J/cm² showing laser processing errors that enhance the visibility of LIPSS, and (i) 1.09 J/cm² with higher magnification of the flake-like structures.</figcaption></figure>

bacterial adhesion or physically damage cells through increased roughness or sharp features [17,18]. Some studies focus solely on topographical effects to deter bacterial colonization [16,17], while others exploit combined chemical and morphological changes induced by laser processing, such as altered oxidation states or elemental distribution, to enhance antimicrobial activity [19,20]. For example, hierarchical micro- and mesoporous copper surfaces created via laser texturing have demonstrated superior bactericidal activity due to a combination of increased surface area, specific oxide phases, and topographic features [20].

Among the various laser structuring techniques, Direct Laser Interference Patterning (DLIP) is a particularly promising approach. DLIP utilizes the interference of coherent laser beams to create controlled surface modifications, with periodical features down to the nanoscale [21]. Recent studies [22,23] have shown that ultrashort pulsed DLIP (USP-DLIP) can significantly enhance bacterial-killing properties in copper and its alloys. Furthermore, Müller et al. [24] investigated various structure periodicities fabricated with USP-DLIP and their effects on different bacteria. It highlights that the impact of surface topography on bacterial adhesion differs among bacterial species, which in turn affects the antibacterial effectiveness of Cu surfaces.

While most research has focused on how surface topography influences antibacterial activity, few studies have examined how material composition in conjunction with laser parameters affect the antibacterial killing via the contact mechanism. This study addresses that gap by investigating the role of tin in ultrashort-pulsed laser-induced surface modifications on bronze.

Bronze containing 94 wt% copper and 6 wt% tin is selected to isolate the influence of tin, excluding other typical alloying elements such as phosphorus, aluminum, zinc, and nickel [8]. Using only tin as the

alloying element allows a focused investigation of its specific effect on laser-material interaction and surface structure. The objective is to fabricate periodic line-like structures with a 3 µm spacing, matching the dimensions of *Escherichia coli* (*E. coli*) cells, using a two-beam DLIP setup [22,23,25].

The first phase of the study involves identifying optimal parameters for fabricating the line-like structures by evaluating the effects of pulse energy (*E*<sub>pulse</sub>) or pulse fluence (*F*<sub>pulse</sub>) on the topography. A fixed accumulated fluence (*F*<sub>acc</sub>)—the total laser energy delivered per unit area—based previous studies [23,25], is used to replicate the successful fabrication of these structures. In the second phase, the fabricated surfaces are characterized to assess how laser parameters influence topography, followed by evaluation of their antibacterial performance via contact-killing tests [26]. Additionally, the wetting behaviour of the surfaces is briefly examined due to its relevance in antibacterial efficacy.

## 2. Results and discussion

### 2.1. Topography of line-like structures

Fig. 1 reveals that the desired periodicity of 3 µm was achieved in all the samples. However, a variation in topography is observed involving the width of the valleys and peaks. Samples a and b were produced with lower pulse irradiation energies, resulting in a significantly higher number of pulses required for the predetermined value of *F*<sub>acc</sub>. In this case, the valleys appear notably slender with a width of approx. 1 µm and broad peaks resembling plateaus. Presumably, the irradiation energy was insufficient to induce ablation in the form of a complete intensity profile. Instead, it appears that the ablation was confined to the maximum of the intensity profile.
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** SEM images of laser treated bronze surfaces with an accumulated fluence of 10 J/cm² and subsequently etched with 5 % citric acid. Each sample varies in their single pulse fluence *F*<sub>pulse</sub>: (a) 0.16 J/cm² (b) 0.62 J/cm² (c) 1.09 J/cm².</figcaption></figure>

Additionally, the valleys in Fig. 1 a and b exhibit roughness at sub- µm scale along the line-like structures. A closer examination reveals that this roughness is not random but periodic in nature. Fig. 1 h of the surface processed at 0.16 J/cm², due to the laser processing error, clearly shows that the structures are perpendicular to the surface, with a spatial periodicity of approximately 575 nm. This can be attributed to the formation of laser induced periodic surface structures (LIPSS), which arise from the irradiation of surfaces with a linearly polarized laser beam and are strongly influenced by factors such as laser fluence, number of pulses, and laser wavelength [27,28]. These observations are consistent with a recent study on copper, where similar periodic structures were observed under the same DLIP setup with an s-polarized laser beam, particularly at low pulse fluences, due to multi-pulse structuring [29]. Given that the copper content in the bronze investigated in this study is also very high, these findings align well with previous observations.

As the pulse fluence increases, the sub- µm periodic structures gradually lose its defined pattern, adopting a more random appearance and less visibility of the LIPSS. This results from the increased amount of melted and redeposited material. This indicates a shift from spallation to phase explosion as the predominant ablation mechanism. This transition occurs due to an increase in pulse energy on the surface, resulting in temperatures exceeding the material's boiling point. Under these conditions, the system approaches the critical temperature, inducing extensive homogeneous nucleation within the irradiated surface region [30].

Although the accumulated number of pulses becomes smaller, the higher pulse energy leads to broader valleys and narrower peaks. The line-like structures in Fig. 1a-e, with a laser pulse fluence ranging from 0.16 J/cm² to 0.72 J/cm², show a rather defined structure. In contrast, the topography in Fig. 1f and g reveals less defined lines, with a notable accumulation of redeposited molten and ablated matter in the valleys and on the peaks. In the case of 1.09 J/cm² (see Fig. 1g), the valleys are a lot wider in comparison to the structures produced with lower pulse fluences. In this case, the melting and ablation mechanisms result in significant variations in both the morphology of the peak tips and the structure depth.

An additional interesting observation is the presence of flake-like structures, initially appearing in the topography created with 0.47 J/cm² and become most prominent on the surface treated with 1.09 J/cm² surface (see Fig. 1i). These structures are linked to the formation of oxides on the surface [7,25]. A recent study [29] demonstrates that the formation of these structures is highly dependent on the laser parameters. The results indicate that the threshold pulse fluence for the formation of flake-like structures lies between 0.31 and 0.47 J/cm², as these structures are already evident at 0.47 J/cm². Furthermore, the occurrence of these flake-like structures correlates with the laser pulse fluence, highlighting an increase in their abundance. Previous studies suggest that working with femtosecond laser pulses should result in a negligible heat-affected zone and cold ablation, reducing the probability

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

Depth of the line-like structures of the samples with following single pulse fluence *F*<sub>pulse</sub>: 0.16 J/cm², 0.62 J/cm² and 1.09 J/cm².

<table><thead><tr><th>Sample set</th><th>a</th><th>d</th><th>g</th></tr></thead><tbody><tr><td><i>F</i><sub>pulse</sub> [J/cm<sup>2</sup>]</td><td>0.16</td><td>0.62</td><td>1.09</td></tr><tr><td>Depth [µm]</td><td>0.8 ± 0.2</td><td>1.0 ± 0.2</td><td>1.2 ± 0.2</td></tr></tbody></table>

of chemical alterations [31,32]. However, some literature indicates that the extent of alteration is heavily affected by the chemical composition of the sample. In the case of copper, it tends to predominantly form oxides [7]. Nevertheless, the presence of alloy elements can significantly impact the degree of alteration, depending on their concentration, leading to additional chemical changes [25].

In this study, topographies exhibit a relatively lower occurrence of flake-like structures compared to previous research [22,25]. This is most likely due to the high copper content in the bronze sample, as copper requires relatively high enthalpy for oxide formation—ranging from approximately -155 to -170 kJ/mol [33,34]. In contrast, tin requires significantly lower enthalpy, with tin oxide formation ranging from approx. -280 to -577 kJ/mol depending on its oxidation state [35]. However, since the tin content in the alloy is much lower than that of copper, the observed behaviour likely results from a combined effect of both the elemental composition and their respective oxide formation energetics [36]. Notably, the surface exposed to 1.09 J/cm² displays the highest concentration of flake-like structures. This is consistent with findings from a previous study on copper [29], which showed that laser fluence is directly proportional to the concentration of these flake-like structures. For further in-depth analysis, three distinct sets representing three diverse topographical trends were selected. These sets comprised samples fabricated using the following laser pulse fluences: 0.16 J/cm², 0.62 J/cm², and 1.09 J/cm².

According to previous studies [25,26], the formation of oxides can influence antibacterial properties significantly. To investigate its possible impact on the antibacterial activity of the bronze samples, these were etched with 5 % citric acid to eliminate any existing oxides. Fig. 2 shows the SEM images of the etched laser-processed samples. The samples exposed to higher laser pulse fluences no longer display flake-like structures.

Subsequently, the depths of the three chosen structures were measured by confocal laser scanning microscopy (CLSM) and are presented in Table 1. It is evident that the samples subjected to higher single-pulse fluences have deeper valleys. To investigate the overall alterations, the samples were characterized in both their as-processed and etched states.
<figure><img src="image_4.png" /><figcaption>Fig. 3. XRD-Diffractograms of laser treated and the polished bronze for 2θ range of (a) 35–55° and (b) 70–120°.</figcaption></figure>

## 2.2. Characterization

### 2.2.1. Phase analyses

First, grazing incidence X-ray diffractometer GI-XRD is utilized to investigate the chemical alterations induced via laser processing. Simultaneously, it is compared with the results of a reference polished bronze surface. The findings are illustrated in the form of diffractograms in Fig. 3. The 2θ-range of 55–70° showed no peaks (see Fig. S1) and, therefore, was not included in Fig. 3. The orange diffractogram in Fig. 3 depicts the polished bronze surface. For this reference sample, only the diffraction peaks corresponding to the fcc phase are evidenced. According to existing literature [37] and the International Center for Diffraction Data (ICDD 01-071-784, ICDD 00-044-1477) on bronze with 5–10 wt% tin, the 2θ-values of the peaks are indicative of α-phase of bronze containing 6 wt% tin. Due to the lower atomic radius of copper (135 p.m.) compared to tin (145 p.m.), an observed trend reveals that as the copper content in bronze increases, there is a visible shift in peak positions towards higher 2θ-values, a phenomenon consistent with literature [38] and ICDD 00-004-0836 values for pure copper samples. The comparison between the polished sample and all structured samples in an as-processed state shows a notable shift in peak positions. Specifically, the (111) and (200) peaks have shifted by 0.2° in their respective 2θ-values. This shift suggests an elevated copper concentration on the surface subsequent to the laser-structuring.

Following the etching process, noticeable alterations in peak intensities are observed, particularly in samples exposed to 0.16 and 0.62 J/cm². The (111) and (200) peaks exhibit a considerable reduction in intensity, approaching nearly a fivefold decrease. On the contrary, the (220) peaks demonstrate an enhanced intensity after etching, while the (311) and (222) peaks remain constant after etching. These results suggest a change in grain orientation on the surface for the samples treated with 0.16 and 0.62 J/m². Additionally, it is evident that the 2θ-values of the (400) peak lack consistency. This inconsistency is likely attributed to the peak's broad form and weak intensity, leading to an indistinct position of the peak maximum. Furthermore, for the GI-XRD geometry, it is not unexpected that the (400) signal is not proportional, as intensity variations can arise from differences in crystal plane orientation and the characteristics of the X-ray diffraction process [39]. Overall, the results show a possible increase in copper content and a change in preferred orientation on the surface through laser processing.

### 2.2.2. Characterization of the oxides

The results of the last section reveal the presence of flake-like structures on the surfaces modified with higher pulse fluences. The existence of these is related to the presence of oxides [7]. However, the XRD results did not show any additional oxide peaks for the 0.62 and

<figure><img src="image_5.png" /><figcaption>Fig. 4. Raman-spectra of bronze surfaces treated with 0.16 J/cm² in as-processed (black spectra) and etched (orange spectra).</figcaption></figure>

1.09 J/cm² samples. To develop an understanding of this occurrence, Raman spectroscopy is applied to investigate oxides. All three sample sets were analyzed and the as-processed had identical spectra. This similarity was also observed for all the etched samples. For that purpose, only one set of samples is compared in their as-processed and etched state.

Fig. 4 displays the spectra of bronze surfaces treated with 0.16 J/cm². Bands are observed at 60, 94, 106, 147, 217, 540, 623, and 653 cm⁻¹ for the as-processed surface (black spectra). Notably, characteristic Raman bands associated with cuprous or cupric oxide are absent. However, the bands from 60 to 147 cm⁻¹ can be attributed to defects, non-stoichiometry and resonant excitation of cuprous oxide [40]. Yet, in cases where a phase has defects in the form of vacancies, or it is doped with other elements, these vibrational modes become visible in Raman [41]. Additionally, a band at 217 cm⁻¹ lacks identification with any known oxides. However, a band in a similar range has been observed in the previous study for fs-laser structured copper-alloy [25].

Although no bands related to bulk SnO₂ are visible, two weak bands at 540 and 623 cm⁻¹ can be assigned to SnO₂/Sn-O vibrations [42,43]. Literature indicates the occurrence of such a band in a range of 500–600 cm⁻¹ for Sn-O particles at a nanoscale [44]. Given that the samples were treated with an ultrashort pulse duration, it is highly possible that there is a chemical alteration at the nanoscale [25,26]. Furthermore, processing under an ultrashort time period at very high local temperature/pressure and rapid cooling afterward can lead to the formation of
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Wetting behavior of various bronze surfaces (0.16 J/cm² (green), 0.62 J/cm² (purple), and 1.09 J/cm² (blue)) in as-processed (● solid line) and etched (▲ dashed line) state. Additionally, polished bronze (dashed orange line) is chosen as reference. The results are presented as calculated average values of the contact angle.</figcaption></figure>

not fully/stably formed oxides [45]. This correlates with the observation of copper oxide bands that only occur as a result of defects in the oxide phase.

After etching (orange spectra), the intensity of cuprous oxide bands reduces by half. Whereas the band at 217 cm⁻¹ remains constant. The Sn oxide bands are no longer visible, particularly the band at 540 cm⁻¹. Since the defect bands of Cu₂O remain evident after etching, the disappearance of the 540 cm⁻¹ band suggests that it is not associated with Cu₂O and is, therefore, specifically related to SnO₂. These findings reveal the removal of cuprous oxide phases partially and tin oxide fully through etching. Furthermore, Raman spectra were also recorded for 0.62 and 1.09 J/cm² samples. The results showed identical spectra for all as-processed as well as etched surfaces.

Comparing the characterization results, it can be concluded that the observed increase in copper content in XRD spectra is possibly a result of copper ions diffusion towards the surface, forming oxides as indicated by the acquired Raman spectra. However, the absence of distinct XRD bands and the presence of only silent bands from copper oxides in Raman spectra suggest that the oxide formation is probably located only on the top surface of the bronze samples. In summary, the formation of a

nanoscale tin oxide phase and a copper-rich oxide phase, possibly including tin ions, may be suggested as seen in previous studies [22,25].

## 2.3. Wetting behavior

The antibacterial test performed in this study involves the placement of the bacterial solution in the form of a droplet on the surfaces. To evaluate the results of the contact-killing tests, it is essential to first examine the wetting behavior of the different surfaces, including the polished bronze reference. For that purpose, the experiment is performed over a period of three weeks utilizing the sessile falling droplet method.

The calculated average values of the static contact angle are shown Fig. 5. Over the course of three weeks, a clear trend is observed for all the structured samples, except for the 0.62 J/cm² etched sample (▲ purple) at one week, which is a result of a processing error. This variation was noted since only the batch used for wettability experiments experienced the error in laser processing (similar to that shown in Fig. 1 h). By including this, the intention was to show how even slight variations in laser structuring, caused by missed laser processing, can affect the wettability. In general, an increasing contact angle is noted. This change from hydrophilic to hydrophobic nature after the laser processing with ultrashort pulses over a period of a few weeks has been noted in previous studies for pure metal surfaces [23,46]. Furthermore, these results are consistent with the findings of a previous study that tested the wetting behaviour of laser-processed phosphor bronze [47]. This study validates that irrespective of the bulk composition this trend is visible for all laser processed samples.

Comparing the initial and last results, the initial hydrophilic nature can be attributed to the micro roughness of the surface, which allows for the droplet to stick to the surface. However, at week 0, a significant difference is visible between the as-processed samples (● solid line) and the etched samples (▲ dashed line). It is evident that the etched samples show a more hydrophilic behavior in contrast. Previous studies [48,49] showed that this difference on the surfaces is closely related to the formation of oxygen, C–C, and C–H bonds, which depend on the atmospheric conditions. In this study, the laser process was conducted under ambient conditions, making it highly likely that such contaminants were present. Since the etched samples were treated with citric acid, most oxides and likely other surface contaminants were removed, which led to a change in the wetting behavior.

As the weeks progress, the samples become increasingly

<figure><img src="image_7.png" /><figcaption>**Fig. 6.** a) Surviving colony forming units (CFU/mL) of all bronze surfaces in comparison to polished references, copper (red graph) and steel (grey graph). b) Increase in contact area after direct laser patterning with different pulse fluences on bronze.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Fig. 7. Micrographs of etched laser-samples with *E. coli*. The samples were coated with Au-Pd to achieve a better contrast in presence of biological matter.</figcaption></figure>

hydrophobic. By week 3, all etched samples display contact angles between 100° and 110°, while the as-processed samples reach around 150°, nearly approaching superhydrophobicity (>150°). However, the 0.16 J/cm² as-processed sample (● green) shows behavior like the etched samples by week 3, possibly due to its unique topography (see Fig. 2a). The plateau-like peaks in the surface structure may facilitate droplet adhesion in a way that reflects a combined effect of both polished and other structured surfaces, although further research is needed to confirm this. Future studies should evaluate droplet adhesion using contact angle hysteresis to gain a better understanding of this phenomenon. Overall, an increase in hydrophobicity is highly related to the absorption of organic matter [7] during the aging period of 3 weeks, as the samples were stored in an ambient atmosphere. Overall, the change in wetting behavior can be attributed to microroughness and surface aging.

## 2.4. Contact-killing and bacteria-substrate interactions

Next, the antibacterial activity of all these samples is examined against *E. coli*. Fig. 6 a displays surviving colony forming units (CFU)/mL over a contact period of 30, 60, 90, and 120 min. As expected, steel (■ grey graph) shows no killing of *E. coli* and, on the other side, the surviving CFU/mL on copper (■ red graph) reduces by 99 % in a contact period of 2 h [23]. In comparison, the polished bronze (■ orange graph) shows a significantly slower killing of *E. coli* than on pure copper. This can be explained by the lower copper content in bronze as previously observed for brass [22].

As this is compared to the laser-treated samples, regardless of the pulse fluence (all graphs with ● solid line), it is visible that within the first 30 min of contact, the killing rate is similar to that of polished bronze and copper. However, after 60 min, a discrepancy becomes visible for the 0.16 J/cm² sample. Due to one of the test results with a low killing rate the standard deviation in this case is bigger. This variation can be ascribed to different experimental errors, i.e., improper placement of the droplet leading to incomplete or insufficient duration of contact between bacteria and substrate, improper retrieval of the bacterial sample from the surface after contact, etc. These minor procedural variations were acknowledged to provide a more transparent and realistic interpretation of the data.

After 90 min, the 1.09 J/cm² sample kills significantly better than polished bronze and is almost similar to that of copper. After an increased contact duration, the killing of *E. coli* 1.09 J/cm² continues at a consistent rate. Whereas, in contrast, copper exhibits a notable surge in the killing rate within the final 30 min of contact. Overall, 0.16 J/cm² (● green graph) and 0.62 J/cm² (● purple graph) as-processed samples have similar killing within the 120 min contact duration as the polished bronze.

Simultaneously, the etched samples are assessed for their antibacterial activity, with the results presented in Fig. 6 (all graphs with ▲ dashed lines). During the initial 60 min, all samples exhibit similar

<figure><img src="image_9.png" /><figcaption>Fig. 8. Micrograph of an etched laser-treated sample with 0.16 J/cm² pulse fluence, showing interaction with *E. coli* and displaying the surface morphology.</figcaption></figure>

behavior, surpassing polished bronze and their respective as-processed counterparts by one magnitude. By the 120-min mark, it becomes apparent that approximately 99 % of *E. coli* is killed on all etched samples. In the case of 0.16 J/cm² (▲ green graph) and 1.09 J/cm² (▲ blue graph) etched, the CFU/mL values are similar to that of copper. This indicates an improved antibacterial activity on the laser-treated samples with etching. This enhancement is likely due to the removal of surface layers formed during laser treatment, as these layers did not contribute as much to the increased antibacterial activity. This correlates with the results seen in previous studies on brass with USP-DLIP [22,25]. Fig. 6 b shows that all surfaces exhibited an increase in contact area between 1.8 and 2.5-fold, suggesting that, in the case of contact-killing, the topographical changes leading to this increase in contact area are as significant as the surface's chemical composition.

Fig. 7 shows the interaction between *E. coli* and all three etched laser-treated surfaces. The surfaces exhibit a noticeable change in morphology after contact with bacteria. This is due to corrosion on the surface, which leads to the release of copper ions into the bacterial solution, as observed in a recent study [22]. It's also worth noting Fig. 7 a provides a valuable visual comparison of bacteria-surface interaction across the different surfaces. While the morphology is less visible due to the slightly thicker Au-Pd coating on the 0.16 J/cm² sample, the higher magnification SEM-image without tilting (see Fig. 8) offers a clearer view of the morphology.
A significant observation from the results is that, irrespective of the topographic enhancements achieved through different laser parameters for 3 µm periodic line-like structures, an improved killing of E. coli is observed for all etched samples. This is most likely due to the dimension of the single bacterial cell, which, in the case of E. coli, ranges between 1 and 3 µm. Since the width of the valleys across the varied sets also falls within this range, and an overall increase in contact area is observed (see Fig. 6 b), bacterial-substrate contact is enhanced as displayed in Fig. 7, resulting in a general increase in activity.

The findings further signify that the line-like structures, without any flake-like structures, facilitate a bacteria-substrate interaction, resulting in higher E. coli killing. The increased bacterial adhesion and killing on the etched surfaces can be attributed to several interacting factors. The removal of oxides reduced surface roughness, which likely enhanced bacterial attachment. Additionally, the oxides present on the as-processed surfaces created a hydrophobic layer, hindering bacterial adhesion. Etching improved wettability by removing this oxide layer, thus promoting better bacterial attachment. This reduction in roughness and improved wettability contribute significantly to the observed antibacterial performance.

Additionally, an important factor promoting this increased killing is the elevated copper content, as indicated by the XRD results, which show an overall higher copper concentration after laser processing. The increased bacterial killing is primarily due to the enhanced contact area and the alignment of the structures with bacterial size. Previous studies have shown that when surface structures match bacterial dimensions, but the chemical composition is unsuitable, reduced or no antibacterial effect occurs [25]. Since copper is known to be antibacterial while tin is not, both surface composition and contact area are critical for effective antibacterial performance.

## 3. Conclusion

This study demonstrates the enhancement of bronze surfaces with 6 wt.-% tin using ultrashort-pulsed direct laser interference patterning (USP-DLIP) to create 3 µm periodic line-like structures, improving antibacterial activity against E. coli.

The key findings from this research confirm the successful fabrication of the desired surface structures on bronze, with pulse fluence playing a significant role in determining the depth and width of valleys, which, in turn, increased the contact area by 1.8–2.5 times. Chemical analyses showed an increase in copper content and the formation of tin- and copper-rich oxide phases after laser processing. These oxide phases were effectively removed by etching with 5 % citric acid. The processed surfaces exhibited increased hydrophobicity over time, and the additional etching of the samples demonstrated E. coli killing rates comparable to pure copper, significantly outperforming polished bronze. Importantly, despite differences in topography, the increased antibacterial activity can be attributed to the larger contact area and the compatibility of the periodic structure dimensions with the size of E. coli bacteria, leading to improved bacterial killing.

The results show significant progress in optimizing the antibacterial properties of bronze for high-contact surface applications. Future research should focus on assessing long-term performance through repeated bacterial colonization, cleaning, and re-colonization cycles, exploring the effects of additional alloying elements and laser parameters, and conducting extensive investigations into wettability and bacterial adhesion to better understand the bacteria-substrate interaction.

Overall, this work shows that USP-DLIP, combined with etching, can effectively enhance the antibacterial performance of bronze. These findings offer a promising approach for developing antimicrobial surfaces suitable for a wide range of applications, including public transport, hospitals, schools, and other high-contact areas where hygiene and bacterial contamination are of significant concern. The insights gained from the binary copper-tin system provide a solid foundation for future work, enabling the exploration of more complex alloy systems for use in

these applications.

# 4. Materials and methods

## 4.1. Materials

Bronze with 6 wt.-% tin (Wieland) was chosen for this study. In addition to that 99 % pure copper (Wieland) and stainless steel 304 (Brio) surfaces were used as references. The samples were received in the form of a 1 mm thin sheet, which is then trimmed to coupons measuring 10 × 25 mm². Subsequently, the sample surfaces were prepared metallographically to obtain a polished mirror finish as described in Ahmed et al. [25].

## 4.2. Direct laser interference patterning

Periodic line-like structures with a periodicity of 3 µm were created on bronze surfaces via direct laser interference patterning. This was achieved using a Ti:sapphire Spitfire laser system from Spectra Physics. A centered wavelength of 800 nm and an ultrashort pulsed duration of 150 fs (full width at half maximum) was utilized. The optical setup consisting of five key elements: a beam diameter aperture, a wave plate, a diffractive optical element, a mask, and a focusing lens system (100 mm focal length), was chosen as described in Ahmed et al. [25]. A spot diameter of 70 µm was achieved as a result of the two-beam DLIP on bronze. For this study, the laser-treated samples were tested in an as-processed and etched state. The samples were etched using a 5 % citric acid solution and subsequently cleaned with ethanol.

## 4.3. Experimental Procedure

In general, the laser process is characterized by scanning parameters aimed at achieving a uniform topographical alteration across the sample surface [50]. In this case, various input laser parameters play a role in defining these calculable scanning parameters. The first one is laser pulse fluence ($F_{pulse}$), which is determined by single pulse energy ($E_{pulse}$) or laser power ($P$). Laser pulse fluence is defined as energy per area for pulse irradiation ($A_{spot}$) and is calculated in Equation (1) according to Fox et al. [51].

$$F_{pulse} = \frac{E_{pulse}}{A_{spot}} \qquad \text{Eq. (1)}$$

Furthermore, this energy can be calculated for a defined area of the processed surface as shown in equation (2) and is known as accumulated fluence ($F_{acc}$) [51].

$$F_{acc} = \frac{E_{pulse}}{\Delta x^* \Delta y} \qquad \text{Eq. (2)}$$

Here, $\Delta x$ is the pulse overlap in the scan direction (x-axis) and $\Delta y$ is the step size also known as hatch distance in the y-axis [51]. Moreover, equations (1) and (2) lead to another calculable parameter (see equation (3)), which is the number of accumulated pulses ($n_{acc}$) for this defined area of the processed surface [51].

$$n_{acc} = \frac{F_{cc}}{F_{pulse}} \qquad \text{Eq. (3)}$$

3 µm periodic line-like structures were fabricated and defined by an identical accumulated fluence ($F_{acc}$) of 10 J/cm². For that purpose, the repetition rate ($f$) and the overlap in y-direction ($\Delta y$: also, the hatch distance between two scan lines) were kept constant at 1000 Hz and 24 µm, respectively. The chosen repetition rate is consistent with previous studies [23,25], and the overlap value was selected accordingly for bronze to produce structures comparable to those earlier results. The input laser power ($P$) was varied intentionally to achieve a wide range of $E_{pulse}$. For each selected power, the number of accumulated pulses ($n_{acc}$)
<caption>**Table 2**</caption>

Laser parameters – pulse energy ($E_{pulse}$), number of accumulated pulses ($n_{acc}$) and pulse fluence ($F_{pulse}$) – for 7 sets of samples with 3 µm line-like structures fabricated with femtosecond (fs)-DLIP technique using constant $F_{acc}$ of 10 J/cm². The values of variables were calculated using equations (1) - (3).

<table><thead><tr><th>Sample set</th><th>a</th><th>b</th><th>c</th><th>d</th><th>e</th><th>f</th><th>g</th></tr></thead><tbody><tr><td>$E_{pulse}$ [µJ]</td><td>6</td><td>12</td><td>18</td><td>24</td><td>30</td><td>36</td><td>42</td></tr><tr><td>$n_{acc}$</td><td>64</td><td>32</td><td>21</td><td>16</td><td>12</td><td>11</td><td>9</td></tr><tr><td>$F_{pulse}$ [J/cm²]</td><td>0.156</td><td>0.311</td><td>0.468</td><td>0.623</td><td>0.779</td><td>0.935</td><td>1.091</td></tr></tbody></table>

had to be adjusted to get the set constant value for $F_{acc}$. This is defined by the overlap and stage speed in the x-direction along the scan line [51]. In total, 7 different sets of samples were prepared. The parameters including the respective $F_{pulse}$ are given in Table 2.

The samples were evaluated for their wetting properties over a period of three weeks following laser structuring. Subsequently, all further characterization and contact-killing experiments were conducted after this three-week aging period.

## 4.4. Wettability

Contact angle experiments were performed using a Drop Shape Analysator DSA 100 (Krüss GmbH) to observe the aging of laser-structured samples during a span of three weeks. After the laser treatment, the samples are stored in a box at a room temperature of 20 ± 2 °C and a humidity of 30 ± 7 %. The first measurement (Week 0) is done 24 h after the laser treatment. The remaining measurements up to the third week were carried out at 7-day intervals. Every week's measurement is performed on a different sample at room temperature of 21.5 ± 0.5 °C and a humidity of 20 ± 1.3 %. A droplet of distilled water with a volume of 3 µL is dosed on the sample with a micro-syringe using the sessile falling droplet method. Each sample is dosed with at least 5 droplets on different regions of the surface. Since the laser-structured samples show an anisotropic topography, the contact angle of each droplet was measured orthogonal and parallel to the line-like structures. To achieve reproducible results, the measurements were done within 1 min of droplet dosage.

## 4.5. Surface characterization

Confocal laser scanning microscopy (CLSM, LEXT OLS4100 3D Measuring Laser Microscope, Olympus) was employed to measure the dimensions of the structures, with the depth calculated based on a sequence of 10 peaks and valleys. Furthermore, scanning electron microscopy (SEM, Helios NanoLab 600™, FEI Company) in secondary contrast mode was utilized to observe the topography. The SEM images were obtained using an acceleration voltage of 5 kV and a current of 1.4 nA, with the sample surface positioned at a 45° tilt.

Phase analysis is carried out using a high-resolution grazing incidence X-ray diffractometer (GIXRD, Cu Kα source with a wavelength of 1.5418 Å at 1° grazing angle, PANalytical X'Pert PRO-MPD). Next, the characterization of oxides is carried out with Raman spectroscopy (Horiba Jobin, HRLab with a 532 nm laser).

## 4.6. Antibacterial efficiency determination

The antibacterial efficiency of the samples is investigated via a contact-killing experiment followed by the wet plating method [52]. For that purpose, a bacterial suspension of E. coli WT K12 (BW25113) is prepared with Phosphate-buffered saline (PBS) according to Luo et al. [26]. A total of three droplets (volume: 40 µL each) of the bacterial suspension are then placed on each sample surface. Each sample is tested at least thrice to achieve reproducible results. After 30 min, 5 µL are retrieved from one of the droplets after repetitive pipetting. The same volume is retrieved from the second and third droplets after 60 min and 120 min, respectively. These 5 µL are then serially diluted, spread on Lysogeny broth (LB) agar plates, and stored in an incubator at 37 °C for

24 h. To conclude, the number of surviving colony forming units is enumerated for all the samples as well as the original bacterial suspension prior to contact with any surface.

## CRediT authorship contribution statement

**Aisha Saddiqa Ahmed:** Writing – original draft, Visualization, Validation, Investigation, Conceptualization. **Diego Sancio:** Writing – review & editing, Validation, Investigation. **Daniel Wyn Müller:** Writing – review & editing. **Jean-François Pierson:** Writing – review & editing, Validation, Supervision, Resources, Investigation. **Frank Mücklich:** Writing – review & editing, Supervision, Resources, Funding acquisition.

## 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).

## 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 authors 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). The authors would also like to thank the technical platform "Optic and Lasers" at Institut Jean Lamour (IJL, Nancy, France) for access to Raman facility. Diego Sancio would like to thank the CUAA-DAHZ for the scholarship in the frame of project I.DEAR-Materials (Project ID: 57597084).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtadv.2025.100596.

## Data availability

Data will be made available on request.
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