# Enhancing implant performance: 20% reduction in *Pseudomonas aeruginosa* bacterial initial formation with Cu\_0.75Ti\_0.25O\_2 coating

A. Yadav, S. Pradhan, M. Khokhlova, O. El Khaloufi, N. Khong, S. Lai, A.

Fouchet, A. David, U. Lüders, H.-Y. Li, et al.

► To cite this version:

A. Yadav, S. Pradhan, M. Khokhlova, O. El Khaloufi, N. Khong, et al. Enhancing im-
plant performance: 20% reduction in *Pseudomonas aeruginosa* bacterial initial formation with
Cu\_0.75Ti\_0.25O\_2 coating. AIP Advances, 2023, 13 (9), pp.095008. 10.1063/5.0166085. hal-
04256492

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# Enhancing implant performance: 20% reduction in *Pseudomonas aeruginosa* bacterial initial formation with Cu\_0.75Ti\_0.25O\_2 coating

A. Yadav, S. Pradhan, M. Khokhlova, O. El Khaloufi, N. Khong, S. Lai, A.

Fouchet, A. David, U. Lüders, H.-Y. Li, et al.

► To cite this version:

A. Yadav, S. Pradhan, M. Khokhlova, O. El Khaloufi, N. Khong, et al.. Enhancing im-
plant performance: 20% reduction in *Pseudomonas aeruginosa* bacterial initial formation with
Cu\_0.75Ti\_0.25O\_2 coating. AIP Advances, 2023, 13 (9), pp.095008. 10.1063/5.0166085. hal-
04256492

HAL Id: hal-04256492

https://hal.science/hal-04256492v1

Submitted on 3 Jun 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
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Distributed under a Creative Commons Attribution 4.0 International License
# Enhancing implant performance: 20% reduction in *Pseudomonas aeruginosa* bacterial initial formation with Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> coating

A. Yadav Ⓔ; S. Pradhan Ⓔ; M. Khokhlova Ⓔ; O. El Khaloufi Ⓔ; N. Z. J. Khong; S. K. Lai Ⓔ; A. Fouchet;

A. David Ⓔ; U. Lüders Ⓔ; H.-Y. Li Ⓔ; M. S. R. Rao Ⓔ; W. Prellier ☐

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# Enhancing implant performance: 20% reduction in *Pseudomonas aeruginosa* bacterial initial formation with Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> coating

A. Yadav Ⓔ; S. Pradhan Ⓔ; M. Khokhlova Ⓔ; O. El Khaloufi Ⓔ; N. Z. J. Khong; S. K. Lai Ⓔ; A. Fouchet;

A. David Ⓔ; U. Lüders Ⓔ; H.-Y. Li Ⓔ; M. S. R. Rao Ⓔ; W. Prellier ☐

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# Enhancing implant performance: 20% reduction in *Pseudomonas aeruginosa* bacterial initial formation with Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> coating EP

Cite as: AIP Advances 13, 095008 (2023); doi: 10.1063/5.0166085

Submitted: 18 July 2023 • Accepted: 19 August 2023 •

Published Online: 7 September 2023

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A. Yadav,<fnref n="1" /> ID S. Pradhan,<fnref n="2" /> ID M. Khokhlova,<fnref n="1" /> ID O. El Khaloufi,<fnref n="1" /> ID N. Z. J. Khong,⁵ S. K. Lai,<fnref n="3" /> ID A. Fouchet,<fnref n="1" />

A. David,<fnref n="1" /> ID U. Lüders,<fnref n="1" /> ID H.-Y. Li,<fnref n="3" /> ID M. S. R. Rao,<fnref n="2" /> ID and W. Prellier<fnref n="1" />,ª ID

## AFFILIATIONS

## ABSTRACT

Bacteria are commonly found in various environmental sources, such as soil and water. Among the different bacterial species, *Pseudomonas aeruginosa* is particularly known for causing infections in humans and can easily colonize surfaces. To address this issue, one approach is to modify the surface with antibacterial coatings. Thus, here, we investigate the effectiveness of Cu-Ti oxide coatings by fabricating Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> (0.25 < x < 0.75) thin films on glass substrates using the pulsed laser deposition technique. Microstructural analysis revealed that the films were amorphous and exhibited a smooth surface. In addition, the contact angle measurements demonstrated high hydrophilicity, as indicated by values below 90°. Subsequently, we examined the biofilm formation of *Pseudomonas aeruginosa* bacteria on both Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> coated and uncoated glass surfaces. The results revealed a 20% reduction in bacterial growth on Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub>-coated samples, as determined by calculating the biomass of the biofilm. These findings were further discussed in relation to the copper content present on the surface of the coating. Ultimately, the results contribute to our understanding of bacterial development on various surfaces. Consequently, this research may have practical implications for enhancing the antibacterial properties, preventing bacterial infections associated with different materials and surfaces, and improving implant performance.

© 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0166085

## I. INTRODUCTION

In healthcare facilities, bacterial infections primarily originate from patients and healthcare workers, although the environment also plays a significant role. The environment can act as a reservoir for potentially infectious micro-organisms, contributing to their transmission. As human lifestyles improve, the importance of creating and maintaining a healthy environment becomes increasingly evident. Without environmental awareness, individuals are naturally exposed to various harmful bacteria that can spread on inanimate surfaces, on equipment, and through the air.

Antimicrobial resistance poses a substantial global health threat, with six major pathogens (*Pseudomonas aeruginosa*,

*Escherichia coli*, *Staphylococcus aureus*, *Klebsiella pneumonia*, *Streptococcus pneumonia*, and *Acinetobacter baumannii*) being responsible for 929 000 deaths worldwide in 2019.<fnref n="1" /> Moreover, bacterial biofilms, which accumulate on surfaces ranging from household water pipes to medical equipment, contribute to persistent health problems and infections.<fnref n="2" />,<fnref n="3" /> Surgical implant-related infections, particularly with the presence of biofilms, remain a challenging and catastrophic complication. In 2017, for instance, multidrug-resistant *Pseudomonas aeruginosa* caused an estimated 32 600 infections and 2700 deaths among hospitalized patients in the United States.

*Pseudomonas aeruginosa*, among various *Pseudomonas* species, is the most common cause of infections in humans, affecting the bloodstream, lungs, and other organs.⁴ Consequently, extensive

<footnote n="1">¹ Laboratoire CRISMAT, CNRS UMR 6508, ENSICAEN, Université de Caen Normandie, Normandie Université, 6 Blvd. Maréchal Juin, F-14050 Caen Cedex 4, France</footnote>

<footnote n="2">²Department of Physics, Materials Science Research Centre and Nano Functional Materials Technology Centre, Indian Institute of Technology Madras, Chennai, India</footnote>

<footnote n="3">³School of Biological Sciences, College of Science, Nanyang Technological University, Singapore</footnote>

<footnote>ª) Author to whom correspondence should be addressed: wilfrid.prellier@ensicaen.fr</footnote>
# Enhancing implant performance: 20% reduction in *Pseudomonas aeruginosa* bacterial initial formation with Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> coating EP

Cite as: AIP Advances 13, 095008 (2023); doi: 10.1063/5.0166085

Submitted: 18 July 2023 • Accepted: 19 August 2023 •

Published Online: 7 September 2023

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A. Yadav,<fnref n="1" /> ID S. Pradhan,<fnref n="2" /> ID M. Khokhlova,<fnref n="1" /> ID O. El Khaloufi,<fnref n="1" /> ID N. Z. J. Khong,⁵ S. K. Lai,<fnref n="3" /> ID A. Fouchet,<fnref n="1" />

A. David,<fnref n="1" /> ID U. Lüders,<fnref n="1" /> ID H.-Y. Li,<fnref n="3" /> ID M. S. R. Rao,<fnref n="2" /> ID and W. Prellier<fnref n="1" />,ª ID

## AFFILIATIONS

## ABSTRACT

Bacteria are commonly found in various environmental sources, such as soil and water. Among the different bacterial species, *Pseudomonas aeruginosa* is particularly known for causing infections in humans and can easily colonize surfaces. To address this issue, one approach is to modify the surface with antibacterial coatings. Thus, here, we investigate the effectiveness of Cu-Ti oxide coatings by fabricating Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> (0.25 < x < 0.75) thin films on glass substrates using the pulsed laser deposition technique. Microstructural analysis revealed that the films were amorphous and exhibited a smooth surface. In addition, the contact angle measurements demonstrated high hydrophilicity, as indicated by values below 90°. Subsequently, we examined the biofilm formation of *Pseudomonas aeruginosa* bacteria on both Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> coated and uncoated glass surfaces. The results revealed a 20% reduction in bacterial growth on Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub>-coated samples, as determined by calculating the biomass of the biofilm. These findings were further discussed in relation to the copper content present on the surface of the coating. Ultimately, the results contribute to our understanding of bacterial development on various surfaces. Consequently, this research may have practical implications for enhancing the antibacterial properties, preventing bacterial infections associated with different materials and surfaces, and improving implant performance.

© 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0166085

## I. INTRODUCTION

In healthcare facilities, bacterial infections primarily originate from patients and healthcare workers, although the environment also plays a significant role. The environment can act as a reservoir for potentially infectious micro-organisms, contributing to their transmission. As human lifestyles improve, the importance of creating and maintaining a healthy environment becomes increasingly evident. Without environmental awareness, individuals are naturally exposed to various harmful bacteria that can spread on inanimate surfaces, on equipment, and through the air.

Antimicrobial resistance poses a substantial global health threat, with six major pathogens (*Pseudomonas aeruginosa*,

*Escherichia coli*, *Staphylococcus aureus*, *Klebsiella pneumonia*, *Streptococcus pneumonia*, and *Acinetobacter baumannii*) being responsible for 929 000 deaths worldwide in 2019.<fnref n="1" /> Moreover, bacterial biofilms, which accumulate on surfaces ranging from household water pipes to medical equipment, contribute to persistent health problems and infections.<fnref n="2" />,<fnref n="3" /> Surgical implant-related infections, particularly with the presence of biofilms, remain a challenging and catastrophic complication. In 2017, for instance, multidrug-resistant *Pseudomonas aeruginosa* caused an estimated 32 600 infections and 2700 deaths among hospitalized patients in the United States.

*Pseudomonas aeruginosa*, among various *Pseudomonas* species, is the most common cause of infections in humans, affecting the bloodstream, lungs, and other organs.⁴ Consequently, extensive

<footnote n="1">¹ Laboratoire CRISMAT, CNRS UMR 6508, ENSICAEN, Université de Caen Normandie, Normandie Université, 6 Blvd. Maréchal Juin, F-14050 Caen Cedex 4, France</footnote>

<footnote n="2">² Department of Physics, Materials Science Research Centre and Nano Functional Materials Technology Centre, Indian Institute of Technology Madras, Chennai, India</footnote>

<footnote n="3">³ School of Biological Sciences, College of Science, Nanyang Technological University, Singapore</footnote>

<footnote>ª) Author to whom correspondence should be addressed: wilfrid.prellier@ensicaen.fr</footnote>
research has been conducted on this particular pathogen and its biofilm formation on surfaces. For instance, Kim et al. demonstrated that *Pseudomonas aeruginosa* forms denser biofilms under microgravity conditions than those on earth.⁵ In addition, *Pseudomonas aeruginosa* biofilms are commonly found in chronic wounds and contribute to conditions such as chronic osteomyelitis, an infection where micro-organisms attach to dead bone.⁶ In general, it is widely accepted that organisms associated with biofilms are accountable for more than 65% of microbial infections. These organisms demonstrate significant resistance to antimicrobial agents as well as components of the host defense system, encompassing both innate and adaptive immunity.⁷

Due to the aforementioned challenges,⁸ effectively controlling germ growth has become a critical concern, and extensive efforts are being directed toward combating biofilm formation.⁹⁻¹¹ Developing methods to modify the surfaces of biomaterials or materials while preserving their mechanical properties is essential in addressing this issue. Various strategies have been employed to combat the detrimental effects of biofilm contamination, including anti-adhesion techniques, contact activation, and the release of biocidal agents.¹² However, these treatments often exhibit limited efficacy as incomplete eradication of bacteria can lead to increased resistance against cleaning agents.¹³ As a result, alternative approaches such as surface coating with anti-biofilm agents and related materials have been developed.¹⁴ Among these approaches, thin film technology has shown significant promise in enhancing antibacterial activity. Furthermore, the preparation of thin films at the molecular level can play a crucial role in determining the interaction between human biology and materials, making it an essential aspect to consider.

In biomedical applications, the choice of coating material remains a significant consideration. Polymer surfaces are widely accessible, cost-effective, and effective in combating biofilm growth.¹⁵ However, they tend to exhibit lower strength and are prone to degradation in the body's biochemical environment.¹⁶

Alternatively, other compounds such as metals, alloys, or ceramics can be utilized as coatings. Various metal ions, including Zn²⁺, Cu²⁺, Al³⁺, Ti⁴⁺, and Ag⁺, have been extensively investigated for their antimicrobial properties.¹⁷⁻¹⁹ Copper-based coatings, for instance, show promise as biocompatible materials for developing anti-biofilm coatings, effectively protecting surfaces against harmful biofilm pathogenesis.²⁰ Several studies have demonstrated the antimicrobial activity of copper or copper oxide against a wide range of bacteria.²¹⁻²³

In addition, Ti-based coatings are frequently employed as bio-materials in implant applications. Doping copper into the TiO₂ host offers several advantages. Copper serves as an antibacterial agent, while TiO₂ helps reduce the recombination of charge carriers, which is crucial for antibacterial activity.²¹ These synergistic effects contribute to the enhanced antibacterial properties of Cu-doped TiO₂ coatings. Overall, the choice of coating material is a critical factor in biomedical applications, with both polymer and metal-based coatings offering distinct advantages in terms of antimicrobial properties and biocompatibility.²¹⁻²³

In this article, we utilized the Pulsed Laser Deposition (PLD) technique to grow (Cu,Ti) oxide films on glass surfaces. These coated surfaces were then subjected to exposure to *Pseudomonas aeruginosa*, a specific bacterium of interest. Our main objective was to

investigate the antimicrobial activity of the coatings against this bacterium, focusing on the influence of surface properties and varying Cu/Ti ratios. Remarkably, we observed a significant 20% reduction in biofilm formation by bacterial cells on Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub> coated glass surfaces compared to the control glass samples in the initial steps. We attribute this reduction to the alteration of copper content present at the surface of the films. Our findings highlight the potential of these coatings to effectively inhibit biofilm formation by *Pseudomonas aeruginosa*, emphasizing the significance of surface modifications and the specific Cu/Ti ratio in achieving such antimicrobial effects, which can be further used for bioimplants.

## II. EXPERIMENTAL DETAILS

In this section, various techniques and methods are employed to synthesize and characterize the Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> thin films, including evaluating the roughness, wettability, surface energy, composition, and antibacterial activity.

### A. Deposition of Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> films

A series of thin films with varying thicknesses were fabricated on a 12 mm diameter glass substrate using PLD (refer to the schematic setup depicted in Fig. 1).²⁴ Prior to introduction into the vacuum chamber, the glass substrate underwent ultrasonic cleaning with ethanol, followed by rinsing in deionized water and subsequent drying with compressed air. Various compositions of Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> (CTO) were tested, specifically x values of 0.25, 0.5, and 0.75. For the preparation of (Cu,Ti)O<sub>2</sub> targets, a conventional solid-state process was employed, which involved mixing CuO and TiO<sub>2</sub> powders to the desired stoichiometry, followed by multiple firing cycles at 800 °C in an air environment. The resulting product was then pressed and sintered for 24 h at 1100 °C. During the film deposition process, a KrF excimer laser (λ = 248 nm, 3 Hz) was scanned over the target surface at a fluence of 2.45 J/cm². The substrate, positioned at a distance of 4.5 cm from the target, was maintained at a temperature of 200 °C under a base pressure of 1.1 × 10<sup>-7</sup> mbar. To achieve different film thicknesses, the number of laser pulses was varied from 4500 to 10 000, while employing a low deposition rate of 0.0024 nm/pulse to prevent splashing or the formation of particulates on the films.²⁵

### B. Thin film investigation

The structural quality of the films was assessed through x-ray reflectance (XRR) utilizing a Bruker D8 Discover diffractometer (CuKα₁, λ = 1.5405 Å) within the 2θ 0°-5° range. The film thickness was determined based on the relative position of the vibrational maxima in relation to the square of their atomic number. The topography analysis was performed using the atomic force microscopy (AFM, PicoSPM) technique, and the squared roughness (rms) representing the square root of the surface height distribution was extracted using WSxM 5.0 software for a 1 μm² area in tapping mode.²⁶ Chemical analysis of Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> thin films was carried out by x-ray photoelectron spectroscopy (XPS) with an Al Kα excitation source (hν = 1486.6 eV) operated at 10 mA and 10 kV using a Specs GmbH system in an analyzer chamber with a base vacuum of 5 × 10<sup>-10</sup> mbar. The XPS spectra were obtained in a
research has been conducted on this particular pathogen and its biofilm formation on surfaces. For instance, Kim et al. demonstrated that *Pseudomonas aeruginosa* forms denser biofilms under microgravity conditions than those on earth.⁵ In addition, *Pseudomonas aeruginosa* biofilms are commonly found in chronic wounds and contribute to conditions such as chronic osteomyelitis, an infection where micro-organisms attach to dead bone.⁶ In general, it is widely accepted that organisms associated with biofilms are accountable for more than 65% of microbial infections. These organisms demonstrate significant resistance to antimicrobial agents as well as components of the host defense system, encompassing both innate and adaptive immunity.⁷

Due to the aforementioned challenges,⁸ effectively controlling germ growth has become a critical concern, and extensive efforts are being directed toward combating biofilm formation.⁹⁻¹¹ Developing methods to modify the surfaces of biomaterials or materials while preserving their mechanical properties is essential in addressing this issue. Various strategies have been employed to combat the detrimental effects of biofilm contamination, including anti-adhesion techniques, contact activation, and the release of biocidal agents.¹² However, these treatments often exhibit limited efficacy as incomplete eradication of bacteria can lead to increased resistance against cleaning agents.¹³ As a result, alternative approaches such as surface coating with anti-biofilm agents and related materials have been developed.¹⁴ Among these approaches, thin film technology has shown significant promise in enhancing antibacterial activity. Furthermore, the preparation of thin films at the molecular level can play a crucial role in determining the interaction between human biology and materials, making it an essential aspect to consider.

In biomedical applications, the choice of coating material remains a significant consideration. Polymer surfaces are widely accessible, cost-effective, and effective in combating biofilm growth.¹⁵ However, they tend to exhibit lower strength and are prone to degradation in the body's biochemical environment.¹⁶

Alternatively, other compounds such as metals, alloys, or ceramics can be utilized as coatings. Various metal ions, including Zn²⁺, Cu²⁺, Al³⁺, Ti⁴⁺, and Ag⁺, have been extensively investigated for their antimicrobial properties.¹⁷⁻¹⁹ Copper-based coatings, for instance, show promise as biocompatible materials for developing anti-biofilm coatings, effectively protecting surfaces against harmful biofilm pathogenesis.²⁰ Several studies have demonstrated the antimicrobial activity of copper or copper oxide against a wide range of bacteria.²¹⁻²³

In addition, Ti-based coatings are frequently employed as bio-materials in implant applications. Doping copper into the TiO₂ host offers several advantages. Copper serves as an antibacterial agent, while TiO₂ helps reduce the recombination of charge carriers, which is crucial for antibacterial activity.²¹ These synergistic effects contribute to the enhanced antibacterial properties of Cu-doped TiO₂ coatings. Overall, the choice of coating material is a critical factor in biomedical applications, with both polymer and metal-based coatings offering distinct advantages in terms of antimicrobial properties and biocompatibility.²¹⁻²³

In this article, we utilized the Pulsed Laser Deposition (PLD) technique to grow (Cu,Ti) oxide films on glass surfaces. These coated surfaces were then subjected to exposure to *Pseudomonas aeruginosa*, a specific bacterium of interest. Our main objective was to

investigate the antimicrobial activity of the coatings against this bacterium, focusing on the influence of surface properties and varying Cu/Ti ratios. Remarkably, we observed a significant 20% reduction in biofilm formation by bacterial cells on Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub> coated glass surfaces compared to the control glass samples in the initial steps. We attribute this reduction to the alteration of copper content present at the surface of the films. Our findings highlight the potential of these coatings to effectively inhibit biofilm formation by *Pseudomonas aeruginosa*, emphasizing the significance of surface modifications and the specific Cu/Ti ratio in achieving such antimicrobial effects, which can be further used for bioimplants.

## II. EXPERIMENTAL DETAILS

In this section, various techniques and methods are employed to synthesize and characterize the Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> thin films, including evaluating the roughness, wettability, surface energy, composition, and antibacterial activity.

### A. Deposition of Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> films

A series of thin films with varying thicknesses were fabricated on a 12 mm diameter glass substrate using PLD (refer to the schematic setup depicted in Fig. 1).²⁴ Prior to introduction into the vacuum chamber, the glass substrate underwent ultrasonic cleaning with ethanol, followed by rinsing in deionized water and subsequent drying with compressed air. Various compositions of Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> (CTO) were tested, specifically x values of 0.25, 0.5, and 0.75. For the preparation of (Cu,Ti)O<sub>2</sub> targets, a conventional solid-state process was employed, which involved mixing CuO and TiO<sub>2</sub> powders to the desired stoichiometry, followed by multiple firing cycles at 800 °C in an air environment. The resulting product was then pressed and sintered for 24 h at 1100 °C. During the film deposition process, a KrF excimer laser (λ = 248 nm, 3 Hz) was scanned over the target surface at a fluence of 2.45 J/cm². The substrate, positioned at a distance of 4.5 cm from the target, was maintained at a temperature of 200 °C under a base pressure of 1.1 × 10<sup>-7</sup> mbar. To achieve different film thicknesses, the number of laser pulses was varied from 4500 to 10 000, while employing a low deposition rate of 0.0024 nm/pulse to prevent splashing or the formation of particulates on the films.²⁵

### B. Thin film investigation

The structural quality of the films was assessed through x-ray reflectance (XRR) utilizing a Bruker D8 Discover diffractometer (CuKα₁, λ = 1.5405 Å) within the 2θ 0°-5° range. The film thickness was determined based on the relative position of the vibrational maxima in relation to the square of their atomic number. The topography analysis was performed using the atomic force microscopy (AFM, PicoSPM) technique, and the squared roughness (rms) representing the square root of the surface height distribution was extracted using WSxM 5.0 software for a 1 µm² area in tapping mode.²⁶ Chemical analysis of Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> thin films was carried out by x-ray photoelectron spectroscopy (XPS) with an Al Kα excitation source (hv = 1486.6 eV) operated at 10 mA and 10 kV using a Specs GmbH system in an analyzer chamber with a base vacuum of 5 × 10<sup>-10</sup> mbar. The XPS spectra were obtained in a
<figure><img src="image_11.png" /><figcaption>FIG. 1. Schematic representation of the pulsed laser deposition (PLD) setup.</figcaption></figure>

large-area mode with a passband energy of 60 eV and a step size of 0.05 eV.

### C. Wettability and surface energy

The contact angle is a crucial parameter commonly employed to assess the wettability of a liquid on a solid surface and to measure the surface energy of the solid surface.²⁷ According to the Young equation, the contact angle, which is the angle formed by a non-reactive liquid droplet on an ideal flat solid surface, is determined by the mechanical force equilibrium of the three interfacial tensions at the three-phase contact line. The relationship between these tensions can be described as follows:²⁸

$$y_{sv} = y_{sl} + y_{lv} \cos \theta_{Y}, \quad (1)$$

where $y_{sv}$, $y_{sl}$, and $y_{lv}$ are the solid-vapor interface tension, solid-liquid interface, and vapor-liquid interface tension, respectively, and $\theta_{Y}$ is called the Young contact angle. Therefore, the contact angle of surfaces is determined using a drop shape analysis system (DSA25B, Kruss, Germany) through the sessile drop technique with a droplet volume of 1 µl. While there are several methods available to calculate the surface energy, such as the Zisman method or the Wu method,²⁹,³⁰ we used the Owens-Wendt method in the present case because it allows for the separate determination of the polar and dispersive components of the surface energy. This enables us to consider the charge distribution on the surface and the interactions between dipole moments through the Columbian force.³¹ In this model, the resulting surface energy ($\sigma^t$) can be divided into two distinct components: the dispersion component ($\sigma^d$) and the polar component ($\sigma^p$). The dispersion component is attributed to the interaction resulting from the transient fluctuations of the charge distribution in the atoms or molecules. The polar component is caused by the Coulomb interaction between

permanent dipoles and induced dipoles, such as hydrogen bonds, as follows:

$$\sigma^t = \sigma^d + \sigma^p. \quad (2)$$

### D. Biofilm formation and imaging of initial attachment of mPAO1

To facilitate biofilm formation, the stained bacteria were first collected and suspended in a specially formulated ABT minimal medium, which contained 2 g of glucose per liter and 2 g of casamino acids per liter (referred to as ABTGC). This bacterial suspension was then introduced into an agarose chamber. Prior to imaging, the bacteria were diluted to an optical density of OD600 = 1.5 and stained with Vybrant DyeCycle Green Stain (Invitrogen) for 30 min while being agitated. After an incubation period of 6 h at 37 °C, the agarose chamber was mounted on the Zeiss light-sheet microscope (Z.1). During the imaging process, the focal plane was adjusted to focus on the glass surface where the bacteria adhered and initiated biofilm formation. The entire experiment was repeated three times independently for accurate quantification analysis. For the preparation of the experimental setup, 12-mm glass coverslips, both coated and uncoated, were positioned in a 24-well flat-bottomed microplate. Next, 200 µl of the bacterial suspension was added to each well, followed by an incubation period of 24 h at 37 °C with the lid closed. Subsequently, the well contents were aspirated and washed, and a 0.1% aqueous solution of CV (Sigma-Aldrich) was added to the wells containing the biofilm. The plate was incubated at room temperature for 30 min. Afterward, the CV solution was removed, and the wells were washed three times using 200 µl of sterile water to avoid disturbing the biofilm. The plate was left to air-dry for 30 min. Finally, 200 µl of a 30% (v/v) glacial acetic acid solution was added to each well to dissolve the dye bound to the
<figure><img src="image_11.png" /><figcaption>FIG. 1. Schematic representation of the pulsed laser deposition (PLD) setup.</figcaption></figure>

large-area mode with a passband energy of 60 eV and a step size of 0.05 eV.

### C. Wettability and surface energy

The contact angle is a crucial parameter commonly employed to assess the wettability of a liquid on a solid surface and to measure the surface energy of the solid surface.²⁷ According to the Young equation, the contact angle, which is the angle formed by a non-reactive liquid droplet on an ideal flat solid surface, is determined by the mechanical force equilibrium of the three interfacial tensions at the three-phase contact line. The relationship between these tensions can be described as follows:²⁸

$$y_{sv} = y_{sl} + y_{lv} \cos \theta_{Y}, \quad (1)$$

where $y_{sv}$, $y_{sl}$, and $y_{lv}$ are the solid-vapor interface tension, solid-liquid interface, and vapor-liquid interface tension, respectively, and $\theta_{Y}$ is called the Young contact angle. Therefore, the contact angle of surfaces is determined using a drop shape analysis system (DSA25B, Kruss, Germany) through the sessile drop technique with a droplet volume of 1 µl. While there are several methods available to calculate the surface energy, such as the Zisman method or the Wu method,²⁹,³⁰ we used the Owens-Wendt method in the present case because it allows for the separate determination of the polar and dispersive components of the surface energy. This enables us to consider the charge distribution on the surface and the interactions between dipole moments through the Columbian force.³¹ In this model, the resulting surface energy ($\sigma^t$) can be divided into two distinct components: the dispersion component ($\sigma^d$) and the polar component ($\sigma^p$). The dispersion component is attributed to the interaction resulting from the transient fluctuations of the charge distribution in the atoms or molecules. The polar component is caused by the Coulomb interaction between

permanent dipoles and induced dipoles, such as hydrogen bonds, as follows:

$$\sigma^t = \sigma^d + \sigma^p. \quad (2)$$

### D. Biofilm formation and imaging of initial attachment of mPAO1

To facilitate biofilm formation, the stained bacteria were first collected and suspended in a specially formulated ABT minimal medium, which contained 2 g of glucose per liter and 2 g of casamino acids per liter (referred to as ABTGC). This bacterial suspension was then introduced into an agarose chamber. Prior to imaging, the bacteria were diluted to an optical density of OD600 = 1.5 and stained with Vybrant DyeCycle Green Stain (Invitrogen) for 30 min while being agitated. After an incubation period of 6 h at 37°C, the agarose chamber was mounted on the Zeiss light-sheet microscope (Z.1). During the imaging process, the focal plane was adjusted to focus on the glass surface where the bacteria adhered and initiated biofilm formation. The entire experiment was repeated three times independently for accurate quantification analysis. For the preparation of the experimental setup, 12-mm glass coverslips, both coated and uncoated, were positioned in a 24-well flat-bottomed microplate. Next, 200 µl of the bacterial suspension was added to each well, followed by an incubation period of 24 h at 37°C with the lid closed. Subsequently, the well contents were aspirated and washed, and a 0.1% aqueous solution of CV (Sigma-Aldrich) was added to the wells containing the biofilm. The plate was incubated at room temperature for 30 min. Afterward, the CV solution was removed, and the wells were washed three times using 200 µl of sterile water to avoid disturbing the biofilm. The plate was left to air-dry for 30 min. Finally, 200 µl of a 30% (v/v) glacial acetic acid solution was added to each well to dissolve the dye bound to the
adherent biofilm, and absorbance readings were taken at 570 nm. To study the initial attachment of mPAO1, the adhesion of individual bacteria to both control and Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub>-coated glass surfaces was visualized using a light-sheet microscope. The mPAO1 cells were incubated in a custom-made light-sheet chamber designed to simulate a three-dimensional environment conducive to natural biofilm formation. The setup involved a bacterial inoculation chamber and a Zeiss light-sheet microscope Z.1 (Carl Zeiss), as previously described by Khong et al.<sup>32</sup> To create the bacterial inoculation chamber, a 1% liquid agarose solution was injected into a 1 ml syringe, and the syringe tip was then sealed with parafilm after removing the tip. A square styrene rod was placed on a yellow tip holder and inserted into the center of the cylindrical agarose column, forming a hollow chamber suitable for bacterial inoculation. Once the agarose solidified, the square styrene rod was removed, and a long glass strip, either coated or uncoated, was positioned against the agarose wall. The imaging chamber was then extended to the desired length (approximately half of the agarose chamber) by pushing the syringe plunger, enabling subsequent light-sheet imaging.

### III. RESULTS AND DISCUSSION

#### A. Structure and surface topography of thin films

The surface characteristics of the deposited thin films of Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> were examined using the AFM (Atomic Force Microscopy) technique. Figures 2(a) and 2(b) present representative images of Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> films with thicknesses of 20 and 24 nm,

respectively. These images illustrate that the films exhibit a uniform coverage with a yellow transparent color, indicating a homogeneous deposition without any presence of pin-hole defects. Moreover, all the films display excellent adhesion to the glass substrate. The roughness measurements of both samples reveal values less than 1 nm, indicating remarkably smooth surfaces. The thicknesses of the films were determined using XRR analysis, as depicted in Fig. 2, and were found to be 11, 16, 20, and 24 nm.

Figure 3 illustrates the variation in the contact angle with respect to film thickness. For distilled water, the contact angles were measured to be 60.4°, 67.5°, 76.1°, and 81.3° for film thicknesses of 11, 16, 20, and 24 nm, respectively. Similarly, for ethylene glycol, the contact angles were found to be 42.2°, 53.7°, 55.5°, and 65.7° for the same respective film thicknesses. Figures 3(b) and 3(c) depict typical images used to measure the contact angle for water and ethylene glycol, respectively (Table I). The obtained data suggest that the thin Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> films exhibit inherent hydrophilicity, as indicated by contact angles of less than 90°. The surface, serving as the point of contact between the bacteria and the material, plays a crucial role in promoting or preventing bacterial adhesion. Surface wettability, a fundamental property, governs the interactions between solid and liquid phases in biological systems as hydrophilic surfaces facilitate adhesion. By utilizing the Owens–Wendt equation and the contact angles of these films, the surface energy was calculated (see Fig. 3 and Table I).

We utilized the dispersive ($y_i^d$) and polar ($y_i^p$) fractions to calculate the surface energy, which were found to be 21.8 and 51 mJ/m<sup>2</sup> for water and 34 and 30.4 mJ/m<sup>2</sup> for ethylene glycol, respectively, as previously reported.<sup>31</sup> By considering these

<figure><img src="image_12.png" /><figcaption>FIG. 2. 1 × 1 µm<sup>2</sup> AFM images of 20 nm (a), and 24 nm (b) thin CTO films. The roughness values are indicated below each respective figure. In addition, typical XRR curves for these films, recorded from 0° to 3°, are shown, with experimental data depicted in black and fitted curves in red. The corresponding calculated thicknesses are 20 nm (c) and 24 nm (d).</figcaption></figure>
adherent biofilm, and absorbance readings were taken at 570 nm. To study the initial attachment of mPAO1, the adhesion of individual bacteria to both control and Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub>-coated glass surfaces was visualized using a light-sheet microscope. The mPAO1 cells were incubated in a custom-made light-sheet chamber designed to simulate a three-dimensional environment conducive to natural biofilm formation. The setup involved a bacterial inoculation chamber and a Zeiss light-sheet microscope Z.1 (Carl Zeiss), as previously described by Khong et al.<sup>32</sup> To create the bacterial inoculation chamber, a 1% liquid agarose solution was injected into a 1 ml syringe, and the syringe tip was then sealed with parafilm after removing the tip. A square styrene rod was placed on a yellow tip holder and inserted into the center of the cylindrical agarose column, forming a hollow chamber suitable for bacterial inoculation. Once the agarose solidified, the square styrene rod was removed, and a long glass strip, either coated or uncoated, was positioned against the agarose wall. The imaging chamber was then extended to the desired length (approximately half of the agarose chamber) by pushing the syringe plunger, enabling subsequent light-sheet imaging.

### III. RESULTS AND DISCUSSION

#### A. Structure and surface topography of thin films

The surface characteristics of the deposited thin films of Cu<sub>1-x</sub>Ti<sub>x</sub>O<sub>2</sub> were examined using the AFM (Atomic Force Microscopy) technique. Figures 2(a) and 2(b) present representative images of Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> films with thicknesses of 20 and 24 nm,

respectively. These images illustrate that the films exhibit a uniform coverage with a yellow transparent color, indicating a homogeneous deposition without any presence of pin-hole defects. Moreover, all the films display excellent adhesion to the glass substrate. The roughness measurements of both samples reveal values less than 1 nm, indicating remarkably smooth surfaces. The thicknesses of the films were determined using XRR analysis, as depicted in Fig. 2, and were found to be 11, 16, 20, and 24 nm.

Figure 3 illustrates the variation in the contact angle with respect to film thickness. For distilled water, the contact angles were measured to be 60.4°, 67.5°, 76.1°, and 81.3° for film thicknesses of 11, 16, 20, and 24 nm, respectively. Similarly, for ethylene glycol, the contact angles were found to be 42.2°, 53.7°, 55.5°, and 65.7° for the same respective film thicknesses. Figures 3(b) and 3(c) depict typical images used to measure the contact angle for water and ethylene glycol, respectively (Table I). The obtained data suggest that the thin Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> films exhibit inherent hydrophilicity, as indicated by contact angles of less than 90°. The surface, serving as the point of contact between the bacteria and the material, plays a crucial role in promoting or preventing bacterial adhesion. Surface wettability, a fundamental property, governs the interactions between solid and liquid phases in biological systems as hydrophilic surfaces facilitate adhesion. By utilizing the Owens–Wendt equation and the contact angles of these films, the surface energy was calculated (see Fig. 3 and Table I).

We utilized the dispersive ($y_i^d$) and polar ($y_i^p$) fractions to calculate the surface energy, which were found to be 21.8 and 51 mJ/m<sup>2</sup> for water and 34 and 30.4 mJ/m<sup>2</sup> for ethylene glycol, respectively, as previously reported.<sup>31</sup> By considering these

<figure><img src="image_12.png" /><figcaption>FIG. 2. 1 × 1 µm<sup>2</sup> AFM images of 20 nm (a), and 24 nm (b) thin CTO films. The roughness values are indicated below each respective figure. In addition, typical XRR curves for these films, recorded from 0° to 3°, are shown, with experimental data depicted in black and fitted curves in red. The corresponding calculated thicknesses are 20 nm (c) and 24 nm (d).</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>FIG. 3. (a) Variation in the surface energy and contact angle with film thickness is depicted, where ■ and ● represent the test liquids water and ethylene glycol, respectively. (b) and (c) Typical contact angle images with water and ethanol, respectively.</figcaption></figure>

<table><caption>TABLE I. Evolution of different parameters for the series of films.</caption><thead><tr><th></th><th>11</th><th>16</th><th>20</th><th>24</th></tr></thead><tbody><tr><td>Thickness (nm)</td><td>60.4</td><td>67.5</td><td>76.1</td><td>81.3</td></tr><tr><td>Contact angle with water (deg)</td><td>42.2</td><td>53.7</td><td>55.5</td><td>65.7</td></tr><tr><td>Contact angle with ethylene glycol (deg)</td><td>40.75</td><td>33.98</td><td>29.79</td><td>24.45</td></tr><tr><td>Surface energy (mJ/m²)</td><td>40.75</td><td>33.98</td><td>29.79</td><td>24.45</td></tr></tbody></table>

components, the total surface energy was determined, resulting in surface energies of 40.75, 33.98, 29.79, and 24.45 mJ/m² for Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> films with thicknesses of 11, 16, 20, and 24 nm, respectively (Fig. 3). Table I clearly demonstrates that increasing the film thickness leads to modifications in both the surface energy and contact angle. In fact, the contact angle and surface energy exhibited an inverse relationship. These changes can be explained by Eq. (1), where an increase in θ leads to a decrease in the term cos(θ). As a result, the surface tension of the solid decreases. The variation in the contact angle strongly depends on surface roughness, which is influenced by factors such as the density of layers, the preparation method of thin films, and geometric factors.<sup>33</sup>

## B. Analysis of the surface state by x-ray photo spectroscopy

XPS analysis was conducted on Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> thin films with two different thicknesses (16 and 24 nm), and the resulting spectra are presented in Fig. 4. The survey scans revealed the presence of Cu, Ti, O, and C elements without any unexpected peaks. The binding energy of all elements was calibrated using the C 1s peak (284.6 eV). Figures 4(b)–4(d) and 4(f)–4(h) exhibit high-resolution spectra for Cu 2p, Ti 2p, and O 1s of the films. In the Cu 2p core-level spectrum, two distinct peaks correspond to

Cu 2p<sub>1/2</sub> and 2p<sub>3/2</sub>, each of which can be further deconvoluted into two peaks, indicating the presence of both Cu(I) and Cu(II) species in the samples. The binding energies (BEs) at 932.14 and 952.09 eV correspond to Cu 2p<sub>3/2</sub> and 2p<sub>1/2</sub> spectra, respectively, of Cu(I) associated with Cu<sub>2</sub>O. Conversely, the BEs at 934.09 and 954.44 eV correspond to Cu 2p<sub>3/2</sub> and 2p<sub>1/2</sub> spectra, respectively, of Cu(II) related to Cu(OH)<sub>2</sub>. In addition, peaks at 942.39 and 962.09 eV represent the shake-up satellite peaks, which are also associated with Cu(II) species. The relative concentration of Cu(I) and Cu(II) species on the surface of the thin film can be calculated using the equation proposed by Ghodselahi et al.,<sup>34</sup>

$$ \mathrm{Cu(II)} = \frac{\mathrm{Cu}_2 + \mathrm{S}}{\mathrm{Cu}_1 + \mathrm{Cu}_2 + \mathrm{S}} \cdot 100\%. \qquad (3) $$

The relative concentration of Cu(I) species, Cu(I), Cu(II) species, and Cu<sub>2</sub> and the total area of the shake-up satellite peak, S, were used to calculate their respective proportions. The calculated relative concentration of Cu(I) species was determined to be 38.03% and 39.28% on the surfaces of the 16 and 24 nm thick films, respectively. The Ti 2p spectrum exhibited two peaks, at ~457.71 eV (2p<sub>3/2</sub>) and 463.21 eV (2p<sub>1/2</sub>), with a difference of ~5.5 eV, closely matching the 5.6 eV difference for Ti(IV) species. Thus, the peak fitting confirmed the presence of only Ti(IV) species on the film surface, ruling out the presence of any Ti(0)-metallic species. This observation suggests the presence of Ti<sup>4+</sup>-O bonds within the system. The experimental Cu/Ti ratio in the samples was determined as 70.32/29.68 for the 16 nm film and 80.11/19.89 for the 24 nm film. This indicates a deviation of ~±5% from the nominal composition of the target used during the deposition. The deviation primarily arises from two factors: the XPS experiment, which relies on proper background selection and accurate peak
<figure><img src="image_13.png" /><figcaption>FIG. 3. (a) Variation in the surface energy and contact angle with film thickness is depicted, where ■ and ● represent the test liquids water and ethylene glycol, respectively. (b) and (c) Typical contact angle images with water and ethanol, respectively.</figcaption></figure>

<table><caption>TABLE I. Evolution of different parameters for the series of films.</caption><thead><tr><th></th><th>11</th><th>16</th><th>20</th><th>24</th></tr></thead><tbody><tr><td>Thickness (nm)</td><td>60.4</td><td>67.5</td><td>76.1</td><td>81.3</td></tr><tr><td>Contact angle with water (deg)</td><td>42.2</td><td>53.7</td><td>55.5</td><td>65.7</td></tr><tr><td>Contact angle with ethylene glycol (deg)</td><td>40.75</td><td>33.98</td><td>29.79</td><td>24.45</td></tr><tr><td>Surface energy (mJ/m²)</td><td>40.75</td><td>33.98</td><td>29.79</td><td>24.45</td></tr></tbody></table>

components, the total surface energy was determined, resulting in surface energies of 40.75, 33.98, 29.79, and 24.45 mJ/m² for Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> films with thicknesses of 11, 16, 20, and 24 nm, respectively (Fig. 3). Table I clearly demonstrates that increasing the film thickness leads to modifications in both the surface energy and contact angle. In fact, the contact angle and surface energy exhibited an inverse relationship. These changes can be explained by Eq. (1), where an increase in θ leads to a decrease in the term cos(θ). As a result, the surface tension of the solid decreases. The variation in the contact angle strongly depends on surface roughness, which is influenced by factors such as the density of layers, the preparation method of thin films, and geometric factors.<sup>33</sup>

## B. Analysis of the surface state by x-ray photo spectroscopy

XPS analysis was conducted on Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> thin films with two different thicknesses (16 and 24 nm), and the resulting spectra are presented in Fig. 4. The survey scans revealed the presence of Cu, Ti, O, and C elements without any unexpected peaks. The binding energy of all elements was calibrated using the C 1s peak (284.6 eV). Figures 4(b)–4(d) and 4(f)–4(h) exhibit high-resolution spectra for Cu 2p, Ti 2p, and O 1s of the films. In the Cu 2p core-level spectrum, two distinct peaks correspond to

Cu 2p<sub>1/2</sub> and 2p<sub>3/2</sub>, each of which can be further deconvoluted into two peaks, indicating the presence of both Cu(I) and Cu(II) species in the samples. The binding energies (BEs) at 932.14 and 952.09 eV correspond to Cu 2p<sub>3/2</sub> and 2p<sub>1/2</sub> spectra, respectively, of Cu(I) associated with Cu<sub>2</sub>O. Conversely, the BEs at 934.09 and 954.44 eV correspond to Cu 2p<sub>3/2</sub> and 2p<sub>1/2</sub> spectra, respectively, of Cu(II) related to Cu(OH)<sub>2</sub>. In addition, peaks at 942.39 and 962.09 eV represent the shake-up satellite peaks, which are also associated with Cu(II) species. The relative concentration of Cu(I) and Cu(II) species on the surface of the thin film can be calculated using the equation proposed by Ghodselahi et al.,<sup>34</sup>

$$ \mathrm{Cu(II)} = \frac{\mathrm{Cu}_2 + \mathrm{S}}{\mathrm{Cu}_1 + \mathrm{Cu}_2 + \mathrm{S}} \cdot 100\%. \qquad (3) $$

The relative concentration of Cu(I) species, Cu(I), Cu(II) species, and Cu<sub>2</sub> and the total area of the shake-up satellite peak, S, were used to calculate their respective proportions. The calculated relative concentration of Cu(I) species was determined to be 38.03% and 39.28% on the surfaces of the 16 and 24 nm thick films, respectively. The Ti 2p spectrum exhibited two peaks, at ~457.71 eV (2p<sub>3/2</sub>) and 463.21 eV (2p<sub>1/2</sub>), with a difference of ~5.5 eV, closely matching the 5.6 eV difference for Ti(IV) species. Thus, the peak fitting confirmed the presence of only Ti(IV) species on the film surface, ruling out the presence of any Ti(0)-metallic species. This observation suggests the presence of Ti<sup>4+</sup>-O bonds within the system. The experimental Cu/Ti ratio in the samples was determined as 70.32/29.68 for the 16 nm film and 80.11/19.89 for the 24 nm film. This indicates a deviation of ~±5% from the nominal composition of the target used during the deposition. The deviation primarily arises from two factors: the XPS experiment, which relies on proper background selection and accurate peak
<figure><img src="image_14.png" /><figcaption>FIG. 4. XPS scan of Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> thin films. (a) Survey scan for 16 nm. (b)–(d) High resolution spectra of Cu 2p, Ti 2p, and O 1s, respectively. (e) Survey scan of 24 nm. (f)–(h) High-resolution spectra of Cu 2p, Ti 2p, and O 1s, respectively.</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>FIG. 5. Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub>-coating reduces bacterial cell initial attachment and resisted biofilm formation. (a) The bar chart shows the normalized mPAO1 biofilm biomass 24 h after incubation under biofilm-inducing conditions. There was larger biofilm biomass than Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub>-coated glass surfaces. (b) The number of planktonic mPAO1 cells that attached to uncoated and Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub>-coated glass surfaces was counted and quantified 6 h after incubation under biofilm-inducing conditions. The bar chart depicts the number of adhered planktonic mPAO1 bacterial cells 6 h after incubation. There were almost 20% more bacterial cell attachments detected on the uncoated glass surface (control) than the Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub>-coated glass surfaces. (c) The light sheet image shows the initial attachment of fluorescence-labeled mPAO1 bacterial cells (green) on the glass surface in the control and Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub>-coated glass surface. Each bacterial cell appears as a green dot on a single plane.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>FIG. 4. XPS scan of Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> thin films. (a) Survey scan for 16 nm. (b)–(d) High resolution spectra of Cu 2p, Ti 2p, and O 1s, respectively. (e) Survey scan of 24 nm. (f)–(h) High-resolution spectra of Cu 2p, Ti 2p, and O 1s, respectively.</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>FIG. 5. Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub>-coating reduces bacterial cell initial attachment and resisted biofilm formation. (a) The bar chart shows the normalized mPAO1 biofilm biomass 24 h after incubation under biofilm-inducing conditions. There was larger biofilm biomass than Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub>-coated glass surfaces. (b) The number of planktonic mPAO1 cells that attached to uncoated and Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub>-coated glass surfaces was counted and quantified 6 h after incubation under biofilm-inducing conditions. The bar chart depicts the number of adhered planktonic mPAO1 bacterial cells 6 h after incubation. There were almost 20% more bacterial cell attachments detected on the uncoated glass surface (control) than the Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub>-coated glass surfaces. (c) The light sheet image shows the initial attachment of fluorescence-labeled mPAO1 bacterial cells (green) on the glass surface in the control and Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>x</sub>-coated glass surface. Each bacterial cell appears as a green dot on a single plane.</figcaption></figure>
<table><caption>TABLE II. Cu–Ti ratio impact on bacterial biofilm growth.</caption><thead><tr><th>No.</th><th>Composition ratio Cu–Ti–O</th><th>Deposition temperature (°C)</th><th>Comparative assessment of antibacterial activity</th></tr></thead><tbody><tr><td>1</td><td>Cu:Ti = 3:1</td><td>300</td><td>20% reduction in growth of bacteria compared to glass control</td></tr><tr><td>2</td><td>Cu:Ti = 1:3</td><td>300</td><td>Active growth of bacteria</td></tr><tr><td>3</td><td>Cu:Ti = 1:1</td><td>300</td><td>Partial growth of bacteria but no significant change compared to glass control</td></tr><tr><td>4</td><td>Glass control</td><td></td><td>Active growth of bacteria</td></tr></tbody></table>

positioning, and the variation in experimental conditions, such as pressure, energy calibration of the laser, scan range, etc., between different depositions. These results confirm the reliability of the experimental composition of the thin films. Furthermore, the O 1s asymmetric peak could be deconvoluted into multiple peaks, where O₁ and O₂ correspond to the lattice oxygen of CuO and Cu₂O, while O₃ and O₄ represent oxygen vacancies and adsorbed oxygen, respectively.

### C. Antibacterial activity of thin films

To investigate the ability of Cu₁-xTiₓO₂ coatings with varying compositions (x = 0.25, 0.50, 0.75) to resist biofilm formation, we conducted an experiment using round glass slides, both coated and uncoated, placed in microtiter plates. The efficiency of biofilm formation was assessed using the crystal violet biofilm detection assay. mPAO1 bacteria were cultured under conditions that promote biofilm formation and incubated for 24 h at 37 °C in the microtiter plates. The bacterial cells adhering to the glass surfaces were stained and quantified to evaluate the extent of biofilm formation. Our findings consistently demonstrated that the biomass of the biofilm was significantly lower on the glass surfaces coated with Cu₁-xTiₓO₂ [specifically, (x = 0.75)] than the uncoated control surfaces [refer to Fig. 5(a)]. It is widely recognized that the initial attachment of bacteria to a surface is crucial for biofilm formation. Therefore, we tested the hypothesis that a Cu₁-xTiₓO₂ coating with x = 0.75 could hinder the initial attachment of mPAO1 bacteria while the other two compositions (x = 0.25, 0.50) did not significantly alter the attachment (see Table II). To examine the initial attachment of mPAO1, we visualized the adhesion of individual bacteria to both the control and Cu₀.75Ti₀.25O₂-coated glass surfaces using a light sheet microscope. The mPAO1 cells were incubated for 6 h in a specially prepared light sheet chamber under conditions favorable for biofilm formation. Our results revealed a significant reduction in the adhesion of mPAO1 bacterial cells to the glass surface coated with Cu₀.75Ti₀.25O₂ compared to that to the uncoated glass surface. The light sheet image illustrates the initial accumulation of fluorescently labeled mPAO1 bacterial cells (depicted in green) on both the control and Cu₀.75Ti₀.25O₂-coated glass surfaces. Each bacterial cell is visualized as a green dot on a single plane [refer to Figs. 5(b) and 5(c)].

### IV. INTERPRETATION OF THE RESULTS

Structural studies initially revealed that the Cu₀.75Ti₀.25O₂ films deposited on glass substrates by PLD are amorphous, as evidenced by their x-ray diffraction patterns. In addition, AFM images displayed a remarkably low surface roughness of ~1 nm. Moreover, all the films exhibited hydrophilic properties, although the thicker ones displayed higher contact angles (81.3° for the 24 nm film compared to 60.4° for the 11 nm film). In a related study, Matlaga et al. reported that biomaterials with contact angles exceeding 65° can be classified as hydrophobic and such surfaces have the potential to inhibit cell attachment.³⁵ Conversely, surfaces with contact angles ranging from ~40° to 80° tend to exhibit adhesive properties, promoting relatively high cell and bacterial attachment.³⁶ This corroborates our findings as the contact angles of the Cu₀.75Ti₀.25O₂ films were less than 90°, indicating their hydrophilic nature. Therefore, we can infer that the surface topography of the Cu–Ti oxide layer facilitates cell adhesion.

Overall, our comprehensive data analysis supports the conclusion that the Cu₀.75Ti₀.25O₂ films possess a hydrophilic surface, making them favorable for promoting cell attachment and potentially enhancing biomedical applications.

Based on the XPS data, our analysis revealed that the films predominantly contain Cu and Ti elements. Notably, the Cu/Ti ratio increases with film thickness, indicating the migration of copper ions toward the film surface. Furthermore, the presence of both Cu⁺ and Cu⁺² ions on the surface was confirmed, with the thicker film exhibiting a slightly higher proportion of Cu⁺ ions. This phenomenon could potentially enhance the penetration of copper ions through bacterial cell membranes, leading to the disruption of cellular processes and eventual cell death. The initial attachment of bacteria plays a critical role in biofilm formation as it establishes the foundation for inter-bacterial interactions and communication within the community.³⁷ Previous studies have attributed the toxicity of copper to the release of copper ions under wetting conditions. Our findings reinforce the significance of copper by confirming its essential presence. The interaction between copper ions on the film surface and proteins in *Pseudomonas aeruginosa* bacteria can be described as follows: One important mechanism involves the formation of copper complexes that generate radicals capable of inactivating viruses. In addition, copper can bind to thiol and other protein groups, leading to the disruption of enzyme structure and
<table><caption>TABLE II. Cu–Ti ratio impact on bacterial biofilm growth.</caption><thead><tr><th>No.</th><th>Composition ratio Cu–Ti–O</th><th>Deposition temperature (°C)</th><th>Comparative assessment of antibacterial activity</th></tr></thead><tbody><tr><td>1</td><td>Cu:Ti = 3:1</td><td>300</td><td>20% reduction in growth of bacteria compared to glass control</td></tr><tr><td>2</td><td>Cu:Ti = 1:3</td><td>300</td><td>Active growth of bacteria</td></tr><tr><td>3</td><td>Cu:Ti = 1:1</td><td>300</td><td>Partial growth of bacteria but no significant change compared to glass control</td></tr><tr><td>4</td><td>Glass control</td><td></td><td>Active growth of bacteria</td></tr></tbody></table>

positioning, and the variation in experimental conditions, such as pressure, energy calibration of the laser, scan range, etc., between different depositions. These results confirm the reliability of the experimental composition of the thin films. Furthermore, the O 1s asymmetric peak could be deconvoluted into multiple peaks, where O₁ and O₂ correspond to the lattice oxygen of CuO and Cu₂O, while O₃ and O₄ represent oxygen vacancies and adsorbed oxygen, respectively.

### C. Antibacterial activity of thin films

To investigate the ability of Cu₁-xTiₓO₂ coatings with varying compositions (x = 0.25, 0.50, 0.75) to resist biofilm formation, we conducted an experiment using round glass slides, both coated and uncoated, placed in microtiter plates. The efficiency of biofilm formation was assessed using the crystal violet biofilm detection assay. mPAO1 bacteria were cultured under conditions that promote biofilm formation and incubated for 24 h at 37 °C in the microtiter plates. The bacterial cells adhering to the glass surfaces were stained and quantified to evaluate the extent of biofilm formation. Our findings consistently demonstrated that the biomass of the biofilm was significantly lower on the glass surfaces coated with Cu₁-xTiₓO₂ [specifically, (x = 0.75)] than the uncoated control surfaces [refer to Fig. 5(a)]. It is widely recognized that the initial attachment of bacteria to a surface is crucial for biofilm formation. Therefore, we tested the hypothesis that a Cu₁-xTiₓO₂ coating with x = 0.75 could hinder the initial attachment of mPAO1 bacteria while the other two compositions (x = 0.25, 0.50) did not significantly alter the attachment (see Table II). To examine the initial attachment of mPAO1, we visualized the adhesion of individual bacteria to both the control and Cu₀.75Ti₀.25O₂-coated glass surfaces using a light sheet microscope. The mPAO1 cells were incubated for 6 h in a specially prepared light sheet chamber under conditions favorable for biofilm formation. Our results revealed a significant reduction in the adhesion of mPAO1 bacterial cells to the glass surface coated with Cu₀.75Ti₀.25O₂ compared to that to the uncoated glass surface. The light sheet image illustrates the initial accumulation of fluorescently labeled mPAO1 bacterial cells (depicted in green) on both the control and Cu₀.75Ti₀.25O₂-coated glass surfaces. Each bacterial cell is visualized as a green dot on a single plane [refer to Figs. 5(b) and 5(c)].

### IV. INTERPRETATION OF THE RESULTS

Structural studies initially revealed that the Cu₀.75Ti₀.25O₂ films deposited on glass substrates by PLD are amorphous, as evidenced by their x-ray diffraction patterns. In addition, AFM images displayed a remarkably low surface roughness of ~1 nm. Moreover, all the films exhibited hydrophilic properties, although the thicker ones displayed higher contact angles (81.3° for the 24 nm film compared to 60.4° for the 11 nm film). In a related study, Matlaga et al. reported that biomaterials with contact angles exceeding 65° can be classified as hydrophobic and such surfaces have the potential to inhibit cell attachment.³⁵ Conversely, surfaces with contact angles ranging from ~40° to 80° tend to exhibit adhesive properties, promoting relatively high cell and bacterial attachment.³⁶ This corroborates our findings as the contact angles of the Cu₀.75Ti₀.25O₂ films were less than 90°, indicating their hydrophilic nature. Therefore, we can infer that the surface topography of the Cu–Ti oxide layer facilitates cell adhesion.

Overall, our comprehensive data analysis supports the conclusion that the Cu₀.75Ti₀.25O₂ films possess a hydrophilic surface, making them favorable for promoting cell attachment and potentially enhancing biomedical applications.

Based on the XPS data, our analysis revealed that the films predominantly contain Cu and Ti elements. Notably, the Cu/Ti ratio increases with film thickness, indicating the migration of copper ions toward the film surface. Furthermore, the presence of both Cu⁺ and Cu⁺² ions on the surface was confirmed, with the thicker film exhibiting a slightly higher proportion of Cu⁺ ions. This phenomenon could potentially enhance the penetration of copper ions through bacterial cell membranes, leading to the disruption of cellular processes and eventual cell death. The initial attachment of bacteria plays a critical role in biofilm formation as it establishes the foundation for inter-bacterial interactions and communication within the community.³⁷ Previous studies have attributed the toxicity of copper to the release of copper ions under wetting conditions. Our findings reinforce the significance of copper by confirming its essential presence. The interaction between copper ions on the film surface and proteins in *Pseudomonas aeruginosa* bacteria can be described as follows: One important mechanism involves the formation of copper complexes that generate radicals capable of inactivating viruses. In addition, copper can bind to thiol and other protein groups, leading to the disruption of enzyme structure and
function, similar to other transition metals. Copper ions can also chelate with proteins by binding to amino and carboxyl groups, resulting in protein inactivation. Thus, our study highlights the presence and role of copper ions in the films. Their ability to interact with proteins in *Pseudomonas aeruginosa* bacteria through complex formation, radical generation, and protein binding contributes to the disruption of bacterial processes, emphasizing their potential as antimicrobial agents.

Furthermore, the antimicrobial activity of copper is closely linked to its interaction with DNA. Copper ions, specifically Cu<sup>+2</sup>, possess the capability to form chelates with phosphate groups, resulting in the disruption of hydrogen bonds within DNA and subsequent microbial death. Copper ions exhibit a distinct affinity to DNA, enabling them to bind and disturb the helical structure of DNA and integrate within and between DNA strands. Moreover, copper can form complexes with mRNA, contributing to the degradation of viruses. This multifaceted interaction of copper with genetic material underscores its potent antimicrobial properties.<sup>38,39</sup>

Copper's antimicrobial effects are not limited to nucleic acids; they also extend to lipids. In the presence of copper, lipids can undergo peroxidation, leading to the formation of gaps in the cell membrane and consequent disruption of cellular integrity. XPS analysis confirmed the presence of Cu<sup>+</sup> ions on the surface, which exhibited higher cytotoxicity against bacteria than Cu<sup>+2</sup> ions. The combination of Cu<sup>+</sup> ions and OH<sup>-</sup> ions (active oxygen) effectively destroyed microbial membranes and DNA by interacting with proteins that contain -CH and -SH groups. This interaction resulted in the disruption of microbial cellular components, further emphasizing copper's antimicrobial properties.

In addition, adding Ti to a thin coating improves adherence with implant-use TiAlV alloy. The thin film and TiAlV alloy are compatible and chemically affine due to their titanium-based chemical compositions. Titanium naturally creates oxide layers, improving its adherence. The thin film's titanium oxide layers interact with the TiAlV alloy's titanium oxide layer to promote adhesion and bonding. Titanium aids interface atomic interdiffusion. Titanium atoms travel between the thin film and alloy, forming intermetallic compounds and strengthening their link. Titanium in the thin coating enhances surface roughness, which increases contact points and surface area, improving mechanical interlocking with the TiAlV alloy.

Furthermore, our light sheet microscope imaging setup is well-suited for studying the initial attachment and behavior of bacterial cells on various surfaces, including walls and tilted surfaces.<sup>32</sup> In line with this, using the light sheet microscope, we demonstrated a reduction in the initial attachment of mPAO1 bacterial cells on Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub>-coated glass surfaces compared to uncoated control surfaces. These findings align with previous reports highlighting the antimicrobial activities of copper-plated surfaces.<sup>40</sup> We observed similar antimicrobial activity of the Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> coating in preventing initial biofilm formation on vertical surfaces. Ongoing efforts are focused on improving the performance of copper coatings through modifications in compound compositions, as reported in previous studies.<sup>38,40</sup> These observations further support the crucial role of copper ions in exerting toxicity against bacteria.<sup>41</sup>

## V. CONCLUSION

In this study, we investigated the growth of *Pseudomonas aeruginosa* bacteria on thin Cu-Ti films deposited on glass coverslips. Our findings shed light on the interaction between bacterial cells and the Cu-Ti films, providing valuable insights into their antimicrobial properties. The deposited films exhibited a hydrophilic and smooth surface, which are favorable characteristics for bacterial cell attachment. XPS analyses confirmed the presence of active Cu<sup>+</sup> and Cu<sup>+2</sup> ions on the film surface. These ions were found to penetrate the bacterial cells, leading to the destruction of their DNA and RNA, effectively inhibiting bacterial growth.

By utilizing our light sheet microscope imaging setup, we observed a remarkable 20% reduction in bacterial growth on the Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> coated surfaces compared to that on uncoated surfaces, as demonstrated by the crystal violet test. This provides evidence that the Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> surfaces exhibit resistance to biofilm formation. We attribute this reduction in biofilm formation to the inhibition of the initial attachment of individual mPAO1 bacterial cells, consistent with previous reports on the antimicrobial activities of copper-plated surfaces.

While our experimental conditions may not fully replicate real-life scenarios, we believe that these findings have significant implications for various applications, particularly in the healthcare industry and in the prevention of surgical implant-related infections. Biofilm formation poses a substantial risk, and our study contributes to the understanding of strategies to mitigate this risk.

Finally, our study provides valuable insights into the antimicrobial properties of Cu-Ti films and their impact on bacterial growth and biofilm formation. These results hold promise for the development of effective approaches to combat bacterial infections, benefiting diverse fields where biofilm-related risks are a concern, such as healthcare and surgical implant applications.

## ACKNOWLEDGMENTS

The authors are thankful for the Indo-French collaboration through the financial support of the Laboratoire Franco-Indien pour le Cooperation Scientifique (LAFICS) from the CNRS and are thankful for the financial support through MERLION Project No. 5.02.18, "Interaction of Living Matter with Oxide Thin films." M.S.R.R. is thankful for a fellowship for Invited Professors from the University of Caen, Normandie. Partial support from the Program Emergence "InCox" supported by the Region Normandie and the RIN ONCOTHERA is also acknowledged.

## AUTHOR DECLARATIONS

### Conflict of Interest

The authors have no conflicts to disclose.

### Author Contributions

**A. Yadav:** Conceptualization (equal); Investigation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). **S. Pradhan:** Methodology (supporting); Visualization (equal). **M. Khokholva:** Visualization (supporting).
function, similar to other transition metals. Copper ions can also chelate with proteins by binding to amino and carboxyl groups, resulting in protein inactivation. Thus, our study highlights the presence and role of copper ions in the films. Their ability to interact with proteins in *Pseudomonas aeruginosa* bacteria through complex formation, radical generation, and protein binding contributes to the disruption of bacterial processes, emphasizing their potential as antimicrobial agents.

Furthermore, the antimicrobial activity of copper is closely linked to its interaction with DNA. Copper ions, specifically Cu<sup>+2</sup>, possess the capability to form chelates with phosphate groups, resulting in the disruption of hydrogen bonds within DNA and subsequent microbial death. Copper ions exhibit a distinct affinity to DNA, enabling them to bind and disturb the helical structure of DNA and integrate within and between DNA strands. Moreover, copper can form complexes with mRNA, contributing to the degradation of viruses. This multifaceted interaction of copper with genetic material underscores its potent antimicrobial properties.<sup>38,39</sup>

Copper's antimicrobial effects are not limited to nucleic acids; they also extend to lipids. In the presence of copper, lipids can undergo peroxidation, leading to the formation of gaps in the cell membrane and consequent disruption of cellular integrity. XPS analysis confirmed the presence of Cu<sup>+</sup> ions on the surface, which exhibited higher cytotoxicity against bacteria than Cu<sup>+2</sup> ions. The combination of Cu<sup>+</sup> ions and OH<sup>-</sup> ions (active oxygen) effectively destroyed microbial membranes and DNA by interacting with proteins that contain -CH and -SH groups. This interaction resulted in the disruption of microbial cellular components, further emphasizing copper's antimicrobial properties.

In addition, adding Ti to a thin coating improves adherence with implant-use TiAlV alloy. The thin film and TiAlV alloy are compatible and chemically affine due to their titanium-based chemical compositions. Titanium naturally creates oxide layers, improving its adherence. The thin film's titanium oxide layers interact with the TiAlV alloy's titanium oxide layer to promote adhesion and bonding. Titanium aids interface atomic interdiffusion. Titanium atoms travel between the thin film and alloy, forming intermetallic compounds and strengthening their link. Titanium in the thin coating enhances surface roughness, which increases contact points and surface area, improving mechanical interlocking with the TiAlV alloy.

Furthermore, our light sheet microscope imaging setup is well-suited for studying the initial attachment and behavior of bacterial cells on various surfaces, including walls and tilted surfaces.<sup>32</sup> In line with this, using the light sheet microscope, we demonstrated a reduction in the initial attachment of mPAO1 bacterial cells on Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub>-coated glass surfaces compared to uncoated control surfaces. These findings align with previous reports highlighting the antimicrobial activities of copper-plated surfaces.<sup>40</sup> We observed similar antimicrobial activity of the Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> coating in preventing initial biofilm formation on vertical surfaces. Ongoing efforts are focused on improving the performance of copper coatings through modifications in compound compositions, as reported in previous studies.<sup>38,40</sup> These observations further support the crucial role of copper ions in exerting toxicity against bacteria.<sup>41</sup>

## V. CONCLUSION

In this study, we investigated the growth of *Pseudomonas aeruginosa* bacteria on thin Cu-Ti films deposited on glass coverslips. Our findings shed light on the interaction between bacterial cells and the Cu-Ti films, providing valuable insights into their antimicrobial properties. The deposited films exhibited a hydrophilic and smooth surface, which are favorable characteristics for bacterial cell attachment. XPS analyses confirmed the presence of active Cu<sup>+</sup> and Cu<sup>+2</sup> ions on the film surface. These ions were found to penetrate the bacterial cells, leading to the destruction of their DNA and RNA, effectively inhibiting bacterial growth.

By utilizing our light sheet microscope imaging setup, we observed a remarkable 20% reduction in bacterial growth on the Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> coated surfaces compared to that on uncoated surfaces, as demonstrated by the crystal violet test. This provides evidence that the Cu<sub>0.75</sub>Ti<sub>0.25</sub>O<sub>2</sub> surfaces exhibit resistance to biofilm formation. We attribute this reduction in biofilm formation to the inhibition of the initial attachment of individual mPAO1 bacterial cells, consistent with previous reports on the antimicrobial activities of copper-plated surfaces.

While our experimental conditions may not fully replicate real-life scenarios, we believe that these findings have significant implications for various applications, particularly in the healthcare industry and in the prevention of surgical implant-related infections. Biofilm formation poses a substantial risk, and our study contributes to the understanding of strategies to mitigate this risk.

Finally, our study provides valuable insights into the antimicrobial properties of Cu-Ti films and their impact on bacterial growth and biofilm formation. These results hold promise for the development of effective approaches to combat bacterial infections, benefiting diverse fields where biofilm-related risks are a concern, such as healthcare and surgical implant applications.

## ACKNOWLEDGMENTS

The authors are thankful for the Indo-French collaboration through the financial support of the Laboratoire Franco-Indien pour le Cooperation Scientifique (LAFICS) from the CNRS and are thankful for the financial support through MERLION Project No. 5.02.18, "Interaction of Living Matter with Oxide Thin films." M.S.R.R. is thankful for a fellowship for Invited Professors from the University of Caen, Normandie. Partial support from the Program Emergence "InCox" supported by the Region Normandie and the RIN ONCOTHERA is also acknowledged.

## AUTHOR DECLARATIONS

### Conflict of Interest

The authors have no conflicts to disclose.

### Author Contributions

**A. Yadav:** Conceptualization (equal); Investigation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). **S. Pradhan:** Methodology (supporting); Visualization (equal). **M. Khokholva:** Visualization (supporting).
**O. El Khaloufi:** Visualization (equal). **N. Z. J. Khong:** Investigation (equal); Methodology (equal); Visualization (equal). **S. K. Lai:** Methodology (equal); Visualization (equal). **A. Fouchet:** Visualization (equal). **A. David:** Visualization (equal). **U. Lüders:** Formal analysis (equal); Visualization (equal). **H.-Y. Li:** Formal analysis (equal); Methodology (equal); Visualization (equal). **M. S. R. Rao:** Visualization (equal). **W. Prellier:** Investigation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal).

DATA AVAILABILITY

The data that support the findings of this study are available
from the corresponding author upon reasonable request.

REFERENCES

¹Antimicrobial Resistance Collaborators, “Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis,” *Lancet* **399**(10325), 629–655 (2022).

²J. D. Bryers, "Medical biofilms," *Biotechnol. Bioeng.* **100**(1), 1–18 (2008).

³A. Burkowska-But, A. Kalwasińska, and M. Swiontek Brzezinska, "Bacterial growth and biofilm formation in household-stored groundwater collected from public wells," *J. Water Health* **13**(2), 353–361 (2015).

⁴G. P. Bodey, R. Bolivar, V. Fainstein, and L. Jadeja, "Infections caused by *Pseudomonas aeruginosa*," *Clin. Infect. Dis.* **5**(2), 279–313 (1983).

⁵W. Kim, F. K. Tengra, Z. Young, J. Shong, N. Marchand, H. K. Chan, R. C. Pangule, M. Parra, J. S. Dordick, J. L. Plawsky, and C. H. Collins, "Spaceflight promotes biofilm formation by *Pseudomonas aeruginosa*," *PLoS One* **8**(4), e62437 (2013).

⁶N. Rabin, Y. Zheng, C. Opoku-Temeng, Y. Du, E. Bonsu, and H. O. Sintim, "Biofilm formation mechanisms and targets for developing antibiofilm agents," *Future Med. Chem.* **7**(4), 493–512 (2015).

⁷M. Jamal, W. Ahmad, S. Andleeb, F. Jalil, M. Imran, M. A. Nawaz, T. Hussain, M. Ali, M. Rafiq, and M. A. Kamil, "Bacterial biofilm and associated infections," *J. Chin. Med. Assoc.* **81**(1), 7–11 (2018).

⁸V. Stanić, D. Janačković, S. Dimitrijević, S. B. Tanasković, M. Mitrić, M. S. Pavlović, A. Krstić, D. Jovanović, and S. Raičević, "Synthesis of antimicrobial monophase silver-doped hydroxyapatite nanopowders for bone tissue engineering," *Appl. Surf. Sci.* **257**(9), 4510–4518 (2011).

⁹J. Palmer, S. Flint, and J. Brooks, "Bacterial cell attachment, the beginning of a biofilm," *J. Ind. Microbiol. Biotechnol.* **34**(9), 577–588 (2007).

¹⁰L. K. Vestby, T. Grønseth, R. Simm, and L. L. Nesse, "Bacterial biofilm and its role in the pathogenesis of disease," *Antibiotics* **9**(2), 59 (2020).

¹¹I. Ostrov, A. Harel, S. Bernstein, D. Steinberg, and M. Shemesh, "Development of a method to determine the effectiveness of cleaning agents in removal of biofilm derived spores in milking system," *Front. Microbiol.* **7**, 1498 (2016).

¹²G. Midelet and B. Carpentier, "Impact of cleaning and disinfection agents on biofilm structure and on microbial transfer to a solid model food," *J. Appl. Microbiol.* **97**(2), 262–270 (2004).

¹³K. Bruellhoff, J. Fiedler, M. Möller, J. Groll, and R. E. Brenner, "Surface coating strategies to prevent biofilm formation on implant surfaces," *Int. J. Artif. Organs* **33**(9), 646–653 (2010).

¹⁴C. Cattò and F. Cappitelli, "Testing anti-biofilm polymeric surfaces: Where to start?," *Int. J. Mol. Sci.* **20**(15), 3794 (2019).

¹⁵K. K. Amirtharaj Mosas, A. R. Chandrasekar, A. Dasan, A. Pakseresht, and D. Galusek, "Recent advancements in materials and coatings for biomedical implants," *Gels* **8**(5), 323 (2022).

¹⁶Y.-H. Kim, Y.-r. Choi, K.-M. Kim, and S.-Y. Choi, "Evaluation of copper ion of antibacterial effect on *Pseudomonas aeruginosa*, *Salmonella typhimurium* and *Helicobacter pylori* and optical, mechanical properties," *Appl. Surf. Sci.* **258**(8), 3823–3828 (2012).

¹⁷M. Mansour lakouraj, G. Rahpaima, and S. M. Mohseni, "Synthesis, characterization, and biological activities of organosoluble and thermally stable xanthone-based polyamides," *J. Mater. Sci.* **48**, 2520–2529 (2013).

¹⁸N. L. Brown, D. A. Rouch, and B. T. Lee, "Copper resistance determinants in bacteria," *Plasmid* **27**(1), 41–51 (1992).

¹⁹Y. Seo, J. Hwang, E. Lee, Y. J. Kim, K. Lee, C. Park, Y. Choi, H. Jeon, and J. Choi, "Engineering copper nanoparticles synthesized on the surface of carbon nanotubes for anti-microbial and anti-biofilm applications," *Nanoscale* **10**(33), 15529–15544 (2018).

²⁰M. Amiri, Z. Etemadifar, A. Daneshkazemi, and M. Nateghi, "Antimicrobial effect of copper oxide nanoparticles on some oral bacteria and *Candida* species," *J. Dent. Biomater.* **4**(1), 347 (2017).

²¹D. Longano, N. Ditaranto, L. Sabbatini, L. Torsi, and N. Cioffi, "Synthesis and antimicrobial activity of copper nanomaterials," in *Nano-Antimicrobials: Progress and Prospects* (Springer, 2012), pp. 85–117.

²²G. Mary, S. Bajpai, and N. Chand, "Copper (II) ions and copper nanoparticles-loaded chemically modified cotton cellulose fibers with fair antibacterial properties," *J. Appl. Polym. Sci.* **113**(2), 757–766 (2009).

²³L. Randolph, M. Banjafar, T. R. Preston, T. Yabuuchi, M. Makita, N. P. Dover, C. Rödel, S. Göde, Y. Inubushi, G. Jakob *et al.*, "Nanoscale subsurface dynamics of solids upon high-intensity femtosecond laser irradiation observed by grazing-incidence x-ray scattering," *Phys. Rev. Res.* **4**(3), 033038 (2022).

²⁴E. Kentzinger, U. Rücker, B. Toperverg, F. Ott, and T. Brückel, "Depth-resolved investigation of the lateral magnetic correlations in a gradient nanocrystalline multilayer," *Phys. Rev. B* **77**(10), 104435 (2008).

²⁵N. Popovici, E. Jimenez, R. Da Silva, W. Branford, L. Cohen, and O. Conde, "Optical and magnetic properties of Co-doped TiO₂ thin films grown by pulsed laser deposition," *J. Non-Cryst. Solids* **352**(9–20), 1486–1489 (2006).

²⁶I. Horcas, R. Fernández, J. M. Gómez-Rodríguez, J. Colchero, J. Gómez-Herrero, and A. M. Baro, "WSXM: A software for scanning probe microscopy and a tool for nanotechnology," *Rev. Sci. Instrum.* **78**(1), 013705 (2007).

²⁷Z. Shi, Y. Zhang, M. Liu, D. A. Hanaor, and Y. Gan, "Dynamic contact angle hysteresis in liquid bridges," *Colloids Surf., A* **555**, 365–371 (2018).

²⁸T. Young, "An essay on the cohesion of fluids," *Philos. Trans. R. Soc. London* **95**, 171–172 (1832).

²⁹W. A. Zisman, *Contact Angle, Wettability, and Adhesion* (American Chemical Society, 1964), p. 1.

³⁰S. Wu, "Calculation of interfacial tension in polymer systems," *J. Polym. Sci., Part C: Polym. Symp.* **34**, 19–30 (1971).

³¹D. K. Owens and R. Wendt, "Estimation of the surface free energy of polymers," *J. Appl. Polym. Sci.* **13**(8), 1741–1747 (1969).

³²N. Z.-J. Khong, Y. Zeng, S.-K. Lai, C.-G. Koh, Z.-X. Liang, K.-H. Chiam, and H.-Y. Li, "Dynamic swimming pattern of *Pseudomonas aeruginosa* near a vertical wall during initial attachment stages of biofilm formation," *Sci. Rep.* **11**(1), 1952 (2021).

³³B. Bhushan and Y. C. Jung, "Micro- and nanoscale characterization of hydrophobic and hydrophilic leaf surfaces," *Nanotechnology* **17**(11), 2758 (2006).

³⁴T. Ghodselahi, M. Vesaghi, A. Shafiekhani, A. Baghizadeh, and M. Lameii, "XPS study of the Cu@Cu₂O core-shell nanoparticles," *Appl. Surf. Sci.* **255**(5), 2730–2734 (2008).

³⁵B. F. Matlaga, L. P. Yasenchak, and T. N. Salthouse, "Tissue response to implanted polymers: The significance of sample shape," *J. Biomed. Mater. Res.* **10**(3), 391–397 (1976).

³⁶A. Taubert, J. F. Mano *et al.*, *Biomaterials Surface Science* (John Wiley & Sons, 2013).

³⁷M. Vincent, R. E. Duval, P. Hartemann, and M. Engels-Deutsch, "Contact killing and antimicrobial properties of copper," *J. Appl. Microbiol.* **124**(5), 1032–1046 (2018).

³⁸I. B. Gomes, M. Simões, and L. C. Simões, "Copper surfaces in biofilm control," *Nanomaterials* **10**(12), 2491 (2020).

³⁹K. P. Pontin, K. A. Borges, T. Q. Furian, D. Carvalho, D. E. Wilsmann, H. R. P. Cardoso, A. K. Alves, G. Z. Chitolina, C. T. P. Salle, H. L. de Souza Moraes, and
**O. El Khaloufi:** Visualization (equal). **N. Z. J. Khong:** Investigation (equal); Methodology (equal); Visualization (equal). **S. K. Lai:** Methodology (equal); Visualization (equal). **A. Fouchet:** Visualization (equal). **A. David:** Visualization (equal). **U. Lüders:** Formal analysis (equal); Visualization (equal). **H.-Y. Li:** Formal analysis (equal); Methodology (equal); Visualization (equal). **M. S. R. Rao:** Visualization (equal). **W. Prellier:** Investigation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal).

DATA AVAILABILITY

The data that support the findings of this study are available
from the corresponding author upon reasonable request.

REFERENCES

¹Antimicrobial Resistance Collaborators, “Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis,” *Lancet* **399**(10325), 629–655 (2022).

²J. D. Bryers, "Medical biofilms," *Biotechnol. Bioeng.* **100**(1), 1–18 (2008).

³A. Burkowska-But, A. Kalwasińska, and M. Swiontek Brzezinska, "Bacterial growth and biofilm formation in household-stored groundwater collected from public wells," *J. Water Health* **13**(2), 353–361 (2015).

⁴G. P. Bodey, R. Bolivar, V. Fainstein, and L. Jadeja, "Infections caused by *Pseudomonas aeruginosa*," *Clin. Infect. Dis.* **5**(2), 279–313 (1983).

⁵W. Kim, F. K. Tengra, Z. Young, J. Shong, N. Marchand, H. K. Chan, R. C. Pangule, M. Parra, J. S. Dordick, J. L. Plawsky, and C. H. Collins, "Spaceflight promotes biofilm formation by *Pseudomonas aeruginosa*," *PLoS One* **8**(4), e62437 (2013).

⁶N. Rabin, Y. Zheng, C. Opoku-Temeng, Y. Du, E. Bonsu, and H. O. Sintim, "Biofilm formation mechanisms and targets for developing antibiofilm agents," *Future Med. Chem.* **7**(4), 493–512 (2015).

⁷M. Jamal, W. Ahmad, S. Andleeb, F. Jalil, M. Imran, M. A. Nawaz, T. Hussain, M. Ali, M. Rafiq, and M. A. Kamil, "Bacterial biofilm and associated infections," *J. Chin. Med. Assoc.* **81**(1), 7–11 (2018).

⁸V. Stanić, D. Janačković, S. Dimitrijević, S. B. Tanasković, M. Mitrić, M. S. Pavlović, A. Krstić, D. Jovanović, and S. Raičević, "Synthesis of antimicrobial monophase silver-doped hydroxyapatite nanopowders for bone tissue engineering," *Appl. Surf. Sci.* **257**(9), 4510–4518 (2011).

⁹J. Palmer, S. Flint, and J. Brooks, "Bacterial cell attachment, the beginning of a biofilm," *J. Ind. Microbiol. Biotechnol.* **34**(9), 577–588 (2007).

¹⁰L. K. Vestby, T. Grønseth, R. Simm, and L. L. Nesse, "Bacterial biofilm and its role in the pathogenesis of disease," *Antibiotics* **9**(2), 59 (2020).

¹¹I. Ostrov, A. Harel, S. Bernstein, D. Steinberg, and M. Shemesh, "Development of a method to determine the effectiveness of cleaning agents in removal of biofilm derived spores in milking system," *Front. Microbiol.* **7**, 1498 (2016).

¹²G. Midelet and B. Carpentier, "Impact of cleaning and disinfection agents on biofilm structure and on microbial transfer to a solid model food," *J. Appl. Microbiol.* **97**(2), 262–270 (2004).

¹³K. Bruellhoff, J. Fiedler, M. Möller, J. Groll, and R. E. Brenner, "Surface coating strategies to prevent biofilm formation on implant surfaces," *Int. J. Artif. Organs* **33**(9), 646–653 (2010).

¹⁴C. Cattò and F. Cappitelli, "Testing anti-biofilm polymeric surfaces: Where to start?," *Int. J. Mol. Sci.* **20**(15), 3794 (2019).

¹⁵K. K. Amirtharaj Mosas, A. R. Chandrasekar, A. Dasan, A. Pakseresht, and D. Galusek, "Recent advancements in materials and coatings for biomedical implants," *Gels* **8**(5), 323 (2022).

¹⁶Y.-H. Kim, Y.-r. Choi, K.-M. Kim, and S.-Y. Choi, "Evaluation of copper ion of antibacterial effect on *Pseudomonas aeruginosa*, *Salmonella typhimurium* and *Helicobacter pylori* and optical, mechanical properties," *Appl. Surf. Sci.* **258**(8), 3823–3828 (2012).

¹⁷M. Mansour lakouraj, G. Rahpaima, and S. M. Mohseni, "Synthesis, characterization, and biological activities of organosoluble and thermally stable xanthone-based polyamides," *J. Mater. Sci.* **48**, 2520–2529 (2013).

¹⁸N. L. Brown, D. A. Rouch, and B. T. Lee, "Copper resistance determinants in bacteria," *Plasmid* **27**(1), 41–51 (1992).

¹⁹Y. Seo, J. Hwang, E. Lee, Y. J. Kim, K. Lee, C. Park, Y. Choi, H. Jeon, and J. Choi, "Engineering copper nanoparticles synthesized on the surface of carbon nanotubes for anti-microbial and anti-biofilm applications," *Nanoscale* **10**(33), 15529–15544 (2018).

²⁰M. Amiri, Z. Etemadifar, A. Daneshkazemi, and M. Nateghi, "Antimicrobial effect of copper oxide nanoparticles on some oral bacteria and *Candida* species," *J. Dent. Biomater.* **4**(1), 347 (2017).

²¹D. Longano, N. Ditaranto, L. Sabbatini, L. Torsi, and N. Cioffi, "Synthesis and antimicrobial activity of copper nanomaterials," in *Nano-Antimicrobials: Progress and Prospects* (Springer, 2012), pp. 85–117.

²²G. Mary, S. Bajpai, and N. Chand, "Copper (II) ions and copper nanoparticles-loaded chemically modified cotton cellulose fibers with fair antibacterial properties," *J. Appl. Polym. Sci.* **113**(2), 757–766 (2009).

²³L. Randolph, M. Banjafar, T. R. Preston, T. Yabuuchi, M. Makita, N. P. Dover, C. Rödel, S. Göde, Y. Inubushi, G. Jakob *et al.*, "Nanoscale subsurface dynamics of solids upon high-intensity femtosecond laser irradiation observed by grazing-incidence x-ray scattering," *Phys. Rev. Res.* **4**(3), 033038 (2022).

²⁴E. Kentzinger, U. Rücker, B. Toperverg, F. Ott, and T. Brückel, "Depth-resolved investigation of the lateral magnetic correlations in a gradient nanocrystalline multilayer," *Phys. Rev. B* **77**(10), 104435 (2008).

²⁵N. Popovici, E. Jimenez, R. Da Silva, W. Branford, L. Cohen, and O. Conde, "Optical and magnetic properties of Co-doped TiO₂ thin films grown by pulsed laser deposition," *J. Non-Cryst. Solids* **352**(9–20), 1486–1489 (2006).

²⁶I. Horcas, R. Fernández, J. M. Gómez-Rodríguez, J. Colchero, J. Gómez-Herrero, and A. M. Baro, "WSXM: A software for scanning probe microscopy and a tool for nanotechnology," *Rev. Sci. Instrum.* **78**(1), 013705 (2007).

²⁷Z. Shi, Y. Zhang, M. Liu, D. A. Hanaor, and Y. Gan, "Dynamic contact angle hysteresis in liquid bridges," *Colloids Surf., A* **555**, 365–371 (2018).

²⁸T. Young, "An essay on the cohesion of fluids," *Philos. Trans. R. Soc. London* **95**, 171–172 (1832).

²⁹W. A. Zisman, *Contact Angle, Wettability, and Adhesion* (American Chemical Society, 1964), p. 1.

³⁰S. Wu, "Calculation of interfacial tension in polymer systems," *J. Polym. Sci., Part C: Polym. Symp.* **34**, 19–30 (1971).

³¹D. K. Owens and R. Wendt, "Estimation of the surface free energy of polymers," *J. Appl. Polym. Sci.* **13**(8), 1741–1747 (1969).

³²N. Z.-J. Khong, Y. Zeng, S.-K. Lai, C.-G. Koh, Z.-X. Liang, K.-H. Chiam, and H.-Y. Li, "Dynamic swimming pattern of *Pseudomonas aeruginosa* near a vertical wall during initial attachment stages of biofilm formation," *Sci. Rep.* **11**(1), 1952 (2021).

³³B. Bhushan and Y. C. Jung, "Micro- and nanoscale characterization of hydrophobic and hydrophilic leaf surfaces," *Nanotechnology* **17**(11), 2758 (2006).

³⁴T. Ghodselahi, M. Vesaghi, A. Shafiekhani, A. Baghizadeh, and M. Lameii, "XPS study of the Cu@Cu₂O core-shell nanoparticles," *Appl. Surf. Sci.* **255**(5), 2730–2734 (2008).

³⁵B. F. Matlaga, L. P. Yasenchak, and T. N. Salthouse, "Tissue response to implanted polymers: The significance of sample shape," *J. Biomed. Mater. Res.* **10**(3), 391–397 (1976).

³⁶A. Taubert, J. F. Mano *et al.*, *Biomaterials Surface Science* (John Wiley & Sons, 2013).

³⁷M. Vincent, R. E. Duval, P. Hartemann, and M. Engels-Deutsch, "Contact killing and antimicrobial properties of copper," *J. Appl. Microbiol.* **124**(5), 1032–1046 (2018).

³⁸I. B. Gomes, M. Simões, and L. C. Simões, "Copper surfaces in biofilm control," *Nanomaterials* **10**(12), 2491 (2020).

³⁹K. P. Pontin, K. A. Borges, T. Q. Furian, D. Carvalho, D. E. Wilsmann, H. R. P. Cardoso, A. K. Alves, G. Z. Chitolina, C. T. P. Salle, H. L. de Souza Moraes, and
V. P. do Nascimento, "Antimicrobial activity of copper surfaces against biofilm formation by *Salmonella enteritidis* and its potential application in the poultry industry," *Food Microbiol.* **94**, 103645 (2021).

<sup>40</sup>M. J. Vargas-Straube, S. Beard, R. Norambuena, A. Paradela, M. Vera, and C. A. Jerez, "High copper concentration reduces biofilm formation in *Acidithiobacillus*

ferrooxidans by decreasing production of extracellular polymeric substances and its adherence to elemental sulfur,” *J. Proteomics* **225**, 103874 (2020).

<sup>41</sup>C. Molteni, H. K. Abicht, and M. Solioz, "Killing of bacteria by copper surfaces involves dissolved copper," *Appl. Environ. Microbiol.* **76**(12), 4099–4101 (2010).
V. P. do Nascimento, "Antimicrobial activity of copper surfaces against biofilm formation by *Salmonella enteritidis* and its potential application in the poultry industry," *Food Microbiol.* **94**, 103645 (2021).

<sup>40</sup>M. J. Vargas-Straube, S. Beard, R. Norambuena, A. Paradela, M. Vera, and C. A. Jerez, "High copper concentration reduces biofilm formation in *Acidithiobacillus*

*ferrooxidans* by decreasing production of extracellular polymeric substances and its adherence to elemental sulfur,” *J. Proteomics* **225**, 103874 (2020).

<sup>41</sup>C. Molteni, H. K. Abicht, and M. Solioz, "Killing of bacteria by copper surfaces involves dissolved copper," *Appl. Environ. Microbiol.* **76**(12), 4099–4101 (2010).