AgCu NP Formation by the Ag NP Catalysis of Cu Ions at Room Temperature and Their Antibacterial Efficacy: A Kinetic Study

Although a facile route to prepare AgCu nanoalloys (NAs) with enhanced antibacterial efficacy using Ag NP catalysis of Cu ions at elevated temperatures was previously developed, its detailed reaction process is still unclear due to the fast reaction process at higher temperatures. This work found that AgCu NAs can also be synthesized by the same process but at room temperature. AgCu NAs formation kinetics have been studied using UV–Visible spectra and Transmission Electron Microscopy (TEM), where formation includes Cu2+ deposition onto the Ag NP surface and Ag+ release, reduction, and agglomeration to form new Ag NPs; this is followed by a redistribution of the NA components and coalescence to form larger AgCu NPs. It is found that SPR absorption is linear with time early in the reaction, as expected for both pseudo-first-order (PFO) and pseudo-second-order (PSO) kinetics; neither model is followed subsequently due to contributions from newly formed Ag NPs and AgCu NAs. The antibacterial efficacy of the AgCu NAs thus formed was estimated, with a continuous increase over the whole alloying process, demonstrating the correlation of antibacterial efficacy with the extent of AgCu NA formation and Ag+ release.


Introduction
Ag-based nanoalloys (NAs), especially AgCu, have been of great interest in recent decades due to their enhanced antibacterial efficacies and the reduced amount of silver they use [1][2][3]. The enhanced antibacterial efficacy of AgCu NAs is greater than that of Ag NPs, Cu NPs, and their mixture [4], although the exact mechanism is still unknown [5,6]. AgCu NAs also exhibit less toxicity to human cells. For example, Thakore et al. [7], using the MTT assay method, reported that AgCu NAs prepared using the fruit latex of Achras sapota Linn showed lower toxicity to mouse embryonic fibroblast 3T3L1 cells. Długosz et al. [3] recently disclosed that AgCu NAs not only exhibit better antibacterial and antiviral efficacies, but they also showed no genotoxicity compared to Ag or Cu NPs, even at concentrations as high as 500 ppm. Al Tamimi et al. [8] found that Ag NPs showed no toxic effects on healthy cells but were toxic to MCF-7 human breast cancer cells at a concentration of 10 ppm. Ashraf et al. [9] showed that AgCu NAs conjugated with wheat germ agglutinin exhibited toxicity to homologous (MCF-7 and MDA-MB 231) breast cancer cells. Kushwah et al. [10] reported that the biosynthesis of AgCu NAs exhibited enhanced methylene blue photocatalytic and antibacterial efficacies. Bello-Lopez et al. [11] used AgCu NAs deposited onto nonwoven polypropylene for protection against SARS-CoV-2 and found no toxicity for human fibroblasts and keratinocyte cells; additionally, AgCu NAs showed better antioxidant and catalytic performances.
A typical color change variation with time, for 10 ppm Ag NPs + 100 ppm Cu 2+ , is shown in Figure S1, in the Supplementary Materials, where it can be seen that the color changes from yellow to almost colorless as a function of time. Note that the color decreases with the increasing Cu 2+ concentration.

UV-Visible Spectra
Representative UV-Visible spectra of 10 ppm Ag NPs + 500 ppm Cu 2+ , evolving as a function of time over 30 min, are found in Figure 1a; the SPR peak, located at~396 nm, is observed. The SPR evolution of 10 ppm Ag NPs + 100 ppm Cu 2+ over a longer period of time is found in Figure 1b,c. It shows that the peak intensity decreases over both the early stage (30 min) and a longer time (e.g., 18 days). However, the peak narrows at the higher wavelength side during the early stage (Figure 1a,b) and at the lower wavelength side over the longer time period (Figure 1c). The SPR peak intensity time variation of 10 ppm Ag NPs + Cu 2+ for different Cu 2+ concentrations can be found in Figure 2a, and the variations of the peak position with time and Cu 2+ concentration are shown in Figure 2b,c, respectively. The SPR intensity decreases with time, especially for higher Cu 2+ concentra- tions, but its effect on the SPR peak position is minimal. However, increasing the Cu 2+ concentration leads to a peak shift to lower wavelengths (blue shift, Figure 2c). stage (30 min) and a longer time (e.g., 18 days). However, the peak narrows at the higher wavelength side during the early stage (Figure 1a,b) and at the lower wavelength side over the longer time period (Figure 1c). The SPR peak intensity time variation of 10 ppm Ag NPs + Cu 2+ for different Cu 2+ concentrations can be found in Figure 2a, and the variations of the peak position with time and Cu 2+ concentration are shown in Figure 2b and 2c, respectively. The SPR intensity decreases with time, especially for higher Cu 2+ concentrations, but its effect on the SPR peak position is minimal. However, increasing the Cu 2+ concentration leads to a peak shift to lower wavelengths (blue shift, Figure 2c).   Figure 3 shows the SPR peak intensity of 10 ppm Ag NPs + Cu 2+ as a function of time, for different Cu 2+ concentrations, during the early stage (e.g., 30 min), indicating that the peak intensity decreases linearly with time for a given Cu 2+ concentration. stage (30 min) and a longer time (e.g., 18 days). However, the peak narrows at the higher wavelength side during the early stage (Figure 1a,b) and at the lower wavelength side over the longer time period (Figure 1c). The SPR peak intensity time variation of 10 ppm Ag NPs + Cu 2+ for different Cu 2+ concentrations can be found in Figure 2a, and the variations of the peak position with time and Cu 2+ concentration are shown in Figure 2b and 2c, respectively. The SPR intensity decreases with time, especially for higher Cu 2+ concentrations, but its effect on the SPR peak position is minimal. However, increasing the Cu 2+ concentration leads to a peak shift to lower wavelengths (blue shift, Figure 2c).   Figure 3 shows the SPR peak intensity of 10 ppm Ag NPs + Cu 2+ as a function of time, for different Cu 2+ concentrations, during the early stage (e.g., 30 min), indicating that the peak intensity decreases linearly with time for a given Cu 2+ concentration.  Figure 3 shows the SPR peak intensity of 10 ppm Ag NPs + Cu 2+ as a function of time, for different Cu 2+ concentrations, during the early stage (e.g., 30 min), indicating that the peak intensity decreases linearly with time for a given Cu 2+ concentration.   Figure 4 shows TEMs and NP size distributions for 10 ppm Ag + 100 ppm Cu 2+ as a function of time. One notes many more small NPs (Figure 4b) compared to pure Ag NPs (Figure 4a). It would appear that the average size of the NPs is first reduced before increasing with time (Figure 4c-e). The smaller NPs in Figure 5 appear to be either Ag (e.g., Figure 5c) or, mostly, AgCu (Figure 5b). It is interesting to note that AgCu appears within   Figure 4 shows TEMs and NP size distributions for 10 ppm Ag + 100 ppm Cu 2+ as a function of time. One notes many more small NPs (Figure 4b) compared to pure Ag NPs (Figure 4a). It would appear that the average size of the NPs is first reduced before increasing with time (Figure 4c-e). The smaller NPs in Figure 5 appear to be either Ag (e.g., Figure 5c) or, mostly, AgCu (Figure 5b). It is interesting to note that AgCu appears within 10 min (e.g., forming the bimetal structure in Figure 5b). For a detailed analysis of size changes, one can choose to group the sizes thus: from 1 to 7 nm (labeled A), 8 to 20 nm (B), and over 22 nm (C). The A component appears on Cu 2+ addition, the B component is made up of the original Ag NPs, and the C component appears over time, following Cu 2+ addition. Their variations with time are seen in Figure 6.        Figure 7 and Table S1 in the Supplementary Materials show the antibacterial activity of the 10 ppm AgNPs + 100 ppm Cu 2+ samples as a function of time, using the agar diffusion method against the two bacterial strains. While they differ significantly between S. aureus and E. coli, both samples show increased efficacy over time (samples a-e). This is also in good agreement with the MIC assay data in Table S2, which means that a longer time produces lower MICs values. Moreover, it is worth noting that sample e (38 days at room temperature) showed approximately the same antibacterial effects as sample f (at an elevated temperature) against the two strains.  Figure 7 and Table S1 in the Supplementary Materials show the antibacterial activity of the 10 ppm AgNPs + 100 ppm Cu 2+ samples as a function of time, using the agar diffusion method against the two bacterial strains. While they differ significantly between S. aureus and E. coli, both samples show increased efficacy over time (samples a-e). This is also in good agreement with the MIC assay data in Table S2, which means that a longer time produces lower MICs values. Moreover, it is worth noting that sample e (38 days at room temperature) showed approximately the same antibacterial effects as sample f (at an elevated temperature) against the two strains.

Color Change and UV-Visible spectra
The color change of the Ag NPs upon adding Cu 2+ , varying with time from yellow to almost colorless after 8 days ( Figure S1), is attributed to the reaction of Cu 2+ with Ag NPs. As in our previous work [5,30], the color of the AgNPs' dispersion is not only related to the size and density of the NPs, but also to the agglomeration and dispersion of the NPs, so it is difficult to directly judge or determine the cause of color changes over time. However, for a given Ag concentration, the color change can be attributed to the AgCu NAs formation and the associated size change [5,30].
The SPRs of Ag NP dispersions have been well studied. They can be affected by NP shape and size [32,33], as well as the dispersion medium [34][35][36]. They have been used to estimate concentration [37][38][39], invoking the Beer-Lambert law. The spectral evolutions in Figure 1; Figure 2 can be summarized as follows: (1) The peak intensity decreases with time, which can be caused by either the deposition of an outer layer of Cu or a size reduction in the Ag NPs. Both effectively reduce the Ag NP surface area, and the SPR peak intensity of the Ag NPs is directly proportional to its surface area [40]; (2) The small blue shift of the SPR peak, as the Cu 2+ concentration increases during the early stage, as seen in Figure 2b, is attributed to the medium refractive index change caused by the change in the dielectric constant [41,42]. Cu 2+ addition increases the refractive index value of water [43]. (3) The initial SPR peak narrowing at the high wavelength side and the later narrowing at the low wavelength side, in Figure 1a,b, are attributed, respectively, to the NP size decrease and AgCu alloying; alloying NPs will lead to an SPR peak red shift [1,5,44]. This is consistent with the TEM data and will be discussed later.

Color Change and UV-Visible Spectra
The color change of the Ag NPs upon adding Cu 2+ , varying with time from yellow to almost colorless after 8 days ( Figure S1), is attributed to the reaction of Cu 2+ with Ag NPs. As in our previous work [5,30], the color of the AgNPs' dispersion is not only related to the size and density of the NPs, but also to the agglomeration and dispersion of the NPs, so it is difficult to directly judge or determine the cause of color changes over time. However, for a given Ag concentration, the color change can be attributed to the AgCu NAs formation and the associated size change [5,30].
The SPRs of Ag NP dispersions have been well studied. They can be affected by NP shape and size [32,33], as well as the dispersion medium [34][35][36]. They have been used to estimate concentration [37][38][39], invoking the Beer-Lambert law. The spectral evolutions in Figure 1; Figure 2 can be summarized as follows: (1) The peak intensity decreases with time, which can be caused by either the deposition of an outer layer of Cu or a size reduction in the Ag NPs. Both effectively reduce the Ag NP surface area, and the SPR peak intensity of the Ag NPs is directly proportional to its surface area [40]; (2) The small blue shift of the SPR peak, as the Cu 2+ concentration increases during the early stage, as seen in Figure 2b, is attributed to the medium refractive index change caused by the change in the dielectric constant [41,42]. Cu 2+ addition increases the refractive index value of water [43]. (3) The initial SPR peak narrowing at the high wavelength side and the later narrowing at the low wavelength side, in Figure 1a,b, are attributed, respectively, to the NP size decrease and AgCu alloying; alloying NPs will lead to an SPR peak red shift [1,5,44]. This is consistent with the TEM data and will be discussed later. (4) There is no aggregation of Ag NPs during the reaction and alloying because the appearance of a new peak at 450-600 nm [45][46][47] signifying aggregation was not observed; this is consistent with the TEM photomicrographs.
(5) The dependence of the SPR peak intensity on time and Cu 2+ concentration (Figures 2a and 3) indicates that the greater the Cu concentration, the greater the SPR intensity decrease. It may indicate that the greater the Cu 2+ concentration, the faster it is deposited onto the Ag NPs, reducing the uncovered Ag NP area, and diminishing the SPR peak intensity. This will be discussed in Section 3.3.

Morphologic Changes
As shown by the TEMs in Figure 4a and our previous work [5], the Ag NPs are almost monodisperse, most of which are distributed in the 10-25 nm range with an average size of 14 nm; there are few NPs below 7 nm. However, substantial changes occur upon adding Cu 2+ . Figures 4 and 5 show that the deposition of Cu is rapid, and AgCu NAs (about 7 nm in Figure 5b) are produced within 10 min; more small NPs (less than 7 nm) appear afterward (Figures 5 and 6). The small NPs in Figure 4b,c are attributed to the aggregation of Ag 0 from the reduction of the Ag + released from the Ag-Cu interface [5,6]. Figure 6 indicates that these small NPs remain stabilized for up to 1000 min before disappearing, while the B component decreases and the C component increases. Eventually, the average size of the dispersion increases, consistent with our previous high-temperature AgCu NA synthesis data [5].

AgCu NA Formation Kinetics
Based on the present data, as well as those from the previous higher-temperature work [5], a possible schematic for forming AgCu NAs at room temperature can be found in Figure 8. The Ag NP stabilizers also reduce Cu 2+ through the catalysis of Ag NPs, depositing Cu 0 onto the Ag NP surface at room temperature. The Cu-Ag interface thus formed enhances Ag + release, similar to the case at higher temperatures [5]; this continues, with the AgCu NAs becoming small. The released Ag + is easily reduced either by PVP aldehyde chain terminations [48]-similar to what occurs in the Tollens reaction-and/or by the PVA secondary hydroxyl group, forming Ag 0 followed by Ag agglomeration, leading to the smaller Ag NPs. It must be mentioned that Ag NP catalysis is essential for the reduction of Ag + and Cu 2+ ; the presence of Cu 2+ will suppress Ag ion reduction by PVA [49]. Cu 0 deposits onto the smaller Ag NP surface because of its higher catalytic activity [50], forming AgCu NAs, followed by coalescence and reorganization to form larger AgCu NAs, consistent with the TEM photomicrographs in Figures 4-6. Molecules 2022, 27, x FOR PEER REVIEW 8 of 14 (4) There is no aggregation of Ag NPs during the reaction and alloying because the appearance of a new peak at 450-600 nm [45][46][47] signifying aggregation was not observed; this is consistent with the TEM photomicrographs. (5) The dependence of the SPR peak intensity on time and Cu 2+ concentration (Figure 2a and Figure 3) indicates that the greater the Cu concentration, the greater the SPR intensity decrease. It may indicate that the greater the Cu 2+ concentration, the faster it is deposited onto the Ag NPs, reducing the uncovered Ag NP area, and diminishing the SPR peak intensity. This will be discussed in Section 3.3.

Morphologic Changes
As shown by the TEMs in Figure 4a and our previous work [5], the Ag NPs are almost monodisperse, most of which are distributed in the 10-25 nm range with an average size of 14 nm; there are few NPs below 7 nm. However, substantial changes occur upon adding Cu 2+ . Figures 4 and 5 show that the deposition of Cu is rapid, and AgCu NAs (about 7 nm in Figure 5b) are produced within 10 min; more small NPs (less than 7 nm) appear afterward ( Figures 5 and 6). The small NPs in Figure 4b,c are attributed to the aggregation of Ag 0 from the reduction of the Ag + released from the Ag-Cu interface [5,6]. Figure 6 indicates that these small NPs remain stabilized for up to 1000 min before disappearing, while the B component decreases and the C component increases. Eventually, the average size of the dispersion increases, consistent with our previous high-temperature AgCu NA synthesis data [5].

AgCu NA Formation Kinetics
Based on the present data, as well as those from the previous higher-temperature work [5], a possible schematic for forming AgCu NAs at room temperature can be found in Figure 8. The Ag NP stabilizers also reduce Cu 2+ through the catalysis of Ag NPs, depositing Cu 0 onto the Ag NP surface at room temperature. The Cu-Ag interface thus formed enhances Ag + release, similar to the case at higher temperatures [5]; this continues, with the AgCu NAs becoming small. The released Ag + is easily reduced either by PVP aldehyde chain terminations [48]-similar to what occurs in the Tollens reaction-and/or by the PVA secondary hydroxyl group, forming Ag 0 followed by Ag agglomeration, leading to the smaller Ag NPs. It must be mentioned that Ag NP catalysis is essential for the reduction of Ag + and Cu 2+ ; the presence of Cu 2+ will suppress Ag ion reduction by PVA [49]. Cu 0 deposits onto the smaller Ag NP surface because of its higher catalytic activity [50], forming AgCu NAs, followed by coalescence and reorganization to form larger AgCu NAs, consistent with the TEM photomicrographs in Figure 4    The SPR spectral absorbance of Ag NPs is expressed by I 1 reflects SPR absorption from the effective surface area of the original Ag NPs (effective surface area = original surface area, treated as a sphere, less that covered by Cu 0 ). At the endpoint of the reaction, the effective surface area of the Ag NPs equals zero. I 2 reflects the effect of the reduction of the original spherical surface area due to Ag + release, reducing the NP surface area until the Ag disappears. I 3 reflects the compensation effect from the newly materialized surface area resulting from the agglomeration of the reduced Ag 0 , the coalescence of the newly formed Ag NPs, and any exposed Ag on the coalescing AgCu NAs.
Cu adsorption on the Ag NP surface is generally considered to fit the pseudo-firstorder (PFO) Lagergren equation or the pseudo-second-order (PSO) equation with nonlinear formation [51][52][53]. The pseudo-first-order kinetic equation is and the pseudo-second-order kinetic equation is where q t and q e are the sorption capacities at time t and at equilibrium, respectively; k 1 and k 2 are the rate constants of the PFO and PSO models, respectively. Considering the Cu deposition to be irreversible, the deposition capacity is equivalent to the surface area covered by Cu 0 . This permits us to modify the PFO Equation (2) to where r t =q e − q t is the exposed Ag NP surface area, while the PSO Equation (3) can be modified to r t = q e 1 1 + k 2 q e (5) The SPR peak intensity is directly related to the uncovered Ag NP surface (effective Ag NP surface area) [40] and is proportional to the effective surface area: Therefore, with Equation (6), Equations (4) and (5) can be simplified to PFO equation: PSO equation: where I 0 is the SPR intensity of Ag NPs before Cu deposition, and I(t) is the SPR intensity at time t after adding Cu 2+ . Figure 9 shows a comparison of experimental, PFO, and PSO data fits from the SPR intensity variations for 10 ppm AgNPs + 100 ppm Cu 2+ and 10 ppm Ag NPs + 500 ppm Cu 2+ . During the first 30 min, the Cu 2+ adsorption onto Ag NPs is accommodated by both PFO and PSO kinetics (Figure 9a), with R 2 > 0.99, although not afterward, affecting our ability to distinguish between equations. Following the initial 30 min, as seen in Figure 9b, the theoretical predictions for both the PFO and PSO models decrease far more rapidly than in the experiment. This is attributed to contributions from the new Ag NPs and AgCu NAs formed, as illustrated in Figure 8. The ∆ 1 and ∆ 2 differences in Figure 9b indicate that new NP formations now play the dominant role. For relatively small ki and t, ki t→0, Equations (7) and (8) can be further simplified as linear approximations: This is a linear relationship with time, consistent with Figure 3.

Antibacterial Efficacy Enhancement of AgCu NAs
Antibacterial efficacy testing, from both the Zone of Inhibition method and MIC test, shows an enhancement of efficacy with time (Figure 7 and Tables S1 and S2). As found in the previous section and illustrated in Figure 8, the AgCu NAs form over time as the Cu is redistributed throughout the Ag NP. This strongly suggests that the enhanced antibacterial efficacy is correlated with the fraction of Ag-Cu interfaces since Ag and Cu are mutually immiscible. That is, aging produces larger NAs with smaller domains and more Ag-Cu interfaces, which implies that the initial step involves bacterial contact with both Ag and Cu surface oxides. The bacteria-NA contact results in enhancing Ag + release [54][55][56][57]; this conclusion is reached by the time dependences of both Ag+ loss and the antibacterial assay. Meanwhile, it is noted that although there are some differences in the details of the two tests (Tables S1 and S2), the general direction of the changes is the same. The difference between the details of Tables S1 and S2 can be attributed to the use of different methods, which are involved in the dilution of an antibacterial agent in a pure liquid process (or liquid-liquid interaction) for MIC testing, while the antibacterial agent is diffused into an agar nutrition process (liquid-solid interaction) for Zone of Inhibition testing.
It is worth noting that the variation with time of the SPR intensity of AgNPs after adding Cu ions, as shown in Equation (9), can be used directly to estimate Cu ion centration, which will be discussed in a forthcoming paper. Although the process of preparing AgCu NAs at room temperature was indeed time-consuming, it has a lower manufacturing cost. It was also demonstrated that AgCu NAs deposited on fibers had excellent antibacterial efficacy and durability and superior wind resistance compared with Ag ions and AgNPs applied to an air conditioning filter. For relatively small k i and t, k i t→0, Equations (7) and (8) can be further simplified as linear approximations: This is a linear relationship with time, consistent with Figure 3.

Antibacterial Efficacy Enhancement of AgCu NAs
Antibacterial efficacy testing, from both the Zone of Inhibition method and MIC test, shows an enhancement of efficacy with time ( Figure 7 and Tables S1 and S2). As found in the previous section and illustrated in Figure 8, the AgCu NAs form over time as the Cu is redistributed throughout the Ag NP. This strongly suggests that the enhanced antibacterial efficacy is correlated with the fraction of Ag-Cu interfaces since Ag and Cu are mutually immiscible. That is, aging produces larger NAs with smaller domains and more Ag-Cu interfaces, which implies that the initial step involves bacterial contact with both Ag and Cu surface oxides. The bacteria-NA contact results in enhancing Ag + release [54][55][56][57]; this conclusion is reached by the time dependences of both Ag+ loss and the antibacterial assay. Meanwhile, it is noted that although there are some differences in the details of the two tests (Tables S1 and S2), the general direction of the changes is the same. The difference between the details of Tables S1 and S2 can be attributed to the use of different methods, which are involved in the dilution of an antibacterial agent in a pure liquid process (or liquid-liquid interaction) for MIC testing, while the antibacterial agent is diffused into an agar nutrition process (liquid-solid interaction) for Zone of Inhibition testing.
It is worth noting that the variation with time of the SPR intensity of AgNPs after adding Cu ions, as shown in Equation (9), can be used directly to estimate Cu ion centration, which will be discussed in a forthcoming paper. Although the process of preparing AgCu NAs at room temperature was indeed time-consuming, it has a lower manufacturing cost. It was also demonstrated that AgCu NAs deposited on fibers had excellent antibacterial efficacy and durability and superior wind resistance compared with Ag ions and AgNPs applied to an air conditioning filter.

Materials
A commercial aqueous dispersion of Ag NPs, 1000 ppm, stabilized by polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA), was provided by Solmont Technologies Co., Ltd. (Wuxi, China). Using both PVA and PVP greatly improves the stability of the Ag NPs, extending their long-term aging time, both at room temperature and 60 • C, enabling their use in commercial applications. Cu(NO 3 ) 2 (Aladdin, AR grade, CAS No 10031-  and Milli Q water were used to synthesize the NAs. All chemicals were used as received. Medium plates were used for bacterial culturing and counting. Microorganisms: the bacteria used were Gram-positive Staphylococcus aureus (ATCC 25923, ATCC 6538) and Gram-negative Escherichia coli (ATCC 25922, ATCC 8099).
AgCu NA preparation: the process can be found in our previous article [5]. Briefly, aqueous Cu 2+ was added to Ag NPs at the appropriate concentration and shaken to mix. All preparations were carried out at room temperature.

Antibacterial Assay
The antibacterial efficacies of the NPs were evaluated against Escherichia coli and Staphylococcus aureus. The inoculums of 0.5 McFarland standards (1.5 × 10 8 CFU/mL) were maintained in nutrient broth by picking up a single colony from the sub-culture plate [1], then diluted to a concentration of 1.5 × 10 6 CFU/mL.
The agar diffusion method is considered to be the gold standard of susceptibility testing or the most accurate way to measure the resistance of bacteria to antibiotics [58]. The method was used according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [59] with minor modifications. The agar plate surface was inoculated by spreading a volume of the microbial inoculum (1.5 × 10 6 CFU/mL) over the entire agar surface. Then, wells with a diameter of 8 mm were prepared on the surface of plates, and 50 µL of the antimicrobial agent, at the desired concentration, was introduced into the wells. The Petri dishes were incubated at 37 • C. The antimicrobial agent diffused into the agar and inhibited germination and growth of the test microorganism. The diameters of the inhibition zones were measured by a vernier caliper at least three times [60].
MIC tests were carried out by the Broth dilution method [60]. MIC (Minimum Inhibitory Concentration) is the lowest bacteriostatic concentration of an antibiotic component or agent preventing the visible growth of bacteria. A variety of nanoparticle dilutions was applied to 96-well plates containing bacteria suspension and cultured at 37 • C overnight. Before and after incubation, the MIC was determined by visual observation and validated by turbidity measurements [61]. Each assay was repeated at least 3 times.

Conclusions
This is the first time SPR has been used to study AgCu NA formation's kinetic process. It has revealed the AgCu NA formation process-from Cu deposition on the AgNPs's surface to nucleation, alloying, etc., using UV-Visible spectra and TEM. Smaller AgCu NAs coalesce to form larger NAs. It was not possible to determine whether Cu adsorption onto Ag NPs followed PFO or PSO kinetics since both were linear with time over the first 30 min, and both deviated from the theoretical predictions afterward due to contributions from newly formed Ag NPs and AgCu NAs. It was found that the enhanced antibacterial efficacies for E. coli and S. aureus using Zone of Inhibition and MIC testing were timedependent, as was the Ag + release. This work promises a new route to prepare low-cost AgCu NAs on a large scale with enhanced antibacterial efficacy comparable to preparation at elevated temperatures. These AgCu NAs can be further used as antibacterial agents loaded on fibers for application in air condition filters.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27206951/s1, Figure S1: The color change of 10 ppm Ag NPs + 100 ppm Cu 2+ over time; Table S1: Zones of Inhibition of samples of Ag NPs + 100 ppm Cu 2+ , against two strains at various times (a-e) at room temperature, and at 85 • C (f); Table S2. MIC of samples with different time of 10 ppm AgNPs + 100 ppm Cu 2+ against two strains.
Author Contributions: Y.T. Investigation UV-Vis spectra, data analysis and results discussion; F.Z. Investigation of antibacterial activity, data analysis, and discussion of results; K.W. investigation of TEM; D.Y. conceptualization, methodology, supervision, and writing-original draft; E.S. conceptualization, methodology, and writing-review and editing. All authors have read and agreed to the published version of the manuscript.