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. 2023 Sep 14;8(38):34919–34927. doi: 10.1021/acsomega.3c04303

Enhanced Antibacterial Activity at Ag–Cu Nanojunctions: Unveiling the Mechanism with Simple Surfaces of CuNPs-on-Ag Films

Weerapat Li †, Supphanat Anantachaisophon †, Thanakrit Vachiraanun †, Worachon Promchaisri †, Pongpop Sangsawang †, Pattarapon Tanalikhit ‡, Somlak Ittisanronnachai §, Thassanant Atithep §, Passapan Sanguanchua †, Arjaree Ratanasangsathien †, Mathus Jirapunyawong †, Siriporn Suntiworapong ∥, Sakol Warintaraporn †,*, Yutichai Mueanngern †,*
PMCID: PMC10536021  PMID: 37779963

Abstract

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Deposition of CuNPs on silver film gives rise to the formation of active Ag–Cu interfaces leading to dramatic enhancements in antibacterial activity against Escherichia coli. Transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDAX) analyses reveal that CuNPs are covered in a thin Cu2O shell, while X-ray photoelectron spectroscopy measurements (XPS) reveal that the Ag film samples contain significant amounts of Ag2O. XPS analyses show that the deposition of CuNPs on Ag films leads to the formation of a photoactive Ag2O–Cu2O heterostructure. Following a Z-scheme mechanism, electrons from the conduction band of Ag2O recombine with photogenerated holes from the valence band of Cu2O. Consequently, electrons at Cu2O’s conduction band render Cu reduced and cause reductive activation of surface oxygen species on Cu forming reactive oxygen species (ROS). Interaction between metallic Cu and ROS species leads to the formation of a Cu(OH)2 phase. Both ROS and Cu(OH)2 species have previously been reported to lead to enhanced antibacterial properties. Holes on Ag2O produce a highly oxidized AgO phase, a phase reported to exhibit excellent antibacterial properties. Quantitative analysis of Cu and Ag high-resolution X-ray photoelectron spectroscopy (HR-XPS) spectra directly reveals several-fold increases in these active phases in full agreement with the observed increase in antibacterial activities. This study provides insight and surface design parameters by elucidating the important roles of Ag and Cu’s bifunctionality as active antibacterial materials.

Introduction

Antibacterial surfaces have garnered much interest in recent years, particularly during the COVID-19 pandemic.1−4 Metal nanoparticles are commonly used in antibacterial surfaces due to their high surface area/size ratio allowing for intimate contact with bacterial cell walls.5,6 Mechanisms behind these antibacterial surfaces range from the formation of reactive oxygen species (ROS) catalyzed by metal surfaces, as well as the release of metal ions resulting in the disruption of ATP production as well as cell wall impairment.7,8 Ag and Cu metal nanoparticles are commonly used for such applications.8−13 In addition to their small penetrable size and oxidative tendency, dissociated ions of both Ag and Cu also exhibit strong affinities toward the thiol groups present on cell walls. This suggests another potential antibacterial mechanism: the interaction of copper and silver ions with the thiol groups found in proteins, which are crucial for enzymatic activity, leading to the inactivation of enzymes. It has been reported that heavy metals react with proteins by combining with thiol groups, which leads to the inactivation of the proteins.14−16 Although the mechanism detailed above is generally accepted in the scientific community, a comprehensive understanding of the driving force behind the release of these ions, which may stem from a combination of structural and electronic properties intrinsic to each metal, remains elusive.17,18 In addition, different types of metal phases have also been reported to exhibit very high antibacterial activity. Reports from Clavier et al. and Karkhanechi et al. indicate that Cu(OH)2 can also act as a highly active antibacterial phase, although claimed origins of this observed effect vary widely from increased Cu ion release to release of surface OH radicals from Cu sites.19,20

Several studies have reported strong interactions across two metal phases leading to enhanced catalytic and antibacterial activity.21−23 Peridakaki et al. reported significantly increased antibacterial activity when Ag/Cu hybrid structures on graphene oxide supports were evaluated against Escherichia coli (E. coli). This was attributed to structural interactions between the Ag and the Cu phases.24 In studies conducted by Hans et al., inductively coupled plasma mass spectrometry (ICP-MS) data reveals a markedly higher release of Cu ions when Ag–Cu alloys are used as antibacterial surfaces as compared to monometallic systems of Ag and Cu alone.25 Another study by Bankier et al. suggested that the synergistic effect between Cu and Ag arises from Cu’s ability to perforate holes in bacterial cell membranes, allowing for Ag to efficiently enter the cell—subsequently damaging cell interior and sabotaging cell division.21 Other types of synergistic effects have also been observed, including those that stem from metal/metal oxide and metal-oxide/metal-oxide systems.26−28 On metal–metal oxide systems, core–shell nanoparticles of Ag@Cu2O have been reported to lead to the formation of a heterostructure at the interface of the two materials allowing for efficient electron–hole pair separation.29 Density functional theory (DFT) calculations by Feng et al. have shown that photoinduced charge transfer can occur when the Fermi energy of Ag lies close to the conduction band minimum of Cu2O, leading to increased antibacterial activity.30 Study on metal oxide/metal oxide system of Ag2O–Cu2O heterostructure by Guleria et al. also reports enhanced performance in dye degradation reactions arising from the formation of reactive hydroxyl and oxygen radicals—which can also have drastic effects on antibacterial activities.31 Even though these studies provide strong evidence that the amplified antibacterial activity is due to strong interactions between Ag and Cu, no surface spectroscopic study has been carried out on simple colloid-thin film model surfaces in order to investigate the origins of these effects.

Recent advances in nanofabrication, colloid synthesis, and lithography have allowed for the fabrication of well-controlled planar surfaces. The ability to control morphological parameters at the nanoscale has led to the widespread use of planar surface systems in studying different types of surface chemical phenomena. These so-called “model surfaces” can be systematically controlled to affect different catalytic and/or antibacterial trends.32−35 These trends can be used to complement existing proposed mechanisms and bridge the gap between experimental observables and computational results. Effects arising from the bifunctionality of two materials are known as strong metal–support interactions (SMSI), usually induced by strong bonding interactions between two different material phases. These interactions can be induced by alloying or exposing two materials to reactive gases at elevated temperatures. However, several reports have demonstrated that mere deposition of colloidal structures on thin film supports can lead to robust bonding interactions, resulting in changes to material activity.36−39 To further investigate the effects of the Ag–Cu interface, we synthesized the Cu nanoparticles (CuNPs) via a robust-simple method and deposited the particles via drop-casting on electron-beam Ag silver thin films and tested the samples against E. coli. Gram-negative E. coli strains were chosen due to their thinner cell walls, which would allow for better interaction with the antibacterial surfaces. As CuNPs utilized in the majority of antibacterial applications are often inconsistent in structural morphologies, we decided to adjust the amount of PVP accordingly to emulate the structural characteristics of these nanoparticles while maintaining uniform particle size distributions.40 We show that the presence of newly formed Ag–Cu interfaces leads to significant enhancement in antibacterial activity relative to Cu and Ag controls. X-ray photoelectron spectroscopy (XPS) studies reveal that Cu exists as Cu2O prior to deposition on Ag film substrates forming Cu2O–Ag2O interfaces. Upon contact with Ag, the Cu 2p spectra begin to exhibit a much stronger metallic character, whereas Ag 3d becomes noticeably more oxidized. The antibacterial mechanisms can be explained by a charge transfer process from the conduction band of Ag2O, under visible light illumination, to Cu2O, which in turn reduces metal surface sites at the Cu phase and diminishes oxygen species near the metal surface forming ROS which are reported to be highly antibacterial. The origins of the observed surface Cu(OH)2 phase, as detected via XPS measurements, could be attributed to reactions of these ROS species with metallic copper. Additionally, the charge transfer process from Ag2O to Cu2O generates a highly oxidized AgO phase, which has been shown in previous studies to have excellent antibacterial properties. We propose that Cu, Cu(OH)2, and AgO are the active phases and the formation of these phases is due to a charge transfer process between the interface of the two materials.

Methods

Surface Preparation

Silver Thin Film Deposition

Silver films were deposited onto a silicon wafer substrate via thermal evaporation deposition using a Kurt J. Lesker system, operating at a deposition rate of 0.5–0.8 nm/s at a base pressure of 5.0 × 10–5 torr. The silver film was deposited at a thickness of 100 nm, the deposition rate was monitored using a quartz crystal microbalance. Individual samples, approximately 0.5 cm × 0.5 cm in size, were diced with a diamond head to obtain suitable samples for Cu deposition.

CuNPs and Cu2O Nanocubes Synthesis40

CuNPs were synthesized by following a previously reported protocol with slight modification. Solutions of poly(vinylpyrrolidone) (PVP) (0.30 M, 3 mL) in anhydrous ethylene glycol (EG), CuSO4·5H2O (0.10 M, 3 mL) in EG, and ascorbic acid (0.25 M, 3 mL) in EG were prepared. The PVP solution was stirred with a magnetic stirrer and was heated to 140 °C in two-neck round-bottom flasks. Solutions of CuSO4·5H2O and ascorbic acid were injected simultaneously, dropwise, into the hot PVP solution at a rate of approx. 0.25 mL/min. The solution was heated and stirred for an hour. Once the solution turned reddish-brown, it was cooled to 80 °C and allowed to age for approx. 1 h. The reaction system was cooled to room temperature, then it was diluted with ethanol and centrifuged (2000 rpm, 15 min) six times. The nanoparticles were deposited by directly drop-casting the solution in ethanol on 0.5 cm × 0.5 cm Ag film samples.

Cu2O nanocubes were synthesized by mixing 0.0576 g of sodium dodecyl sulfate and 8410 μL of 0.1 M CuSO4 with continuous stirring. After 5 min, 40 μL of a 1 M NaOH solution and 1450 μL of a 0.2 M sodium ascorbate solution were added, and the solution was left to stir for 10 min. The final product was centrifuged at 12,000 rpm and purified by water/ethanol wash steps.41

CuNPs Deposition on Silver Thin Film

CuNPs were drop-cast onto the silver thin films. The process was done by pipetting 7.78 μL/mm2 of CuNP suspension onto a silver thin film. The silver thin film was then dried under ambient conditions for 1 h. The process is repeated for 3 samples to obtain statistically significant measurements. The process was replicated on a 300-mesh copper grid to characterize the surface coverage of the CuNPs on Ag film samples.

Antibacterial Activity Assessment42

The antibacterial efficiency of the film samples was assessed using E. coli. This assessment involved measuring the optical density (OD) at 620 nm of the bacteria solution and comparing it with control experiments—these were reference experiments conducted without any samples in the bacterial medium, and others conducted without any bacterial medium. Agar media for E. coli was prepared using the appropriate composition in an Erlenmeyer flask. Once all of the necessary equipment was autoclaved, the prepared media was poured into a Petri dish and left to solidify for 1.5 h. Using an inoculating loop, the bacterial solution was transferred from the stock to the agar media. The dish was incubated for 24 h to allow for bacteria to proliferate. After 24 h, colonies of bacteria were observed. A colony was then selected from the dish and transferred into a prepared Luria-Bertani broth. The solution was subsequently mixed with a vortex mixer. Using a pipette, the solution was transferred into the sterile 96-well plate with 200 μL pipetted into each well for OD 620 measurement. A control solution, which was broth without bacteria, was also added to serve as a reference for antibacterial activity analysis. The film samples—CuNPs on a silicon wafer, blank silver thin film, and CuNPs on silver thin film of various percentages of coverage—were put into the specified wells. The 96-well plate was incubated at 37 °C and subjected to an orbital shake at 150 rpm for 24 h. During incubation, the plate was removed twice, at 12 and 18 h, for OD 620 measurement of each well using the Microplate Reader (Halo LED 96). It should be noted that the films were removed from the wells prior to each measurement to prevent interference with the OD 620 value of the solution. The films were then placed back after the measurement.

Characterization

Various characterization techniques were utilized to analyze the prepared samples and ascertain their properties.

AFM Imaging

The surface roughness of the prepared silver thin film was determined using an atomic force microscope (AFM) (Park NX10).

TEM and EDAX Imaging

Bright-field high-resolution transmission electron microscopy (TEM) images and energy-dispersive X-ray spectroscopy (EDAX) plots were collected using a JEOL-ARM300F TEM microscope with an accelerating voltage of 200 kV. The morphology and surface coverage of the nanoparticles, which had been drop-cast on the 300-mesh copper TEM grids, were characterized through these means. Samples were degassed in a pumping station for 30 min prior to analysis.

XPS Surface Analysis

XPS measurements were performed using a ULVAC-PHI 5000 Versa Probe II, equipped with a micro-focused Mg Kα X-ray source (1253.6 eV). The probe operated at 25 W (with a voltage of 15 kV) and featured a hemispherical analyzer with a multichannel detector. The base pressure for the operation was at 10–10 mbar. Atomic fractions are retrieved from peak area fitting of Ag 3d and Cu 2p normalized to relative sensitivity factors.

Image Analysis

Images retrieved from both TEM and AFM instruments were analyzed using ImageJ software. Through this analysis, the size distribution of CuNPs and the coverage of oxide surfaces were determined, respectively.

Results and Discussion

Characterization of Ag and Cu

CuNPs were synthesized and deposited on Ag thin films. Figure 1A shows the TEM characterization of synthesized colloidal CuNPs. CuNP size distribution was calculated from Figure S1. As depicted in Figure 1B, the synthesized CuNPs have a diameter of 200 nm. XPS characterization was performed on CuNPs and Ag film samples to elucidate the surface states of the two phases prior to the formation of active Ag–Cu interfaces.

Figure 1.

Figure 1

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(A, B) TEM images of colloidal CuNPs. (C) Quantified size distribution of synthesized CuNPs, around 206 nm.

Figure 2A shows the high-resolution X-ray photoelectron spectroscopy (HR-XPS) spectra of Ag 3d. Features at 367.65, 367.95, and 368.30 eV correspond to AgO, Ag2O, and Ag phases, respectively.43 It is clear from Figure 2A that the surface of Ag consists mainly of Ag followed by Ag+ (Ag2O). Previous XPS studies on thermally evaporated Ag thin films have also reported similar surface compositions. Figure 2B shows HR-XPS spectra of Cu 2p, with a dominant feature at 932.0 eV corresponding to Cu2O observed followed by 932.5, 933.59, and 934.88 eV corresponding to Cu, CuO, and Cu(OH)2, respectively.43 Results from Figure 2 indicate the presence of two important phases: Ag2O and Cu2O.

Figure 2.

Figure 2

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(A) Ag 3d HR-XPS spectra of 100 nm electron-beam-deposited Ag film. Features at 367.65, 367.95, and 368.30 eV corresponding to AgO, Ag2O, and Ag, respectively. (B) HR-XPS spectrum of Cu 2p. Features at 932.0, 932.5, 933.59, and 934.88 eV corresponding to Cu2O, Cu metal, CuO, and Cu(OH)x, respectively.

AFM characterization of Ag film indicates an rms roughness of 6.754 nm, as depicted in Figure 3A. Ag nanofilm roughness is in agreement with the reported AFM characterization of Ag thin films prepared via thermal evaporation. Based on previous AFM studies, which have shown that the coalescence of grains on thin film metal surfaces is indicative of oxide defects, this information was utilized to calculate the composition of the silver oxide islands on the silver film samples.44Figure S2 and Table S1 indicate that surface defects account for 10.23% of the entire plot. Although this does not prove that these grains are in fact Ag2O, it is interesting to note that the percentage of oxide defects correlates with the surface concentration of silver(I) oxide quantified from Ag XPS spectra analysis. The presence of an Ag2O can be detrimental in the formation of a heterostructure with other metal oxide materials.

Figure 3.

Figure 3

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(A) Atomic force microscopy (AFM) image of the Ag film showing the surface topology. (B) Defects on the surface of Ag film calculated from ImageJ software, along with the histogram on the left depicting the area distribution of each surface height and the threshold set for the height of surface defects (highlighted in red box).

The nature of the Cu2O was further investigated using EDAX mapping on CuNPs. Figure 4A reveals a thin oxide shell covering the structure of the CuNPs. Further analysis along different profiles of the nanoparticles is shown in Figure 4B and Table S2 in the Supporting Information, which reveals a Cu content of 70% and an O of 30% localized on the nanoparticle shell on analysis spots 4, 9, and 10 (see Supporting Information Table S2 corresponding to values for each spot). It is interesting to note that this corresponds to the ratios of Cu and O in Cu2O. Most analysis spots at the center of the CuNPs reveal a 90+% Cu content. To further verify that the nanoparticles do, in fact, have a Cu2O shell and that there exists no bias from EDAX mapping, a separate set of EDAX analyses was conducted on Cu2O nanocubes control where the Cu2O phase should be evenly distributed throughout the nanoparticle. The results shown in Figure 4C,D reveal a uniform distribution of Cu and O content across these nanoparticles, which confirms that the Cu2O is indeed intrinsic to the outermost shell of the CuNPs used in this study.

Figure 4.

Figure 4

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(A) EDAX mapping of oxygen and copper on CuNP, where green represents O regions and blue represents Cu regions. (B) HAADF image of two isolated CuNPs: EDAX analysis at the shell reveals a Cu/O ratio of 80, 20%, while analysis through the center of the nanoparticle reveals a 99–100% Cu content. (C, D) HAADF and EDAX mapping of separately synthesized Cu2O nanoparticles.

Depositing CuNPs on Ag films leads to the formation of an active Ag2O–Cu2O interface. C. Tseng reports significantly increased photocurrent measurements on Ag2O–Cu2O. Additionally, Guleria showed that Ag2O–Cu2O prepared via ultrasonic wet impregnation leads to significantly enhanced catalytic activity toward methylene blue dye degradation.31 Ag2O narrow band gap at 1.3 eV along with Cu2O band gap of 2.0 eV, which sits in the infrared range, can lead to better charge separation processes at the material interfaces.45,46 The synergy between these two interfacial contacts can lead to drastic changes in the antibacterial activity of the material and is the center of this investigation.

Antibacterial Assessment of Cu-on-Ag Planar Samples

Bacterial cell culture was cultivated in multiple well plates after CuNPs were drop-cast onto Ag thin film samples, and the cultures were then exposed to ambient light conditions. The OD values of the bacteria solutions were then measured and used for assessing the antibacterial efficiencies of the film samples based on the following equation

graphic file with name ao3c04303_m001.jpg

where ODfull denotes the value of the control well, representing the bacterial solution without any film samples, ODmeasured is the measured value of the well of interest, ODnull is the value of the blank control, which is the broth without bacteria, and area denotes the surface area of the film samples, expressed in cm2 and calculated using ImageJ software.

Figure 5 illustrates the results of the antibacterial assessment on Ag–Cu samples at 6 h intervals, all measurements were conducted in triplicates to obtain error bars, and the activity of each sample is normalized to the planar surface area of each sample. Refer to Table S3 for the exact values of the measured OD from each sample. Figure 5A shows the antibacterial activity of CuNPs deposited on Si wafer, CuNPs deposited on Ag thin films, and bare Ag thin film samples at 12 h. It is clear that the presence of Ag–Cu leads to significant enhancement in the antibacterial activity of the sample, whereas the activity of bare Ag films and CuNPs controls are both lower than Ag–Cu samples. Figure S7 in the Supporting Information shows SEM analysis of E. coli on Ag–Cu and bare Ag surfaces. Ag–Cu shows a significant increase in E. coli death relative to bare Ag. XPS atomic fraction analysis reveals a surface coverage comprising 45% CuNPs, in which both copper and silver phases are reasonably exposed. Figure 5B shows results collected at 18 h, and Figure 5C at 24 h, respectively. The effect of the Ag–Cu interface remains observable even at 24 h. The activity of Ag is relatively higher at longer time durations (18 and 24 h) because the Ag film becomes unstable at longer time intervals. This instability was visually evidenced by areas of missing Ag films on the sample. This can result in additional Ag being released from the bulk into the bacterial medium. Given that Ag films were deposited via electron-beam deposition at 100 nm, deterioration of these films could expose the underlying bulk Ag to the bacterial medium. Ag–Cu however remains as the sample with the highest activity. While it is clear that the synergistic effect between Ag2O–Cu2O heterostructure exists, a mechanistic picture of how these two metal phases can induce such potent antibacterial activity has not yet been fully established. Thus, further spectroscopic analysis is required to elucidate the origins of these effects.

Figure 5.

Figure 5

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Results of antibacterial assessment for sample sets including Ag, Ag–Cu, and Cu samples at (A) 12 h, (B) 18 h, and (C) 24 h.

XPS Analysis of Ag–Cu and Proposed Mechanism

To fully understand the observed enhancement, we performed XPS analysis on Ag–Cu samples, as shown in Figure 6. Figure 6A displays the Ag 3d spectra of the Ag–Cu sample, with binding energies of 367.65, 367.95, and 368.30 eV corresponding to AgO, Ag2O, and Ag, respectively.43 It is clear that upon the deposition of CuNPs on Ag, metallic Ag dramatically decreases. Figure 6B depicts Cu 2p XPS spectra with features at 932.0, 932.5, 933.59, and 934.88 eV, which correspond to Cu2O, Cu, CuO, and Cu(OH)2 phases.43 The oxidization of Ag is followed by a dramatic increase in metallic Cu and a significant rise in the Cu(OH)2 phase. Oxidation of Ag, evident in the large fraction of Ag2+ present on the surface and the reduction of Cu, is indicative of a charge transfer process between the Cu and Ag phases upon the formation of Ag–Cu interface sites. The most pronounced alteration in the Cu phase is the emergence of an increased Cu(OH)2 phase at 934.88 eV. The observed phases upon the formation of Ag–Cu interfaces can be directly correlated to the observed antibacterial activity, which arises when Ag and Cu are placed in close proximity.

Figure 6.

Figure 6

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(A) Ag 3d HR-XPS spectra of Ag film in Ag–Cu samples. Features at 367.65, 367.95, and 368. 30 eV correspond to AgO, Ag2O, and Ag, respectively. (B) HR-XPS spectrum of Cu 2p. Features at 932.0, 932.5, 933.59, and 934.88 eV correspond to Cu2O, Cu metal, CuO, and Cu(OH)x, respectively.

The formation of interface sites between Cu2O (VB +0.6 eV, CB −1.4 eV, band gap 2.0 eV) and Ag2O (VB +1.5 eV, CB +0.2 eV, band gap 1.3 eV) has been implicated in leading to enhanced photoinduced charge separation via a direct Z-scheme mechanism.45,46 At close interfacial contact, band bending occurs, allowing for charge carrier recombination to take place between the two oxides.7Figure 7 depicts a proposed mechanism scheme that represents the different phases involved in the antibacterial process on Ag–Cu film samples.

Figure 7.

Figure 7

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Proposed mechanism scheme demonstrating how the Ag–Cu interface mediates the enhanced antibacterial activity.

As seen in Figure 7, under visible light illumination, electrons are excited from the valence band of Ag2O to the conduction band and recombine with photogenerated holes in the valence band of Cu2O. Over time, excited electrons build up on the copper surface, rendering the surface of Cu reduced, as evidenced by XPS analysis, while electrons in the conduction band of Cu2O can also induce the formation of ROS, such as O2–, and OH•, which has been reported to form on similar systems.31,47−49 Consequently, the formation of ROS species might also lead to Cu(OH)2 as hydrogen abstraction from other oxygen-based surface species may occur to help yield the antibacterial.O2–, OH•, and other reactive species. Holes that build up on the surface of Ag can render the surface of Ag film more oxidized forming the antibacterial phase AgO, as shown in XPS analysis. The formation of Cu(OH)2 is quite significant since several reports have indicated that the formation of Cu(OH)2 is directly related to enhanced release of ROS as well as increased amounts of Cu ions, as shown in previous studies, to exhibit the antibacterial effects by disrupting ATP production and impairing bacterial cell wall.8,9,16,19,20 The formation of AgO is also significant as AgO has been reported to exhibit superior antibacterial activity relative to other Ag phases.50

To further confirm the production of ROS species, we performed methylene blue degradation experiments on both bare Ag and Ag–Cu samples. MB degradation occurs when ROS species form at the valence band and conduction band of the material. These species then react directly with the dye molecules; for more information, refer to Scheme 1 in the Supporting Information. A faster decrease in absorption signifies the formation of ROS species, which react with the MB dye resulting in dye degradation. Figure S6 in the Supporting Information reveals faster decay rates for Ag–Cu versus bare Ag, indicating enhanced formation of ROS species at the material interface.

To further substantiate the important role of the interface between the two materials, additional experiments were conducted on an inverse surface system where the coverage of Ag nanocubes was controlled on thermally evaporated Cu films using Langmuir–Blodgett deposition (LB); the procedure for sample fabrication is provided in the Supporting Information. TEM images of Ag nanocubes are shown in Figure S3, and SEM images of various coverages of Ag nanocube samples on Cu films are shown in Figure S4. The antibacterial activity of these samples was assessed, with results revealing the highest activity in samples where the dispersion of Ag nanocubes was maximized and good interfacial contact can be established between the Ag nanocubes and the Cu surface as shown in Figure S5.

Figure 8 presents the XPS quantification of the phases involved in this process. From Figure 8B, the surface concentration of Ag2O remains largely unchanged between bare Ag films and Ag in Ag–Cu samples, indicating the high stability of the Ag2O phase. This phase is important as its presence on the surface of the support film allows for the formation of a heterostructure with Cu2O. Following the previously proposed Z-scheme mechanism, electrons from the conduction band of Ag2O and holes from the Cu2O’s valence band recombine at the interface of the two materials. This is followed by the buildup of surface holes on Ag, consequently leading to an increase in AgO (Ag2+) by over 14-fold. Dellasega et al. have reported that AgO exhibits remarkable antibacterial activity, which contributes to the Ag–Cu synergistic antibacterial enhancement.50 Electrons that build up on the CuNPs are injected into the Cu2O shell of the CuNPs, leading to a decrease in the surface concentration of Cu2O (Cu1+) by 2-fold as seen in Figure 8A. Despite this decrease, Cu2O still remains a major proponent of the nanoparticle surface as its percent surface coverage still accounts for up to 30% of the nanoparticle surface. As expected, the surface concentration of CuO (Cu2+) also experiences a decrease by 2-fold. The decrease in both Cu2O and CuO results in a 3- to 4-fold increase in metallic Cu and Cu(OH)2. We hypothesize that electrons transferred to the Cu nanoparticles are responsible for forming metallic Cu sites and Cu(OH)2 via the previously proposed Z-scheme mechanism. Both of these phases have been reported to be able to contribute substantially to the intrinsic antibacterial activity of the material by inducing the release of Cu ions and ROS species. In addition, the highly oxidized AgO phase also contributes substantially to the antibacterial activity of the material. The correlation of these active phases involved, as well as the activity correlation with the Ag–Cu interface, provides strong insights into the origins of the antibacterial activity in a bifunctional surface system, which is otherwise not yet fully explored.

Figure 8.

Figure 8

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Detailed information about the relative concentrations of all surface species at active Ag–Cu samples. (A) Transfer of electrons across the different copper phases. Cu2O remains the primary Cu phase; however, both Cu2O and CuO are markedly reduced due to electrons transferred from silver. The presence of metallic copper is enhanced 2-fold, and a pronounced Cu(OH)x phase is observed. (B) Ag2O remains stable both pre- and post-deposition of Cu nanoparticles on Ag surfaces. Metallic silver is dramatically oxidized to Ag2+, as shown above.

Conclusions

We investigated in this study the enhanced antibacterial activity induced by the contact of two metals: copper and silver. Colloidal CuNPs were synthesized, deposited on silver thin films, and investigated for antibacterial activity against Escherichia coli. TEM and EDAX analyses indicate that CuNPs are enclosed in a Cu2O shell. XPS measurements demonstrate that depositing CuNPs on Ag films forms a photoactive Cu2O–Ag2O heterostructure. A Z-scheme mechanism was proposed to explain Ag–Cu’s bifunctionality. First, electrons in the Ag2O conduction band recombine with holes in the Cu2O valence band. Electrons from the conduction band of Cu2O can be transferred to nearby Cu1+/2+ sites, thereby rendering a reduced Cu surface. Simultaneously, these electrons can also be transferred to surface oxygen species, leading to the formation of ROS and Cu(OH)2. Cu and Cu(OH)2 have previously been proposed in the literature as active antibacterial phases and have been directly correlated to the release of ROS species and Cu ions. The buildup of holes on Ag2O’s valence band leads to the oxidation of Ag, resulting in the production of the highly antibacterial AgO phase. Quantitative analysis of HR-XPS spectra reveals an upsurge in the Cu(OH)2 phase by 4-fold and AgO by 14-fold. For the first time, this study provides strong evidence that the antibacterial activity of simple Ag–Cu systems may originate from a Z-scheme charge transfer process occurring at the interface of the Cu2O and Ag2O phases present on both Ag and Cu surfaces. Prior to this study, several reports have shown increased antibacterial activity when Ag and Cu are utilized together as antibacterial materials. The results of this study are insightful as they address the mechanistic question of the origins of the antibacterial activity observed in simple Ag–Cu colloid thin film systems.

Acknowledgments

This work was fully funded by Kamnoetvidya Science Academy. Sample characterization was performed at Vidyasirimedhi Institute of Science and Technology. The authors also thank Dr. Taweesak Sudyodsuk at Vidyasirimedhi Institute of Science and Technology for assisting in the preparation of thermal evaporated thin film samples.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c04303.

  • TEM images of Cu nanoparticles and Ag nanocubes; data tables for antibacterial activity calculations and image analyses; antibacterial activities of Ag Nanocubes on Cu films; accompanying SEM images of Ag nanocube coverages on Cu film; methylene blue degradation rates for Ag–Cu and Cu samples; SEM images of E. coli on samples; and methods for Cu thin film deposition and Ag nanocube synthesis (PDF)

Author Contributions

⊥ W.L., S.A., T.V., W.P., and P.S. contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

ao3c04303_si_001.pdf (759.7KB, pdf)

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