Nanoparticle‐based nanocomposite coatings with postprocessing for enhanced antimicrobial capacity of polymeric film

Bacterial adhesion and biofilm formation on surfaces pose a significant risk of microbial contamination and chronic diseases, leading to potential health complications. To mitigate this concern, the implementation of antibacterial coatings becomes paramount in reducing pathogen propagation on contaminated surfaces. To address this requirement, our study focuses on developing cost‐effective and sustainable methods using polymer composite coatings. Copper and titanium dioxide nanoparticles were used to assess their active antimicrobial functions. After coating the surface with nanoparticles, four different combinations of two postprocessing treatments were performed. Intense pulsed light was utilized to sinter the coatings further, and plasma etching was applied to manipulate the physical properties of the nanocomposite‐coated sheet surface. Bacterial viability was comparatively analyzed at four different time points (0, 30, 60, and 120 min) upon contact with the nanocomposite coatings. The samples with nanoparticle coatings and postprocessing treatments showed an above‐average 84.82% mortality rate at 30 min and an average of 89.77% mortality rate at 120 min of contact. In contrast, the control sample, without nanoparticle coatings and postprocessing treatments, showed a 95% microbe viability after 120 min of contact. Through this study, we gained critical insights into effective strategies for preventing the spread of microorganisms on high‐touch surfaces, thereby contributing to the advancement of sustainable antimicrobial coatings.


| INTRODUCTION
The pandemic outbreak of COVID-19 since January 2020 has highlighted the importance of sterilization for eliminating bacteria and viruses (Roy et al., 2020).Coronavirus, or SARS-CoV-2, and various other pathogens typically spread between individuals via respiratory droplets of varying sizes ejected from the mouths of those infected when speaking, coughing, or sneezing (Bhardwaj & Agrawal, 2020).Small droplets can float in the air, especially in poorly ventilated indoor environments, and may cause airborne transmission.Large droplets adhere to various surfaces in the spaces while falling to the ground by gravity (Bahl et al., 2022).There are severe public concerns about pathogen transmission via high-touched surfaces, such as transit handlebars, elevator button, food packaging, touchpads of automated teller machines at the bank, etc.In addition, SARS-CoV-2 has been found with significant viability ranging from 2 h to 9 days (Mallakpour et al., 2021).Applying an antimicrobial coating to frequently touched surfaces and infection-prone areas within public spaces has the potential to effectively reduce the transmission rate of pathogens.Approaches based on a polymeric material with embedded antimicrobial components, including metallic nanoparticles and organic compounds, have already been applied to various industries, including food processing, biotechnology, and life sciences (Jain et al., 2014;Mangaraj et al., 2019;Nagaraja et al., 2019).Although polymeric nanocomposite materials have been utilized by numerous studies and shown considerable growth, available technologies are still limited in terms of microbial inactivation, method of application, and life span, when processing the coating.Current coating technologies lack the essential properties critically needed for mitigating the spread of microbes.Over time, the efficacy of such coatings decreases and necessitates the repetition of the application for sustained antimicrobial action.Conventional coatings might be struggling with antimicrobial effectiveness or the difficulty of coating application.Therefore, coating technology to be developed in the future should have both simplicity and flexibility for diverse surfaces.
Antimicrobial coating refers to incorporating chemical agents on the surface to inhibit the growth of disease-causing microorganisms, such as bacteria, viruses, protozoans, and fungi.Until now, antimicrobial coatings were widely known for being applied to various medical devices, such as catheters, surgery curettes, and surgical instruments, to minimize the risk of infection during surgery or medical processes (Monteiro et al., 2009;Roe et al., 2008).
COVID-19 has naturally pushed antimicrobial coating technology to evolve, whereby copper coating has been highlighted to significantly reduce the virus and bacteria (Govind et al., 2021;Hutasoit et al., 2020;Yang et al., 2017).The research conducted by Munster et al. (2020) reported the survival time of SARS-CoV-2 can be reached up to 9 days and varies depending on various materials, including aerosols, cardboard, plastic, stainless steel, and copper.In contrast, viruses and bacteria remain only for up to 4 h on the surface treated by copper nanoparticles, suggesting the great potential use of copper for antimicrobial use.
Copper nanoparticles (Cu-NPs) are widely used in wood preservatives, catalysts, conductive inks, printable electronics, and even antimicrobial products (Abhinav K et al., 2015;Civardi et al., 2015;Din & Rehan, 2017).Moreover, Cu-NPs can heal wounds due to their high redox potential and superior antibacterial activity against a broad spectrum of bacterial species, such as grampositive and gram-negative bacteria, as well as viruses and fungi (Tiwari et al., 2014).The Cu-NPs are copper-based particles ranging in size from 1 to 100 nm (Li et al., 2007).Coating surfaces with Cu-NPs could offer a solution for sterilization due to copper's natural disinfectant properties.Copper oxide atoms weaken bacteria when they pull these electrons from the atoms that make up the cell walls.
Therefore, bacteria are quickly killed on the surface coated with Cu-NPs, and copper ions released from the surface are important in killing bacteria.
This study also included titanium dioxide nanoparticles (TiO 2 -NPs) as another type of nanoparticle.TiO 2 -NPs exhibit photocatalytic properties and are characterized by chemical stability, non-toxicity, and cost-effectiveness, which make them an attractive choice for antibacterial applications (de Dicastillo et al., 2020).Hence, TiO 2 -NPs are currently used in applications of antibacterial sanitization, deodorizing, wastewater treatment, and air purification (Haider et al., 2019).TiO 2 -NPs have three crystalline phases: anatase, rutile, and brookite (Mo & Ching, 1995).Among them, rutile is the most thermodynamically stable, and anatase and brookite are metastable.
Rutile has a tetragonal crystal structure similar to anatase but with longer axes and it is known for its excellent optical properties and high refractive index, making it widely used in various applications (Allen et al., 2018).In this study, we used TiO 2 Degussa P25 which contains a mixture of different crystal phases, including anatase and rutile to improve the photocatalytic activity.The bacterial killing mechanism of TiO 2 -NPs is the breakdown of cell walls and cytoplasmic membranes due to the production of reactive oxygen species (ROS), such as hydroxyl radicals and hydrogen peroxide (Wu et al., 2016).Furthermore, TiO 2 -NPs have been proven to carry antibacterial function when evaluated against Escherichia coli (E.coli), Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus (Khashan et al., 2021).
This study explored the combinations of postprocessing treatments with polymeric Cu-NPs and TiO 2 -NPs coatings for rapid inactivation of microbes on contact.Two postprocessing treatments were used to modify coating surfaces to identify their relationship with biocidal efficacy (Kudrna et al., 2022;Marković et al., 2020).First, we used intense pulse light (IPL) treatment that sinter the nanoparticles present in the coating, resulting in the formation of a more cohesive and dense structure.This leads to improved mechanical strength, adhesion, and durability of the nanocomposite-coated surface (Jang et al., 2021).Second, we used plasma etching, which involves exposing the coated surface to a plasma discharge.During plasma etching, the ions and reactive species in the plasma interact with the surface, causing the removal of surface material through chemical reactions (Naebe et al., 2022).
We selected E. coli as a model organism for testing the antimicrobial coating for food packaging industries in which E. coli bacterial contamination is a serious problem (Chawengkijwanich & Hayata, 2008;Huang et al., 2019).E. coli contamination occurs at various stages of the process, from the initial production of food products to their packaging and distribution.Additionally, E. coli is known to exhibit higher resistance to antimicrobial agents due to the presence of an outer membrane, making them a challenging and representative target for effective coatings.Therefore, five physical characterizations (fourier transform infrared [FTIR], scanning electron microscopy [SEM], coating transparency, surface roughness, and water wettability measurement) were explored to understand material and surface properties, which are essential components of optimal product and process.Collectively, the results were analyzed based on E. coli bacteria viability at four time points (0, 30, 60, and 120 min).

| Sample preparation
The nanomaterials-based coatings were prepared using compositions of Cu-NPs (TEKNA), formic acid (FA) (VWR International) and TiO 2 -NPs (Evonik), as listed in Table 1.The mean diameter of the utilized Cu-NPs measured at less than 100 nm (information provided by the manufacturer), and the average particle dimension for TiO 2 Degussa P25 typically approximates 25 nm.A total of 20 combinations of the coating composition and postprocessing treatment were obtained.
Ethylene-vinyl acetate (EVA) (VWR International) was used as the polymer matrix for the coatings (Zhao et al., 2016).First, a 5% wt/vol EVA solution (0.2 g) was prepared with methyl ethyl ketone (MEK, 1 g) (VWR International) as the solvent (Chou & Li, 2010).Cu-NPs (20 mg), FA (40 mg), and TiO 2 (20 mg) were then added to the EVA solution and mixed in a 5 mL vial.The polymer solution was kept at 70°C for 24 h, continuously stirring at 600 revolutions per minute.Once the solution was prepared, the subsequent process of making the coating sheet using a mechanical coater followed by postprocessing steps is shown in Figure 1.
Polyethylene terephthalate (PET) was used as the substrate for the coatings (Shlar et al., 2018).The PET sheet was attached to a hot plate, ensuring a flat surface.The hot plate was set to 70°C, facilitating smooth coating printing without agglomerations.Next, the polymer solution with nanoparticle suspension was dispensed onto the PET sheet using a transfer pipette along its width.A cylindrical coating bar wrapped with a 4 μm diameter wire was then pressed down onto the width of the PET sheet and pulled down along its length to create a coating with uniform thickness.This manual coating procedure was conducted on a rigid surface, resulting in uniform coatings without visually noticeable deformities.Subsequently, the PET sheets were allowed to cool down at room temperature for 24 h to cast the polymer solution.
Two postprocessing treatments were carried out to assess their relationship with biocidal efficacy, as shown in Figure 1.The first treatment used an IPL flash setup to sinter the nanoparticles on the surface of the coating, as described earlier (Ryu et al., 2011).The IPL flash samples were treated using an IPL system (S2300, XENON Corporation).The flash intensity was setup with 3 kV at the first stage and 2 kV at the second stage, and both stages pulsed for 3 ms.
The wavelength of the IPL system is from 500 to 1200 nm.Coated samples were placed underneath the Xenon lamp at its focal point at a measured distance of 5 cm.The exposed samples were removed after completely discharging the flash system.The second treatment involved plasma etching the surface of the coating to reduce oxides on the surface.A handheld corona charger (BD-20AC, Electro-Technic Products) was scanned across the surface of the coating while maintaining a separation of 1 cm from the surface of the layer.
The scanning rate was approximately 1 cm/s for six scans.A set of control samples without any postprocessing treatments was also prepared to compare the efficacy of postprocessing techniques.After finishing postprocessing, the formed coating samples were cut into disks for further characterization.

| Fourier transform infrared
The FTIR spectroscopy was conducted to delineate the functional groups present within the chemical composition of the T A B L E 1 All sample combinations between coating compositions and postprocessing (n = 3).

Sample no.
Coating | 509 nanocomposite coatings and the influence of the postprocessing treatments, utilizing the FTIR instrument (FTIR-620).Ten individual scans of the coated circular substrates were recorded across a wavenumber span ranging from 400 to 4000 cm −1 .

| Scanning electron microscopy
The SEM was employed to examine the surface characteristics of the nanocomposite coating samples in our study, and the images were captured using an SEM (Quanta FEG 250 FESEM, Thermofisher Scientific).Before imaging, the samples were sputter coated with a 10 nm thin layer of gold to enhance conductivity.The coated samples were mounted on 12.7 mm SEM stubs using conductive adhesive tape.The images were taken at both lower magnification (e.g., ×1000) to provide an overview of the sample's surface, and higher magnifications (e.g., ×30,000) to capture finer structural details.The accelerating voltage used during imaging was 10 kV, and the working distance was 10 mm.The choice of operating parameters ensured optimal imaging conditions while preserving the integrity of the coated samples.

| Coating transparency and surface roughness
The antimicrobial coating method developed in this study is intended to be applied directly to manufacture antimicrobial packaging products or attach to touch surfaces in public areas.Therefore, we focused on making antimicrobial coating sheets as transparent as possible.To test the transparency of samples, images were taken by placing samples on the university logo sheets to increase the clarity at a fixed height and under stable light conditions (C.Wang et al., 2017).
The surface roughness of the coatings was measured by a 3D optical profiler (Zeta-20, KLA Co.) to assess the relationship between surface roughness and antimicrobial efficiency (Abbott & Zhu, 2019).
Roughness is expressed in Sa (arithmetical mean of the surface), which is the extension of Ra (arithmetical mean of the line) to a surface.The Sa parameter indicates the difference in the height of each point compared to the arithmetical mean of the surface, and it is generally used to evaluate surface roughness (Charles et al., 2019).
Each sample was mounted on the microscope platform and fastened flush with the platform tray.The lens was set to ×20 magnification, and the focus was adjusted using controls in the software.A z-stack mode was used for imaging the samples to obtain images spread at equal intervals in the specified z-axis range.The images were combined with the manufacturer-provided software to generate a 3D topography of the coating sample surface.Three distinct regions were selected in a single coated sample, and three surface roughness were averaged to obtain a single surface roughness value.

| Water wettability measurement
The water wettability of the coated samples was measured by determining the static contact angle of droplets formed on surfaces (Edachery et al., 2021).Contact angles were optically measured by using a goniometer (SmartDrop, Femtobiomed).The contact angle, θ (theta), is a quantitative measure of the wetting of a solid by a liquid Schematic of the coating sample preparation and postprocessing steps.(Huhtamäki et al., 2018).The contact angle is geometrically defined as the angle formed by a liquid at the three-phase boundary where a liquid, gas, and solid intersect.The sample disk was placed on the goniometer's level surface, and a computer-controlled lever dropped 5 μL of water out of a syringe and onto the surface of the disk.The goniometer's camera captured a zoomed-in image of the water droplet and numerically calculated the droplet's right and left contact angles.

| Antibacterial activity testing
For antimicrobial efficiency, the percentage of bacterial viability for coated samples was measured and calculated using ImageJ (1)

| Statistical analysis
The two chosen independent variables were coating composition and postprocessing treatments, whereas surface roughness, contact angle, and bacteria viability were appointed as dependent variables, respectively.Therefore, a two-way analysis of variance (ANOVA) test was carried out to compare each dependent variable based on two independent variables, such as coating compositions and postprocessing treatments.Normality tests were carried out to ensure that variables followed a normal distribution, and constant variance of the error terms and independence were confirmed through randomization.Also, a 95% confidence interval was used in this study, as shown in Figure S2.An F-value and a p-value were determined using Minitab Statistical Software (Minitab, LLC).If there was a significant difference in both main effects and interaction effects, the data were analyzed using multiple means comparison for interaction between coating composition and postprocessing treatments.Tukey's test was used to compare the difference between each pair of means since the lowest experimental error was used.
This test will determine which means amongst a set of means are different from the rest.In addition, grouping information was carried out to determine whether the mean difference between any pair of groups is statistically significant.The grouping contains letters that group the factor levels, herein groups that do not share a letter have a statistically significant mean difference.| 511 incorporated into the TiO 2 , a discernable reduction in the intensity of the 2918.39cm −1 C-H stretching peak is apparent, while the peaks at 1010 cm −1 (C-O-C) and 714 cm −1 (C-H) exhibit an amplification in their intensity.In addition, with postprocessing treatment and the presence of nanoparticles, the peaks of the carboxyl group range of 1700, 2900, and 1230 cm −1 and the amino group of 1650 cm −1 was very prominent.High intensity of carboxyl and amino groups would affect the antimicrobial efficacy since when carboxyl or amino groups come into contact with an antimicrobial agent, the interaction with the bacterial cell membrane is improved (Kalachyova et al., 2017).
Figure S4 illustrates the impact of coating composition and postprocessing on antimicrobial performance and surface characteristics.In terms of surface roughness, it is observed that nanoparticles disperse more evenly in the TiO 2 Control sample (resulting in the highest biocidal efficacy) (Figure S4A).On the other hand, the Cu Corona sample (resulting in the lowest biocidal efficacy) shows a bumpier surface due to irregular dispersion (Figure S4B).This indicates an inverse relationship between surface roughness and antimicrobial efficacy (Allion et al., 2006;Bagherifard et al., 2015a).
The TiO 2 + Cu Flash sample displays the largest contact angle of 137.94 degrees, indicating a hydrophobic characteristic of the coating surface (Figure S4C).Conversely, the Cu+FA FlashCorona sample exhibits hydrophilic characteristics, as evidenced by welldispersed nanoparticles and a relatively small contact angle of 61.28 degrees (Figure S4D).
The intended application for the nanocomposite coatings developed in this study is for frequent contact surfaces in public and food packaging with ambient environments.The PET used is acknowledged for its thermal stability and notable attributes, encompassing commendable mechanical strength, toughness, and a discernible melting temperature, typically in the range of 270°C (Huang et al., 2019;Kawai et al., 2020).Thermal stability characterization with thermal gravimetrical analysis is more relevant to the applications for extreme environments, which may be unnecessary for the intended application at room temperature.

| Transparency
A small disk diameter of about 1.5 cm for all combinations between coating compositions and postprocessing treatments was prepared, This was a result of the sintering of the Cu-NPs.However, the effect was enhanced by the presence of TiO 2 -NPs, which are known to act as visible light photocatalysts (Nakata & Fujishima, 2012).The application of corona discharge did not introduce any visible changes in the coatings.

| Surface roughness
As shown in Figure 3a, the postprocessing treatments affected the surface morphology change of the coated samples due to the localized effect of the nanoparticles.As shown in Figure 3b, most Cu-NP-coated samples with postprocessing treatments increased the surface roughness of the coatings compared to the no-nanoparticle samples.The results from the two-way ANOVA indicate that coating composition and postprocessing treatment, respectively, were significant factors (p < 0.05) for the surface roughness (Table 2).Also, the coating composition and postprocessing treatments had significant interaction effects.Table 3, Table S1 (reorganized based on postprocessing treatments), and Table S2 (reorganized based on coating composition) show the multiple means comparison for twoway interaction effects using Tukey's test to compare the difference between each interaction.
As shown in Table S1, the interaction effect between the Cu-NPs and IPL flash (letters with red color) is generally statistically significant on the surface roughness increment.This is attributed to the fact that the IPL flash irradiation caused localized heating on the Cu-NPs by translating the IPL energy into thermal energy.Also, the IPL irradiation on the coatings resulted in the formation of pores in the polymer layer.However, the samples discharged by corona on the surface did not cause significant changes in the coatings' porosity compared to those under IPL flash.This is expected as, in this study, IPL irradiation was more energy-intensive than the corona discharge.
IPL irradiation did not cause significant warping of the samples.This implies the polymer melting was a localized phenomenon and was possibly assisted by various nanoparticles, which may have absorbed the energy from the IPL and partially imparted it to the surrounding polymer.
The antimicrobial efficacy of metal ion-based coatings is an area of significant interest, but it can sometimes be limited by the hindered transport of the encapsulated metal ions through the polymer layer to reach adherent surface microbes.This phenomenon can be attributed to the nature of the polymer matrix, which may act as a barrier, impeding the efficient release and diffusion of the metal ions.However, emerging research suggests that introducing pores within the coating structure can address this limitation and enhance the transportation of metal ions to the surface.Studies conducted by (P.Wang et al., 2018), have demonstrated that the presence of pores in the coating facilitates the controlled release of metal ions, enabling them to effectively interact with and combat adherent surface microbes.Incorporating pores into the coating creates a more dynamic environment, allowing for improved metal ion diffusion and mobility.These pores serve as pathways through which the metal Transparency of coating sheets according to the coating compositions and postprocessing treatments.Scale bar = 5 mm.The blue dots in some images were marked to differentiate the surface direction.
ions can permeate the coating matrix and access the surface, thereby bolstering the antimicrobial action of the coating.
The coating with only TiO 2 -NPs and the postprocessing treatments did not significantly affect the surface roughness increment, as shown in Figure 3, Table 2, and Table S1 (letters with blue color).In addition, the influence of corona treatment was dependent on the composition of nanomaterials and additives used in the coatings.This can be attributed to the dangling terminal bonds generated by corona treatment and their interaction with the embedded nanomaterial and chemical species.In the presence of Cu-NPs, strong interaction with corona charge treatment was observed, where a statistically significant increase in surface roughness was obtained compared to untreated coatings (Figure 3b and Table 3).
Nanoscale surface roughness is a critical factor that significantly influences bacterial adhesion and microcolony formation (Gharechahi et al., 2012).The relationship between surface roughness and bacterial adhesion has been extensively studied (Preedy et al., 2014).Some scholars have argued that increasing surface roughness leads to more bacterial adhesion (Sharma et al., 2016;Truong et al., 2010).In contrast, others have stated that surface roughness is not correlated with bacterial adhesion and that increasing surface roughness decreases the number of bacteria (Bagherifard et al., 2015a;Liu et al., 2016).This is because surface roughness allows biophysical modifications of the material surface to increase or reduce bacterial adhesion, depending on the bacterium types or treatment materials.
It is important to recognize that the impact of surface of roughness on bacterial adhesion is highly context-dependent and can vary depending on the type of bacteria and the properties of the materials used.In light of these complexities, understanding the biophysical interactions between bacteria and surfaces with different roughness levels is crucial for interpreting the implications of altered surface roughness on bacterial adhesion and growth (Sharma et al., 2016).

| Contact angle analysis
The contact angle evaluation with distilled water revealed the influence of the postprocessing methods employed.The control material, a standard PET sheet, was hydrophilic.Both postprocessing methods altered the surface roughness, which, in turn, manipulated the surface energy of the coatings.The results from the two-way ANOVA indicate that coating composition and postprocessing treatment, respectively, were significant factors (p < 0.05) for the contact angles (Table 4).Also, the coating composition and postprocessing treatments had significant interaction effects.
Table 5, Table S3 (reorganized based on postprocessing treatments), and Table S4 (reorganized based on coating composition) show the multiple means comparison for two-way interaction effects using Tukey's test to compare the difference between each interaction.
The combination of the TiO 2 + Cu × Flash shows the highest contact angle value, indicating the surface has low wetting; therefore, the liquid droplet did not spread much onto the surface (Table 5 and Figure 4a).
As shown in Table S3, upon treatment with a corona charger, the contact angle decreased (letters with red color), forming a more hydrophilic surface when compared with the contact angles with no postprocessing (Figure 4a,b).Irrespective of the surface roughness and the composition of the coatings, corona charge treatment resulted in a decrease in the contact angle for the coatings.This was expected due to dangling hydroxyl groups on the coating surface.In contrast, IPL flashing treatment typically increased the contact angle, forming hydrophobic surfaces as shown in Table S3 T A B L E 2 Analysis of variance of coating compositions and postprocessing treatments for surface roughness (n = 5).(letters with blue color).This can be attributed to the formation of air pockets on rough surfaces.However, the samples coated with Cu-NPs and Cu+FA showed low contact angles because copper is naturally hydrophilic, with less than 90 degrees of liquid contact angles (letter with green color in Table S3).In addition, a threshold surface roughness can be expected, which governs the hydrophilicity and hydrophobicity of the surface of the coatings in some cases.The coated samples with TiO 2 -NPs exhibited hydrophobic surfaces despite low surface roughness.A net positive charge indicated by TiO 2 -NPs could be a possible reason behind this observation.
The hydrophobic or hydrophilic characteristics of bacteria are influenced by the structure and residues on the cell surface (Maikranz et al., 2020).The hydrophobic or hydrophilic surfaces are desired depending on the type of target microbes (Bayoudh et al., 2006).E.
coli in this study favor hydrophilic surfaces for their growth and have been reported to be hydrophilic (Sepehrnia et al., 2023).

| Antimicrobial effect
The antimicrobial assessment of fabricated nanoparticle coatings was performed with E. coli following a standard antibacterial test method.
In Figure 5a, the green channel shows the entire population of bacteria stained with DMAO (5 mM in DMSO) and the red channel shows the dead bacteria selectively stained with EthD-III (2 mM in DMSO/H 2 O).After loading the uniform bacteria solution on the surface of the coated samples, the viability of E. coli was measured at four-time points of 0, 30, 60, and 120 min, as shown in Figure 5a.The number of bacteria gradually increased from the start to 120 min, showing an average survival rate of 94.44% (Figure 5b).When the antimicrobial activity was characterized with a standard PET sheet, which is the substrate used for the coatings without either coating compositions or postprocessing treatments, it showed exponential growth of E. coli over time, up to 95.62% at 120 min.
The random combination images were chosen to show bacteria live and dead assay results under a fluorescence microscope at different time points (30 and 120 min), as shown in Figure 5c,d.In Figure 5e, bacteria viability rapidly decreased until 30 min, which is the first measured timepoint, in all combinations and continued to 120 min.Although there seems to be a degree of difference, it was found that most of them had similar effects.
Statistical analysis performed the combinations that showed the best biocidal performance in the quickest time of 30 min and the most long-lasting of 120 min within the experimental conditions.In Table 6, there was statistical significance in the main and interaction T A B L E 4 Analysis of coating compositions and postprocessing treatments for contact angle (n = 5).T A B L E 5 Tukey pairwise comparisons for coating compositions and postprocessing treatments (n = 5) interaction effect on contact angle.S5) and based on coating compositions (Table S6) are prepared.
In Table 8, there was a statistically significant interaction between coating composition and postprocessing in biocidal effectiveness in 120 min.Therefore, multiple mean comparisons were performed in the same way as mentioned above.In  established.Considering the control samples had a 5% mortality rate, the effect of nanoparticles and postprocessing was strongly supported.However, the results were not correlated with the roughness and wettability of coating and postprocessing combinations.To promote a better understanding, results of Tukey pairwise comparisons for the interaction effect based on postprocessing treatments (Table S7) and based on coating compositions (Table S8) are prepared.
Coatings with increased hydrophilicity provided a suitable surface for bacteria adhesion and growth while improving the interaction between the ROS and ionic species with the microbes in the water droplet.This increased microbicidal action, evidenced by the high bacteria, kills the efficiency of corona-treated coatings.
Usually, the antimicrobial action of metal ion-based coatings is limited due to the hindered transport of the encapsulated metal ions through the polymer layer to reach the adherent microbes on the surface.The postprocessing methods, such as IPL flashing and corona treatment, increased surface roughness and facilitated the transport of ionic species and ROS (Jeong et al., 2021).Overall, with the developed coating compositions and the postprocessing methods, antibacterial coatings with different microbicidal mechanisms were achieved with the capability to kill microbes quickly on contact.This observation can be ascribed to the photocatalytic behavior of TiO 2 -NPs in the presence of visible light, which results in the generation of a higher concentration of ROS.Applying various postprocessing treatments altered the surface characteristics of the coatings, as described in the previous sections.These changes further influenced the microbicidal activity of the coatings.As the hydrophobic coating surfaces prevented the adhesion of bacteria, the microbicidal effect was achieved through two different modes.This microbe population was exposed to various ionic and free-radical species generated in the coatings to cause damage to the cell components.
The most common strategy for developing antimicrobial surfaces is embedding antibiotics.Many studies and reviews by Kaur and Liu have reported quaternary ammonium or other biocidal compounds grafted on polymeric chains (Kaur & Liu, 2016).A common disadvantage of such an approach is its biocidal mechanism.The quaternary ammonium compounds act by denaturing the proteins in bacteria to cause cell contents to leak out of the membrane and eventually kill the bacteria.The biocide's antimicrobial enzymes disrupt cellular functions, eventually killing the bacteria.Surfacebound radical-generating species have been extensively explored in the past decade and present a more robust contact-kill approach (Chouirfa et al., 2019).The radical generation is either continual or triggered by stimulants such as pH, light, or heat (Yue et al., 2016).
Several organic compounds and inorganic nanomaterials have been studied to form contact-kill antibacterial surfaces.However, significant limitations associated with this approach include a limited supply of the radical-generating compounds and the generated radical species available for interacting with the bacteria cells.Surface wear T A B L E 6 Analysis of variance of coating compositions and postprocessing treatments for bacteria viability at 30 min (n = 3).T A B L E 7 Tukey pairwise comparisons for the interaction effect of coating compositions and postprocessing treatments on bacteria viability at 30 min (n = 3).In this study, coated samples were prepared with a combination of Cu-NPs, FA, and TiO 2 -NPs, and IPL flash and plasma etching were performed as postprocessing treatments on the prepared samples.
The first process entailed sintering nanoparticles on the coating surface using IPL flash, significantly enhancing the antibacterial effect T A B L E 8 Analysis of variance of coating compositions and postprocessing treatments for bacterial viability at 120 min (n = 3).T A B L E 9 Tukey pairwise comparisons for the interaction effect of coating compositions and postprocessing treatments on bacterial viability at 120 min (n = 3).

(
National Institutes of Health).First, a concentrated bacteria solution was incubated with a live (DMAO)/dead (EthD-III) (Biotium) assay for 15 min at 37°C.DMAO is a bright green fluorescent nucleic acid dye that stains both live and dead bacteria (Weisman et al., 2015).EthD-III is a red fluorescent nucleic acid dye that stains only dead bacteria with damaged cell membranes.Then, the 3 μL of stained bacterial broth was dispensed on the coated samples to cover the entire disk surface by mounting glass coverslips uniformly.Covering the bacteria solutions by fixing them with glass coverslips prevented bacteria from flowing and drying out while testing antibacterial efficacy.Next, the stained samples were imaged under a fluorescence microscope at ×100 magnification with an enhanced green fluorescent protein (EGFP) (green) and mCherry (red) channels.The live (Nlive) and dead (Ndead) bacteria counts were obtained by processing captured images in ImageJ using the cell counter plugin, and the bacteria viability was calculated using Equation (1).It should be noted that the green channel showed both dead and alive bacteria stained by DMAO.The red channel shows dead bacteria selectively stained by EthD-III, as shown in Figure S1.TiO 2 -NPs especially have been widely used for photocatalytic antibacterial applications.Antibacterial activity testing was performed under light illumination to assess the antibacterial efficacy of TiO 2 -NP-coated samples.

3
Figure S3 is provided to elucidate the existence and efficiency of coating composition and postprocessing treatments, aiming to establish their correlation with antimicrobial efficacy in this research study.The control samples without postprocessing treatment refer to a substance solely coated with EVA on a PET sheet.The distinct peak at 2918.39 cm −1 (C-H) stretching frequency on our EVA-coated samples provided evidence for forming EVA in Figure S3A (Jiang et al., 2017).Additionally, our sample has confirmed the presence of peaks at 1732.81 cm −1 (C = O) and 1235.72 cm −1 (C-O-C) in the no nanoparticle without postprocessing treatment samples (noted as control-control in the graph).Next, control samples treated with the corona discharge resulted in the emergence of specific peaks, including a peak at 3272.28 cm −1 (OH) and another at 848 cm −1(bending C = O inorganic carbonate).Following corona discharge treatment, the broadening of peaks at around 1732, 1235, and 1022 cm −1 was observed.This broadening corresponds to the presence of carboxylic acid, carbon-carbon double bonds, aromatic ether, amino and nitrous acid ester moieties, indicative of intricate reactions that occur during the corona discharge processing(Ding et al., 2014).In FigureS3B, the copper nanoparticle-coated sample exhibits functional groups similar to prior studies conducted by(Betancourt- Galindo et al., 2014;Das et al., 2020).Specifically, pronounced spectral features emerge at 1700 cm −1 , indicative of the presence of carbonyl (C = O) groups, within the copper-coated samples.Next, the sample coated with TiO 2 nanoparticles displays distinctive spectral characteristics at 714 cm −1 , representing the presence of methylene (C-H) groups, and at 1010 cm −1 , indicating the existence of ether (C-O-C) groups, as corroborated by(Ghann et al., 2017;Madani et al., 2017).In the case of the sample where copper was shown in Figure 2. The camera focused on the disk under constant illumination while acquiring image data.Most samples retained their optical transparency after corona discharge treatments.Coatings with Cu-NPs appeared to have a blackish-brown tint while not compromising the transparency completely.The blackish-brown color indicated the formation of CuO from the Cu-NPs in the coating upon exposure to the ambient atmosphere.The transparency of the coating is an essential factor for various industrial applications.In particular, the transparent layer can be used in cases where the contents inside should be seen, such as food packaging(Thuy et al., 2021).Most samples' transparency did not change significantly with the combined corona discharge and the IPL flash operation compared to the transparency only with the IPL flash operation.The Cu-NPs combined with FA appeared blackish brown like the Cu-NPs-only samples.The color of the coatings intensified and turned dark brown after IPL irradiation.The IPL also caused the sintering of Cu-NPs, which, in turn, resulted in a translucent appearance of the coatings.Copper can react with FA to form copper formate, which imparts a brown color to the coatings(Motokura et al., 2012).The IPL further causes a reduction of Cu2+   to Cu and results in the formation of sintered Cu-NPs, which should maintain the brown color of the coatings.However, IPL irradiation also causes localized melting of the coating polymer poly-EVA and forms microscale pores.Due to the increased porosity of the polymer coating, diffusion-controlled oxidation of Cu-NPs takes place over time to generate Cu 1+ or Cu 2+ oxides and causes the coating color to darken.The TiO 2 -NPs coating samples exhibited an off-white color preand postprocessing(Weir et al., 2012).These coatings had high transparency but were slightly lower than the control (EVA only) coating samples.In contrast to Cu-NPs only and Cu-NPs+FA coating samples, IPL irradiation of TiO 2 -NPs coatings did not affect their transparency.In the case of the TiO 2 -NPs coatings, the TiO 2 -NPs were not sintered by IPL; therefore, their transparency was not changed.The inclusion of corona discharge in postprocessing did not introduce any visible changes in the coatings.On the other hand, the combination of Cu and TiO 2 -NPs coating demonstrated a similar behavior as Cu-NPs-only coating.Without any postprocessing, the TiO 2 + Cu-NPs coatings had an off-white color with high transparency, similar to TiO 2 -NPs-only coatings.Upon IPL irradiation, the appearance of the coatings changed drastically into opaque coatings.

F
I G U R E 3 Surface roughness after coating with the coating compositions and postprocessing treatments.(a) Isometric view of the Characterization.Three white boxes in the image for no nanoparticle with corona treatment were included to show the data collection location.Scale bar = 100 μm.(b) The plot of roughness values with materials and postprocessing treatments.It should be noted that the roughness value is zero for no nanoparticle with no postprocessing treatment.
effects of the coating composition and postprocessing concerning the biocidal effectiveness of various combinations.Multiple means comparison using Tukey's method was carried out to find out which means of combinations are statistically different.There was no statistically significant difference in biocidal performance between the 12 combinations from TiO 2 Control to Cu Flash in Table7because they all share a letter A in the grouping result.The Cu Flash showed the lowest level (82.8%) in A grouping.The overall lowest F I G U R E 4 Contact angle after coating with the coating compositions and postprocessing treatments.(a) Surface wettability of Characterization.Scale bar = 100 μm (b) Plot of contact angle values with different compositions and postprocessing treatments.effect at 30 min showing 64.32% biocidal performance was Cu Corona.To promote a better understanding, results of Tukey pairwise comparisons for the interaction effect based on postprocessing treatments (Table

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I G U R E 5 (a) Bacterial growth on the samples of no coating compositions without postprocessing treatments.No coating compositions with no postprocessing; 0, 30, 60, and 120 min.Scale bar = 10 μm, (b) Bacteria viability (%) of control samples, which is no coating compositions and no postprocessing treatments (n = 3), (c) Resulting in antimicrobial effects.Representative fluorescent microscopy images of bacteria live/dead assay of TiO 2 + Cu Control at 30 min.Scale bar = 10 μm, (d) TiO 2 + Cu Flash to Corona at 120 min, and (e) Bacterial viability (%) of each timepoint for various nanocomposite coating samples (n = 3).
The Cu-TiO 2 composition shifts the peak absorption of TiO 2 towards the visible light spectrum and close to 400 nm.This results in the generation of additional ROS when the visible light is irradiated on these coatings, making them suitable for prolonged disinfection in the presence of sunlight or artificial illumination sources.Therefore, we expected that combining the Cu and TiO 2 -NPs coatings would have a synergistic effect in killing the bacteria because most Cu-NPs exist in oxides, and a small fraction of Cu acts as a dopant for TiO 2 .However, no clear evidence of the synergistic effect was observed.4| CONCLUSIONSThe unexpected emergence of coronavirus disease has accelerated the need for research on antimicrobial coatings.Simultaneously, the demand for establishing a sustainable and cost-effective antimicrobial method to complement existing disinfection and prevention protocols across various industries has become imperative.In this study, we harnessed the potential of Cu and TiO 2 -NPs as coating nanocomposites, both well-established for their natural antibacterial function.Cu exhibits a microscopic effect known as oligodynamic action, rendering it naturally adept at sterilizing viruses, microorganisms, and mold.TiO 2 can create free oxygen radicals that attack anything on its surface when exposed to light by breaking down water vapor in the air.When bacteria or fungi come into contact with surfaces treated with Cu or TiO 2 , these coating molecules impede microbial metabolism, leading to the demise of bacteria and viruses within a few hours.
by converting the copper oxide into pure copper.The second method was a plasma etching performed while scanning the surface with a handheld corona charger to remove any oxides from the surface.The corona charge treatment increased the coating's antibacterial effect by simultaneously reducing the contact angle and increasing the surface charge.Following the sample preparation, five types of physical characterizations were performed: FTIR, SEM, coating transparency, surface roughness imaging, and wettability measurement.In addition, an antibacterial test was demonstrated to validate the successful antibacterial effectiveness of the developed coating method.The results revealed an average mortality rate of 84.82% in 30 min and an average mortality rate of 89.77% in 120 min, providing compelling evidence of its sustainable antimicrobial efficacy.Antimicrobial experiments would further investigate more types of bacteria, including phages, and broaden their generalization and utilization against other unknown viruses.

Table 9 ,
there is no statistically significant difference in biocidal effectiveness between the 12 combinations of Cu Flash and TiO 2 Control.Overall, in 30 min, an average of 84.82% mortality rate was shown in the biocidal performance with nanoparticle coatings and postprocessing treatment, and in 120 min, an average of 89.77% mortality rate was Because these nanomaterials are embedded in the polymer, ROS generation is the dominant process.In the case of Cu-NPs and FA composition, IPL irradiation converts Cu-formate to Cu, which undergoes diffusionlimited oxidation to form Cu 1+ or Cu 2+ species.The primary action of Cu 1+ species is to form complexes with the cell membrane proteins to cause denaturation membrane rupture.TiO 2 -NPs have been reported to have photocatalytic activity, generating ROS when light exposure.
In contrast, the developed coatings in this study resulted in bactericidal action through different mechanisms.The Cu-NPs coated disks contained an oxidized form of Cu in the Cu 2+ state, although a minor proportion of Cu 1+ could have also been present.Cu 2+ species can contribute to ROS generation or enter cells to denature proteins and disrupt cellular functions.
Note: The grouping information is acquired using the Tukey test at a 95% confidence level, and they are significantly different if the grouping does not share a letter.Abbreviations: Cu-NP, copper nanoparticle; IPL, intense pulse light; TiO 2 -NP, titanium dioxide nanoparticles.