Dual activity of copper modified nanotextured stainless steel

Abstract

An exemplary embodiment of the present disclosure provides a method of forming an antibacterial material, comprising: providing a stainless-steel material; etching the stainless-steel material to render a plurality of nanopores and / or nanoprotrusions on at least a portion of a surface of the stainless steel material to create a nanotextured stainless steel material; and depositing an antimicrobial material on at least a portion of the nanotextured stainless steel.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 63 / 682,007, filed on 12 Aug. 2024, which is incorporated herein by reference in its entirety as if fully set forth below.FIELD OF THE DISCLOSURE

[0002] The various embodiments of the present disclosure relate generally to copper-coated nanotextured stainless steel and methods of making the same.BACKGROUND

[0003] The presence of harmful microorganisms on the surfaces we touch every day, especially in healthcare settings with vulnerable patients, necessitates new approaches to control bacterial contamination and the spread of infection. Modifying surfaces with antibiotics has addressed this concern to a certain extent, but continuous usage of antibacterial agents can lead to development of drug-resistant bacteria. In 2019, drug-resistant bacterial infections caused ≈1.27 million deaths worldwide. Both Gram-negative and Gram-positive bacteria are health threats, but the outer membrane of Gram-negative bacteria makes them more difficult to kill and more likely to develop resistance. Therefore, there is a significant need to develop antibacterial surfaces effective for both Gram-positive and Gram-negative bacteria.

[0004] Various approaches have been explored to create bactericidal surfaces, such as coating with antifouling materials, which prevents the adhesion and growth of fouling pathogen substances. However, these coatings often contain toxic biocides such as tributyltin, dichlofluanid, diuron, causing serious health and environmental concerns. Another approach focuses on developing antibacterial surfaces with inherently antibacterial materials, such as silver and copper, which have antifungal, anti-inflammatory, antiviral, and antimicrobial properties. Previous studies have investigated antibacterial activities of Cu including contact killing, DNA damage, membrane depolarization, and reactive oxygen species generation. Nevertheless, due to its high cost, copper cannot be extensively employed in bulk. Moreover, using a Cu-coated surface faces the challenge of gradual copper leaching, which diminishes its antibacterial properties over time.

[0005] Recently, natural antibacterial surfaces, such as microtopographical lotus leaves, have inspired polymer, metal, and metalloids nanotextured surfaces. These micro- or nanostructures can effectively prevent bacterial adhesion and colonization through direct contact. One such example used reactive ion etching to create black silicon (bSi) nanowires with diameters ranging from 20 to 80 nm, heights of 500 nm, and inter-wire spacings varying between 200 and 1800 nm. The surfaces were highly bactericidal against both Gram-negative and Gram-positive bacteria, with an average killing rates of up to ≈450 000 cells min−1 cm−2. Other studies have also explored nanostructured surfaces by employing different cleanroom fabrication techniques such as atomic layer deposition for SiO2—ZnO nanowires, plasma-treated copper and fluorocarbon-coated PMMA etching to develop Cu-PDMS nanostructured materials, chemical vapor deposition of amino methyl styrene and vinyl pyrrolidone, photo lithography-electron beam lithography and vapor-liquid growth process for SiO2 nanowire fabrication, and electron beam evaporation for Cr / ZnO thin films. The impact of nanostructured materials' surface roughness on various nanopatterns (e.g., nanocone, nanopillar, nanowire, nanor-ings, nanodaggers, nanograss, nanoblades, and nanospheres) made of different materials (metals, metal oxides, polymers, and metalloids) have also been studied. These studies emphasized the importance of changing surface topography, height or pitch, and roughness, concluding that not all nanopatterns exert sufficient mechanical forces to eliminate bacterial cells, but altering topography is crucial for creating a bactericidal surface.

[0006] Several conventional approaches have been studies. One study implemented PLA in creating nanostructures via plasma etching with Ag and Cu, achieving a remarkable 95% killing rate. Nonetheless, PLA's inherent biodegradability raises concerns about its long-term structural integrity and durability. Another conventional approach demonstrated commendable antibacterial properties by utilizing Cu nanostructures directly on Cu foil. However, relying solely on Cu as an antimicrobial agent might render susceptibility to oxidation, compromising its efficacy. Still another approach developed a composite using PDMS and Cu to create an antibacterial agent. Yet, PDMS's mechanical properties present challenges, potentially leading to the loss of antibacterial efficacy under high mechanical stress. In another study, PMMA was employed in an antibacterial agent's development. However, PMMA's tendency to become brittle in thin or highly stressed conditions, coupled with its sensitivity to specific chemicals, raises concerns about its durability and surface integrity. While these materials demonstrated effective antibacterial properties, the feasibility of microfabrication technologies is hindered by complex fabrication processes, lengthy processing times, and high costs. Therefore, it is crucial to identify cost-effective, scalable nanofabrication methods and materials for widespread implementation in combating bacterial infections caused by surface contamination.

[0007] Stainless steel 316L (SS316L) is widely used in public settings, including sinks, toilets, surgical tools, and cardiovascular and orthopedic implants, owing to its favorable mechanical strength, corrosion resistance, and biocompatibility. Despite its extensive use, there have been limited studies on the influence of stainless-steel surface topography on bacterial adhesion. Previous investigation revealed that shot peened SS316L surfaces with root mean square (RMS) surface roughness of 29 nm reduced adhesion of Gram-positive bacteria Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) but had no significant impact on adhesion of Gram-negative Escherichia coli (E. coli). Other surface types with similar roughness parameters showed varying effects on bacterial growth and adhesion. For example, a smooth polymer surface with RMS roughness of 1.4 nm did not have any antibacterial properties. In contrast, a nanotextured polymer surface with an RMS roughness of 13.8 nm effectively inhibited the growth of S. aureus, but had less influence on E. coli adhesion. These results highlight the significance of surface types, bacterial species, and surface finishing methods in determining bacterial adhesion and growth behavior.

[0008] Accordingly, there is still a need for improved materials for inhibiting bacterial adhesion and growth. Embodiments of the present disclosure address these needs.BRIEF SUMMARY

[0009] An exemplary embodiment of the present disclosure provides a method of forming an antibacterial material, comprising: providing a stainless steel material; etching the stainless steel material to render a plurality of nanopores and / or nanoprotrusions on at least a portion of a surface of the stainless steel material to create a nanotextured stainless steel material; and depositing an antimicrobial material on at least a portion of the nanotextured stainless steel.

[0010] In any of the embodiments disclosed herein, the antimicrobial material can comprise copper.

[0011] In any of the embodiments disclosed herein, the etching can occur over a period of at least 30 seconds, 30 seconds to 10 minutes, 30 seconds to 5 minutes, 30 seconds to 2 minutes, or 30 seconds to 1 minute.

[0012] In any of the embodiments disclosed herein, the nanotextured stainless steel material can have a pore size distribution of from 10-50 nm, from 10-40 nm, or from 20-30 nm.

[0013] In any of the embodiments disclosed herein, the plurality of nanopores and / or nanoprotrusions can have a vertical depth / height of 10-50 nm, 20-40 nm, 25-35 nm, or about 30 nm.

[0014] In any of the embodiments disclosed herein, depositing the antibacterial material can occur for 4 minutes to 15 minutes and with at least 0.3 amps of current.

[0015] In any of the embodiments disclosed herein, the antibacterial material can form a coating which a thickness of at least 1 micron, at least 1.5 microns, at least 2 microns, at least 3 microns, at least 5 microns, between 1 and 10 microns, between 1 and 8 microns, or between about 1 and about 6 microns.

[0016] In any of the embodiments disclosed herein, the stainless steel material can be SS316L.

[0017] In any of the embodiments disclosed herein, the method can further comprise sonicating the stainless steel material in an organic solvent.

[0018] In any of the embodiments disclosed herein, etching the stainless steel material can comprise an electrochemical etching process.

[0019] Another embodiment of the present disclosure provides an antibacterial material, comprising a nanotextured stainless steel material, a coating of copper on at least a portion of the nanotextured stainless steel material.

[0020] In any of the embodiments disclosed herein, antibacterial material can be made from any of the processes disclosed herein.

[0021] Another embodiment of the present disclosure provides a method of treating a contaminated substance, comprising placing the substance in contact with any of the antibacterial materials disclosed herein.

[0022] In any of the embodiments disclosed herein, the substance can comprise bacteria, and the material can cause at least a portion of the bacteria to die.

[0023] In any of the embodiments disclosed herein, the bacteria can comprise Gram-negative and Gram-positive bacteria, and the material can cause at least a portion of the Gram-negative and Gram-positive bacteria to die.

[0024] In any of the embodiments disclosed herein, the substance can comprise yeast, and the material can cause at least a portion of the yeast to die.

[0025] These and other aspects of the present disclosure are described in the Detailed Description below and the accompanying drawings. Other aspects and features of embodiments will become apparent to those of ordinary skill in the art upon reviewing the following description of specific, exemplary embodiments in concert with the drawings. While features of the present disclosure may be discussed relative to certain embodiments and figures, all embodiments of the present disclosure can include one or more of the features discussed herein. Further, while one or more embodiments may be discussed as having certain advantageous features, one or more of such features may also be used with the various embodiments discussed herein. In similar fashion, while exemplary embodiments may be discussed below as device, system, or method embodiments, it is to be understood that such exemplary embodiments can be implemented in various devices, systems, and methods of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following detailed description of specific embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, specific embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0027] FIG. 1A illustrates a nanotextured stainless steel fabrication process and its modification using Cu by electrochemical techniques, in accordance with some embodiments of the present disclosure. FIGS. 1B-C and FIGS. 1D-E provide SEM and AFM images, respectively, of pristine stainless steel (FIGS. 1B & 1D) and nanotextured stainless steel (FIGS. 1C & 1E) etched for 30 s at 8 V, in accordance with some embodiments of the present disclosure.

[0028] FIGS. 2A-D provide XPS spectra of stainless steel (SS), nSS, and copper-coated nSS for 4 and 15 min, in accordance with some embodiments of the present disclosure.

[0029] FIGS. 3A-C provide steady-state kinetic results using Lineweaver-Burk plots for (FIG. 3A) nSS, (FIG. 3B) Cu_4 min, and (FIG. 3C) Cu_15 min, with varying concentrations of TMB at ambient conditions, in which the reaction time was 15 min and absorbance values were measured at 652 nm, (n=3). FIG. 3D illustrates leached Cu for Cu_4 min and Cu_15 min samples in TS and LB media after 24 h incubation at 37° C. (n=3).

[0030] FIGS. 4A-B provides plots showing the number of adhered E. coli and S. epidermis on control (SS), nSS, Cu_4 min, and Cu_15 min following 24 h of incubation, (n=5); *p<0.05, **p<0.01, **p<0.001, ****p<0.0001 (one way ANOVA). FIGS. 4C-D provides bacterial growth curves of E. coli in LB media (FIG. 4C) and S. epidermis in TS media (FIG. 4D) in the presence of control, nSS, Cu_4 min, and Cu_15 min, (n=3).

[0031] FIGS. 5A-B illustrate mean fluorescence intensity (MFI) of (FIG. 5A) E. coli and (FIG. 5B) S. epidermidis labeled for ROS after incubating with nSS, Cu_4 min, and Cu_15 min. Untreated cells, peroxide-added cells, and SS incubated were taken as control samples. FIG. 5C provides % depolarized S. epidermidis cells with Cu_4 min and Cu_15 min. Untreated cells and treatment with carbonyl cyanide 3-chlorophenylhydrazone (CCCP, a membrane depolarizing agent) were used as negative and positive control samples, respectively, (n=3); *p<0.05, *p<0.01, **p<0.001, ****p<0.0001 (one way ANOVA).DETAILED DESCRIPTION

[0032] Although preferred exemplary embodiments of the disclosure are explained in detail, it is to be understood that other exemplary embodiments are contemplated. Accordingly, it is not intended that the disclosure is limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other exemplary embodiments and of being practiced or carried out in various ways. Also, in describing the preferred exemplary embodiments, specific terminology will be resorted to for the sake of clarity.

[0033] To facilitate an understanding of the principles and features of the present disclosure, various illustrative embodiments are explained below. The components, steps, and materials described hereinafter as making up various elements of the embodiments disclosed herein are intended to be illustrative and not restrictive. Many suitable components, steps, and materials that would perform the same or similar functions as the components, steps, and materials described herein are intended to be embraced within the scope of the disclosure. Such other components, steps, and materials not described herein can include, but are not limited to, similar components or steps that are developed after development of the embodiments disclosed herein.

[0034] As used in the specification and the appended claims, the singular forms “a,”“an” and “the” include plural referents unless the context clearly dictates otherwise.

[0035] Also, in describing the preferred exemplary embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.

[0036] Ranges can be expressed herein as from “about” or “approximately” one particular value and / or to “about” or “approximately” another particular value. When such a range is expressed, another exemplary embodiment includes from the one particular value and / or to the other particular value.

[0037] Similarly, as used herein, “substantially free” of something, or “substantially pure”, and like characterizations, can include both being “at least substantially free” of something, or “at least substantially pure”, and being “completely free” of something, or “completely pure”.

[0038] By “comprising” or “containing” or “including” is meant that at least the named compound, member, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.

[0039] Mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a device or system does not preclude the presence of additional components or intervening components between those components expressly identified.

[0040] The materials described as making up the various members of the invention are intended to be illustrative and not restrictive. Many suitable materials that would perform the same or a similar function as the materials described herein are intended to be embraced within the scope of the invention. Such other materials not described herein can include, but are not limited to, for example, materials that are developed after the time of the development of the invention.

[0041] Reference will now be made in detail to exemplary embodiments of the disclosed technology, examples of which are illustrated in the accompanying drawings and disclosed herein. Wherever convenient, the same references numbers will be used throughout the drawings to refer to the same or like parts.

[0042] The inventors previously demonstrated the antibacterial properties of nanotextured stainless steel with an RMS roughness of 6.51 nm made by electrochemical etching. This nanotextured stainless steel exhibited reduced S. aureus and E. coli adhesion and viability while maintaining cytocompatibility with mammalian cells. The present disclosure improves upon this material through the incorporation of antibacterial materials.

[0043] Exemplary embodiments of the present disclosure provide nanotextured stainless-steel coated with antibacterial materials, e.g., copper. Such materials can exhibit dual antibacterial properties. The dual antibacterial activity can be due to the small grooves and ridges on the nanotextured surface, which make it difficult for bacteria to attach and spread, and release of copper ions that kill or inhibit the growth of bacteria. Accordingly, the present disclosure discloses methods of making nanotextured stainless steel and subsequently coated with copper using inexpensive, scalable electrochemical techniques for both steps. This approach offers several advantages, including dual-antibacterial properties, affordability, scalability, eco-friendliness, and precise control of surface structures through electrochemical parameters such as potential and current density.

[0044] The nanotextured stainless-steel can have a plurliary of nanopores and / or nanoprotrusions defining its nanotextured surface. As those skilled in the art would understand, the nanotextured surface can be created by etching stainless-steel over varying periods of time. The resulting surface can have varying pore / protrusion sizes and depths / heights based on, for example, the etching duration. In some embodiments, the pore sizes (e.g., average diameter of the pores) can range from 10 nm or smaller to at least 50 nm. Similarly, the vertical depths / heights can range from less than 10 nm to at least 50 nm.

[0045] An antibacterial material, such as copper, can be coated on the surfaced of the nanotextured stainless-steel. This can occur many ways known in the art, including, but not limited to, an electrochemical deposition process. Depending on the parameters of the deposition process, e.g., duration, voltage, amperage, etc., the coating can have varying thicknesses, e.g., from 1-10 microns, though the disclosure is not so limited.

[0046] The copper-coated, nanotextured stainless-steel can be used to kill or inhibit growth of bacteria and / or yeast in substances. Thus, in some embodiments, the stainless-steel can be used to manufacture components likely to come into contact with such substances. Additionally, embodiments of the present disclosure have the ability to kill / inhibit growth of both Gram-negative and Gram-positive bacteria.Examples

[0047] The examples below provide details of certain exemplary embodiments of the present disclosure. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the present disclosure.Results and DiscussionFabrication and Characterization of Nanotextured Stainless Steel

[0048] Nanotextured stainless steel (nSS) samples were prepared by etching stainless steel at 8 V for 10 to 90 s (FIG. 1A), and the resulting morphologies are shown in FIG. 1B-E. The electrochemical etching time interval was optimized for the desired nanostructure formation, resulting in a distinct morphology of the developed electrochemically treated steel compared to the control sample. No noticeable etching was observed within 10 s, but the etching amount significantly increased as the time progressed to 30 s and beyond. Nanopores were distributed regularly over the surface of the steel. For 30 s etching time, the pore size distribution of 20-30 nm and vertical structure of ≈30 nm high sharp nano protrusions, which shows a similar morphology as fabricated using clean room techniques.

[0049] Next, Cu was deposited on nSS for various time intervals ranging from 4 to 15 min at 0.3 A current. The current causes the positively charged Cu ions to migrate to the cathode (nSS), resulting in different oxidation states of Cu deposited on nSS, as given in the reactions below. Initially, at a coating time of 4 min, scanning electron microscopy (SEM) and atomic force microscopy (AFM) images revealed a copper deposited on the textured steel surface. The coating appeared as small islands or particles, indicating the initial nucleation and growth of the coating of thickness 1.73 (±0.37) μm. The longest deposition time of 15 min resulted in a highly uniform and dense layer of copper, completely covering the nSS surface of thickness 6.08 (±1.03) μm. Hence, two time intervals were chosen for further characterization of Cu deposited nSS. Cu_4 min is a 4-min coating with a noticeable amount of copper islands but majority of exposed nSS surface. Cu_15 min is a 15 min coating with a near continuous, layer on the nSS surface.C⁢u=>C⁢ u2++2⁢e-Equation⁢ 1C⁢ u2++e-=>C⁢ u+Equation⁢ 22⁢C⁢ u2++H2⁢0=>C⁢ u2⁢O+2⁢H+Equation⁢ 3

[0050] The crystal phase structure of bulk copper was investigated using X-ray diffraction (XRD) in the 20°-80° range. An XRD pattern showed peaks at 2Θ=43.4°, 50.4°, and 74.2°. These peaks correspond to the (111), (200), and (220) crystallographic planes, respectively. Since Cu and Austenite coexist within identical planes, in the case of SS and nSS, these planes might align with Austenite. The increased intensity of the diffraction peaks for Cu_4 min and Cu_15 min confirms Cu existence, and a larger amount of copper deposition over time. The dominant (111) peak shows that the Cu grains have undergone preferential reorientation in one direction during electrochemical deposition. Furthermore, FTIR spectrum of Cu_4 min and Cu_15 min also showed the stretching vibrations of Cu2O at 677 cm-1, which indicates the Cu / Cu2O presence on nSS.

[0051] The surface chemical composition and elemental valence states of the Cu were investigated by X-ray photoelectron spectroscopy (XPS), and the results are shown in FIG. 2. We compared the Cu elemental state between pristine SS and electrochemically treated nSS, Cu_4 min, and Cu_15 min. Pronounced peaks for Cu0 are present in all samples at 932.4 and 952.8 eV. SS and nSS showed similar spectra and the low intensity of Cu0 present in SS / nSS indicates trace amounts of Cu. The high intensity of Cu0 peaks demonstrates the copper-coated surface using electrochemistry. The distinct oxidation states were also observed at 934.6 and 954.5 eV, corresponding to Cu2+ and Cu+ respectively, along with their associated satellite peaks at 942.1, 944.2, and 962.9 eV. The charges on Cu were formed during the electrochemical Cu deposition, as given in reactions (1)-(3).

[0052] To analyze the wetting behavior, contact angle tests were performed on all surfaces. Among them, the hydrophilic stainless-steel surface exhibited the lowest contact angle (60.8°±1.21), indicating a strong attraction to water molecules. On the other hand, nSS surfaces demonstrate a much higher contact angle (108.95°±0.42) due to increased surface hydrophobicity caused by the nanoprotrusive features. Coating with copper reduced the contact angle somewhat, but the Cu_4 min (98.97°±1.32) and Cu_15 min (96.76°±1.84) surfaces still displayed a hydrophobic nature.

[0053] Hydrophobic surfaces can create an inhospitable environment for bacteria to thrive, as their growth often depends on the presence of moisture.Generation of Reactive Species

[0054] To understand the surface generation of reactive oxygen species (ROS), a subset of free radicals, kinetic studies utilizing TMB dye were conducted. TMB dye can undergo oxidation, transitioning from a colorless state to a blue one in the presence of ROS. Some metal surfaces can catalyze the conversion of dissolved oxygen in water to ROS. These ROS can cross the membranes of bacteria and damage DNA, lipids, and proteins. To measure how well the nSS and Cu-coated nSS surfaces catalyze the production of ROS, the maximum initial velocity of the reaction as a measure of the catalytic activity was determined. Here, the reaction occurs in two steps: 1) The catalytic active sites of metal surface can catalyze ROS generation, 2) ROS oxidize colorless TMB to blue-colored TMB, which can be measured at 652 nm. At low substrate (dye) concentrations, the reaction rate (V) showed a linear relationship with the substrate concentration [TMB], indicating first-order kinetics (FIG. 3). Conversely, at high concentrations, the reaction rate (Vmax) was limited by the availability of active sites on the catalyst, following zero-order kinetics where V is independent of substrate concentration. A catalyst with high effectiveness exhibits a high Vmax, representing a rapid reaction rate, indicating a high number of reactive species generated during the catalytic process. In FIG. 3, kinetic studies were performed to compare the catalytic activities of nSS, Cu_4 min and Cu_15 min. The Michaelis-Menten constant (Km) and Vmax were obtained by fitting the absorbance values for the catalytic reaction products versus time with varying concentrations of TMB in ambient air. While SS did not demonstrate significant catalytic activity (Vmax=1.1 nm), nSS (Vmax=15 nM s−1), Cu_4 min (Vmax=52 nvi s−1), and Cu_15 min (Vmax=55 nvi s−1) all show strong surface properties for producing reactive species. The surface of conventional stainless steel is relatively inert, indicating a limited interaction with other substances. In contrast, nanostructured stainless-steel exhibits superior catalytic activity compared to regular stainless steel, owing to various factors associated with its nanoscale features such as defects, edges, corners, and other irregularities on its surfaces, which serve as active sites for catalysis. These imperfections facilitate the adsorption and reaction of reactant molecules, contributing to an enhanced overall catalytic activity. Overall, this suggests that the nanoprotrusive features of nSS have significantly higher catalytic activity than SS, which was further improved by the presence of Cu on nSS.

[0055] Next, the Cu leaching was measured in luria broth (LB) media and tryptic soy (TS) media after 24 h incubation using a Cu calibration for each media, as shown in FIG. 3D. As expected, Cu_4 min leaches less Cu (68.5±10 ppm and 60.6±20.2 ppm) than Cu_15 min (333.5±15.98 and 1154±38.47 ppm) for both LB and TS media. This demonstrates that Cu ions desorb and are released into media, which corresponds with the occurrence of bacterial death, as evident from a prior study that reported Cu can kill bacteria at concentrations of 90 ppm or higher. Additionally, the Cu leaching was measured using ICP-MS in LB media and TS media after 24 h incubation using a Cu calibration curve in 2% nitric acid. All samples were digested in 2% nitric acid. Cu_15 min (261.58±16.29 and 225.43±14.92 ppm) leaches more Cu than Cu_4 min (221.06±56.7 and 134.04±68.02 ppm) for both LB and TS media. We extended incubation to 80 days in water to monitor loss of Cu via the catalytic activity changes of the metal surfaces. As expected, both SS and nSS surfaces displayed negligible change in their activity over the 80-day period due to no contact with air or contamination. However, Cu_4 min and Cu_15 min demonstrated slow decline in their catalytic activity of 27.5% and 14.7%, respectively, attributed to the desorption of Cu from the SS surface. Cu leaching from the non-coated steels surfaces is not likely as the composition of steel is not superficial but rather a physical amalgamation, and copper is integral to alloy's structure. Steel also contains chromium, which is known for its effective antibacterial properties. However, chromium is susceptible to leaching only under specific conditions like heating, exposure to acidity, or mechanical stress.Bacterial Inhibition on Metal Surfaces

[0056] A comparative analysis was conducted to evaluate the % reduction in bacterial adhesion for nSS, Cu_4 min, and Cu_15 min and viability of Gram-negative E. coli and Gram-positive S. epidermidis, with SS as the control. After incubating metal surfaces in bacteria solution for 24 h, adherent bacteria were collected to determine colony formation and growth (FIG. 4). The SS surface exhibited high bacterial growth for both Gram-negative and Gram-positive bacteria, indicating its lack of antibacterial properties. On the other hand, nSS, Cu_4 min, and Cu_15 min surfaces showed either low bacterial growth or no growth at all. The % reduction in bacterial adhesion for nSS was 92.3% for S. epidermidis and 84.5% for E. coli, which were enhanced when nSS was modified with Cu for different durations. Cu_4 min exhibited 94.6% against E. coli and 99.2% against S. epidermidis. Similarly, Cu_15 min displayed further enhanced % reduction, achieving 96.8% against E. coli and 99.6% against S. epidermidis. Like the 24 h data, nSS showed significantly reduced bacterial adhesion than SS, though the additional benefit of Cu coating at this time was minimal, likely due to less Cu desorption into solution in 6 h compared to 24 h. The notable improvements in dual-antibacterial surfaces can be attributed to the combined properties of nanoscale roughness and Cu release. Nanotextures have the capacity to physically harm bacterial cells by either causing physical damage or penetrating the cell wall. Additionally, they can generate ROS, which are highly reactive molecules capable of inflicting damage to bacterial cells. Cu toxicity has been attributed to ROS generation and membrane depolarization, which can induce bacterial stress and disrupt outer membranes.

[0057] The bacterial membrane plays a crucial role in generating a proton gradient for adenine triphosphate synthesis, the primary energy carrier of cells. The disruption of the membrane interface can result in reduced energy production and physical damage to the bacterial cells. This can be observed in the negative effects on bacterial growth patterns following removal from the nanotexture and copper (FIGS. 4C-D). For SS, E. coli and S. epidermidis both exhibited growth after a lag time. However, after exposure to nSS, the growth of both E. coli and S. epidermidis was inhibited, resulting in increased lag times, and lower OD600 at the end of growth. Exposure to Cu_4 min further inhibited growth for E. coli and S. epidermidis and no growth was observed for exposure to Cu_15 min. Altogether, this data indicates that both nanotexture and copper can inhibit bacterial growth by both delaying the onset of exponential growth and reducing the growth rate. These growth curve patterns align with previously reported literature on antibacterial activities of Rh nanoplates. This is consistent with prior findings, reporting that decreasing bacterial growth with increasing Cu and no bacterial growth was observed with copper nanoparticles at a concentration of 60 jig Cu / mL or higher. Moreover, SEM imaging was performed to examine E. coli adhesion on SS, nSS, Cu_4 min, and Cu_15 min. No morphological alterations were observed in the adhered bacteria on SS, suggesting that there was no bacterial damage. Conversely, wrinkled morphology and a reduced number of bacteria were observed on nSS.Assessment of ROS Production in Bacteria

[0058] As stated earlier, ROS generation from nSS and copper-coated nSS surfaces were detected and could be a cause of the decrease in bacteria adhesion and growth. To quantitatively determine the intracellular generation of ROS induced by nSS and Cu-coated nSS, the CellROX Deep Red fluorescence intensity was measured using a flow cytometer. Untreated bacteria and bacteria treated with peroxide, SS, and copper foil were included as controls. Both E. coli and S. epidermidis exhibited high ROS production on nSS surfaces, but not on SS or either copper coated surface (FIGS. 5A-B). While E. coli had a ROS-negative population and ROS-positive population that was lower than the peroxide-positive control, S. epidermidis cells all produced more ROS than peroxide-treated cells. This is consistent with greater reduction in S. epidermidis CFU by nSS compared to E. coli. These results indicate that the sharp features of nSS promoted induction of oxidative stress in bacteria, resulting in ROS production, which could cause the reduced CFU and growth observed. The mechanism of antibacterial activity mediated by Cu remains incompletely defined due to conflicting reports on whether Cu ions induce bacterial generation of ROS. Despite evidence of free radical production by all modified surfaces (FIG. 3), no ROS generation was observed in bacteria cultured on Cu foil, Cu_4 min, and Cu_15 min (FIGS. 5A-B). This suggests that the antibacterial activity of copper-modified nSS is not just ROS-dependent, and maybe mostly promoted by membrane depolarization. In FIG. 2, Cu2+ and Cu+ were detected by XPS, implying that the positively charged ions may bind to negatively charged domains on the bacterial cell membrane, both outer and inner, thereby reducing the electrical potential difference across the membrane, which could rupture the membrane. It is possible that the process of membrane depolarization occurs more rapidly than the nSS-induced cellular production of ROS, which would require physical interaction of the bacteria with the surface, so ROS is only produced in the presence of nSS surfaces without copper. To validate this, the change in membrane potential, which is essential for energy transduction during respiration, was measured after incubating in Cu-modified nSS. A BacLight membrane potential kit, which contains DiOC2(3) (3,3′-diethyoxacarbocyanineiodide), a dye that initially emits green fluorescence in polarized cells was used. However, this green fluorescence decreases upon loss of polarization. It is important to note that the dye is not as accurate in measuring membrane potential in Gram-negative bacteria as it is Gram-positive bacteria, which is why this experiment was performed only on S. epidermidis. A decrease in green fluorescence was observed for Cu_15 min modified nSS. This shift suggests that the interaction between bacteria and metal allows Cu ions to penetrate the cells, potentially facilitating access to cellular components. Subsequently, intercellular Cu ions may cause irreversible damage and cell death. FIG. 5C shows that Cu_15 min exhibited a significant population of cells with membrane depolarization in comparison to Cu_4 min, likely due to the higher Cu concentration. Additionally, Cu contact killing might have damaged the outer and / or bacterial membrane, led to accumulation of Cu in the cells, and degraded bacterial DNA. Also, accumulation of intracellular copper ions can scavenge ROS, reducing our ability to measure it. While more studies will be needed to clarify the mechanism underlying antibacterial activity, these results demonstrate that different mechanisms are involved for antibacterial activity via nSS and Cu-coated nSS surfaces.

[0059] The present disclosure demonstrates dual-activity surfaces for killing bacteria using copper-coated nanotextured stainless steel. This can be achieved by employing an inexpensive electrochemical technique to first create nanotextured surfaces on SS316L, featuring nanopores and nanoprotrusions measuring 20-30 nm, then depositing copper onto the nanotextured surface, resulting in globular or thin film morphology. Both the copper-coated nanotextured stainless steel displayed enhanced antibacterial qualities compared to regular stainless-steel surfaces. ROS generation from nSS and copper coated nSS surface for E. coli and S. epidermidis were also investigated using CellROX Deep Red fluorescence intensity. Copper coated nSS surfaces demonstrated excellent antibacterial activity. Although copper will gradually leach out from the metal over time, the underlying nanotextured stainless steel structure will remain intact, ensuring that the surface retains antibacterial attributes even after the copper has been released. This advancement has significant potential for practical usage, as it offers a method to prevent bacterial adhesion and surface contamination without antibiotics, and a very similar process, electrochemical polishing, is already used at scale to smooth large and small steel products. Further, the dual function may not contribute to development of drug-resistant bacteria like antibiotics do. The cost-effectiveness and scalability of this surface modification approach may potentially make it practically relevant for larger-scale surfaces in public or healthcare settings.Experimental Section

[0060] Materials: E. coli (BL 21) and S. epidermidis (PCI 1200) were purchased from ATCC. Triton X-100, luria broth (LB), tryptic soy (TS) media, copper foil, copper nitrate, and nitric acid (ACS reagent, 70%) were purchased from Sigma-Aldrich. Formaldehyde and hexamethyldisilazane were purchased from VWR and TCI chemicals, respectively. Agar and 3,3′,5,5′ tetramethylbenzidine (TMB) were purchased from Fisher-Scientific. SS316L plates (30×20×0.05 cm3) and vinyl insulating tape were purchased from Maudlin Products and 3M, respectively.

[0061] Nanotextured Steel Preparation: The electrochemical production of nanotextured stainless steel and its subsequent modification with copper is shown in FIG. 1A. SS316L was cut into two sizes: 2.5×1.5×0.05 and 2.5×2.5×0.05 cm3. These served as working and counter electrodes, respectively. The samples were sonicated for 7 min each in acetone, methanol, and isopropanol to eliminate organic contaminants. The samples were rinsed with water to remove organic solvents. A stainless-steel wire was spot-welded onto the SS316L samples to establish electrical connections to the cathode and anode. The working electrode was covered with insulating tape, exposing only an active area of 0.19 cm2 for electrochemical surface modification. Diluted nitric acid solution (48 wt. %) was used as an electrolyte. The working and counter electrodes were separated by 6 cm. Electrochemical surface modification was carried out using a power supply set at 8 V for 30 s. After electrochemical etching, the sample was removed from the electrochemical cell, washed with deionized water, and dried at room temperature.

[0062] For the electrochemically deposited Cu on nanotextured stainless steel, an electrochemically etched steel was used as the working electrode and a copper foil as the counter electrode. A voltage of 1 V was applied for different durations of time in 1 M copper nitrate solution. After completing the electrochemical treatment, the coated sample was thoroughly rinsed with deionized water and dried at room temperature.

[0063] Surface Characterization: Surface morphologies of SS316L samples were characterized by scanning electron microscopy (SEM, Hitachi SEM SU8010) at 5 kV acceleration potential, and topographical information was acquired by atomic force microscopy (AFM, Veeco Dimension 3100) using AppNano ACT tapping mode AFM probes (Applied Nanosciences). The surface roughness parameters of stainless steel, nanotextured stainless steel, and copper coated nanotextured stainless steel surfaces were obtained by AFM measurements from scanning a surface area of 4 μm2 while avoiding artificial defects. Chemical composition of the sample surfaces was analyzed by X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific K-Alpha XPS) with a 400 μm microfocused monochormatic Al Kα X-ray source, which has analysis depth of less than 5 nm. The phase composition of the Cu coated nanotextured steel was determined by X-ray powder diffractometry (Rigaku XRD). The diffraction patterns of samples were recorded in the range of 2θ=20°-80° using a Cu radiation source with fixed power (40 kV, 44 mA). Contact angles of purified water droplets on metal surfaces were monitored using a First Ten Angstrom contact angle goniometer (FTA-200). The Thermo Nicolet 6700 Fourier Transform Infrared Spectrometer (FTIR) was used to measure the infrared energy of Cu. Inductively Coupled plasma mass spectrometry (ICP-MS, Thermo iCAP-RQ) was used to measure the Cu leaching concentration using Argon plasma. 1.5 mL of sample was injected in the ICP-MS with fixed rinse time and measurement time of 45 s in each case.

[0064] Bacterial Cultures and Adhesion Assays: E. coli and S. epidermis were used as model microorganisms for bacterial assays. All metal samples were sterilized in an autoclave at 15 psi and 121° C. for 20 min. The unmodified surface of samples was masked with tape to prevent contamination. These samples were then transferred into 6-well cell culture plates and incubated with 5 mL of bacterial solution. E. coli and S. epidermis were cultured in LB media and TS media, respectively. The working bacterial solutions had an optical density (OD) of 0.3 at 600 nm unless otherwise mentioned, approximately equivalent to 5×107 cells / mL. The samples were cultured with the bacteria for 24 h in a static incubator at 37° C. in a humidified environment. To quantify the number of adhered E. coli and S. epidermidis cells on each metal surface, colony-forming units (CFUs) were counted using the spread plate method. After the 24 h incubation period concluded, the samples underwent five rinses with phosphate-buffered saline (PBS). Following the removal of the tape, they were washed once more before being transferred into a 50 mL tube containing 5 mL of fresh PBS. Each sample was sonicated for 7 min and vortexed for 20 s to release bacteria from the sample surface into the solution. A series of diluted solutions in PBS was prepared by transferring 25 μL of the resuspended cell solution into 225 μL of fresh PBS, resulting in a 10−1 dilution. 25 μL from first dilution was added to 225 uL of nutrient media on a 96-well plate to yield a total volume of 250 μl. The plate was covered with its lid and incubated in a plate reader (Synergy 2, Multi-Mode Microplate Reader, BioTeck) at 30° C. The plate was shaken for 10 s before reading optical density at 600 nm every 5 min over a period of 20 h. Further dilutions (10−1 to 10−1) were made in PBS in 96-well plates. Then, 30 μL of each diluted solution of E. coli and S. epidermidis was spread onto LB or TS agar plates, respectively. After 24 h of incubation at 37° C., the bacterial colonies on each plate were counted. The number of bacteria (CFU) per sample was calculated by dividing the number of colonies by the dilution factor, multiplying by the amount of cell suspension plated to agar (30 μL). The percentage reduction of bacterial adhesion was calculated by subtracting the number of bacterial colonies on the test surface from the number of bacterial colonies on the control surface, dividing this number by the number of bacterial colonies on the control surface, and multiplying by 100. To visualize bacterial adhesion on the metal surface using scanning electron microscopy (SEM), all metal samples underwent the same procedure described above for incubation with bacteria. After incubation, samples were washed three times with PBS, fixed with a 2.5% glutaraldehyde solution for 1 h, and dehydrated using a series of ethanol concentrations in distilled water (50%, 70%, 90%, and 100% ethanol for 20 min each). The dehydrated samples were dried overnight using hexamethyldisilazane (HMDS) and then coated with gold (≈7 nm thickness) using a Quorum Q-150T ES Sputter Coater. The samples were examined using a Hitachi SEM SU8010 at a 5 kV acceleration potential.

[0065] Surface Reactivity Analysis: Steady-state catalysis kinetics assessment was performed on nanotextured steel and copper modified nanotextured steel via catalytic reaction of varying concentrations of TMB under ambient conditions for 15 min. The absorbance values were recorded at 652 nm, and the kinetic catalysis parameters were obtained by linear fitting of the Lineweaver-Burk double-reciprocal plot. The maximal reaction velocity (vmax) was extracted from:1V=KmVmax[TMB]+1VmaxEquation⁢ 4where, V is the initial reaction velocity, and [TMB] refers to the substrate concentration.Flow Cytometry for Reactive Oxygen Species Analysis: To assess the effect of metal on the generation of bacterial reactive oxygen species (ROS), bacteria were incubated on metal surfaces as described above for 24 h at 37° C. Then, the metal surfaces were rinsed with 1 mL of PBS before being scraped to detach and collect E. coli and S. epidermis in 200 μl of PBS. Detached bacteria were loaded onto a 96-well plate, and the cells were stained with CellROX Deep Red reagent at a final concentration of 250 μM to detect ROS as per manufacturer's instructions (ThermoFisher Scientific). Following incubation for 45 min at 37° C., the cells were analyzed by Cytoflex flow cytometer (Beckman Coulter).

[0067] Statistical Analysis: For bacteria growth study, reactive oxygen species analysis, and % depolarized cells, one-way ANOVA was performed to determine the statistical differences between groups, followed by Turkey's post-hoc multiple comparison analysis. Statistical significance was denoted as follows: * for p≤0.05, ** for p≤0.01, *** for p≤0.001, **** for p≤0.0001. All statistical analyses were conducted using GraphPad Prism 10. All data plotted with error bars represent mean values t standard deviation when applicable.

[0068] It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purposes of description and should not be regarded as limiting the claims.

[0069] Accordingly, those skilled in the art will appreciate that the conception upon which the application and claims are based may be readily utilized as a basis for the design of other structures, methods, and systems for carrying out the several purposes of the embodiments and claims presented in this application. It is important, therefore, that the claims be regarded as including such equivalent constructions.

[0070] Furthermore, the purpose of the foregoing Abstract is to enable the United States Patent and Trademark Office and the public generally, and especially including the practitioners in the art who are not familiar with patent and legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The Abstract is neither intended to define the claims of the application, nor is it intended to be limiting to the scope of the claims in any way.

Claims

1. A method of forming an antibacterial material, comprising:providing a stainless steel material;etching the stainless steel material to render a plurality of nanopores and / or nanoprotrusions on at least a portion of a surface of the stainless steel material to create a nanotextured stainless steel material; anddepositing an antimicrobial material on at least a portion of the nanotextured stainless steel.

2. The method of claim 1, wherein the antimicrobial material comprises copper.

3. The method of claim 1, wherein the etching occurs over a period of at least 30 seconds.

4. The method of claim 1, wherein the nanotextured stainless steel material has a pore size distribution of from 10-50 nm.

5. The method of claim 1, wherein the plurality of nanopores and / or nanoprotrusions have a vertical depth / height of 10-50 nm.

6. The method of claim 1, wherein depositing the antibacterial material occurs for 4 minutes to 15 minutes and with at least 0.3 amps of current.

7. The method of claim 1, wherein the antibacterial material forms a coating with a thickness of between 1 and 10 microns.

8. The method of claim 1, wherein the stainless steel material is SS316L.

9. The method of claim 1, further comprising sonicating the stainless steel material in an organic solvent.

10. The method of claim 1, wherein etching the stainless steel material comprises electrochemical etching.

11. An antibacterial material, comprising:nanotextured stainless steel; anda copper coating on at least a portion of the nanotextured stainless steel.

12. The antibacterial material of claim 11, wherein the nanotextured stainless steel comprises a plurality of nanopores and / or nanoprotrusions.

13. The antibacterial material of claim 12, wherein the nanotextured stainless steel material has a pore size distribution of from 10-50 nm.

14. The antibacterial material of claim 12, wherein the plurality of nanopores and / or nanoprotrusions have a vertical depth / height of 10-50 nm.

15. The antibacterial material of claim 11, wherein the copper coating has a thickness of between 1 and 10 microns.

16. The antibacterial material of any of claim 11, wherein the stainless steel material is SS316L.

17. A method of treating a contaminated substance, comprising placing the substance in contact with the antibacterial material of claim 11.

18. The method of claim 17, wherein the substance comprises bacteria, and wherein the material causes at least a portion of the bacteria to die.

19. The method of claim 18, wherein the bacteria comprises Gram-negative and Gram-positive bacteria, and wherein, the material causes at least a portion of the Gram-negative and Gram-positive bacteria to die.

20. The method of claim 17, wherein the substance comprises yeast, and wherein the material causes at least a portion of the yeast to die.

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