A modified polyol method was used to determine the optimum temperature of reduction (i.e. single Cu and AgNPs). Using ethylene glycol (anhydrous, 99.8%, Sigma-Aldrich) as solvent. The reaction mixture was prepared with Sodium Chloride 0.6 µM (ACS, ≥99%, Sigma-Aldrich), polyvinylpyrrolidone 0.005 µM (average mol wt 40,000, Sigma-Aldrich), Sodium Hydroxide 0.8 M (ACS Reagent, Sigma-Aldrich) and Silver Nitrate 1.6 µM (ACS, ≥99%, Sigma-Aldrich) to synthesize AgNP. Alternatively, for the CuNP synthesis, Copper Acetate 1.6 µM (99.99%, Sigma-Aldrich) was added to the mixture, instead of Silver Nitrate. The solutions were heated for 2 min at 155˚C, 160˚C, 165˚C, 170˚C, 175˚C, and 180˚C in a Teflon vessel using a laboratory microwave furnace Mars6 Xtraction, CEM with a power of 1000 W. The nanoparticles were coagulated using 2-propanol (Certified ACS; ≥99.5 Fisher Scientific) and dried for subsequent characterization.

The co-reduction of Silver-Copper (so-called AgCu) were synthesized preparing a mixture of Sodium Chloride 1.2 µM (ACS, ≥99%, Sigma-Aldrich), polyvinylpyrrolidone 0.01 µM (average mol wt 40,000, Sigma-Aldrich), Sodium Hydroxide 1.6 M (ACS Reagent, Sigma-Aldrich), Silver Nitrate 1.6 µM (ACS, ≥99%, Sigma-Aldrich), and Copper Acetate 1.6 µM (99.99%, Sigma-Aldrich), using ethylene glycol (anhydrous, 99.8%, Sigma-Aldrich) as the solvent, resulting in an Ag:Cu = 1:1 molar ratio solution. The synthesis was performed in a laboratory microwave furnace Mars6 Xtraction, CEM with a power of 1000 W for 2 min at 175˚C. The obtained nanoparticles were coagulated using 2-propanol (Certified ACS; ≥99.5 Fisher Scientific).

The crystal structure properties of the synthesized Ag, Cu, and AgCu nanoparticles were determined using powder X-ray diffraction (XRD, SIEMENS D 500 diffractometer with Cu-Kα radiation). For the AgCuNP, the crystalline structure was corroborated using a Rigaku XtalLab Supernova single-crystal X-ray Diffraction system with single micro-focus Cu source and Hypix Hybrid Array detection. The optical properties were analyzed performed UV-Vis absorption measurements using a Beckman DU 800 Spectrophotometer. The particle morphology and size distribution were determined by HRTEM (JEOL-2011 and JEM-ARM200F units). Elemental composition analysis of the AgCuNP was conducted using Energy Dispersive Spectroscopy (EDS) with an EDAX Si(Li) 30 mm² energy dispersive X-ray spectroscopy detector.

To evaluate the stability of nanoparticles in suspension through time, the UV-Vis absorption properties of Ag, Cu, and AgCu nanoparticles, synthesized at 175˚C, in ethylene glycol were monitored every 24 hours for a period of 12 days. For AgNP, a characteristic plasmon peak was expected to be observed around 400 nm, and for Cu nanoparticles around 650 nm. Measurements were performed using a Beckman DU 800 Spectrophotometer.

The antimicrobial capability of Ag, Cu and AgCu nanoparticles was evaluated against Gram-negative bacteria, Escherichia coli (ATCC 25922) and Salmonella typhimurium (ATCC 14020), Gram-positive bacteria Staphylococcus aureus (ATCC 25923), and a yeast, Candida albicans (ATCC 10211) using a broth microdilution method to determine the minimum inhibitory concentration (MIC) [18]. For this assay, nanoparticle suspensions at different concentrations (20 to 2200 µg/mL) were evaluated in 96-well microplates with a bacterial suspension of about 10⁶ CFU/mL, incubated under agitation at 37˚C for 24 h. After the incubation period, a resazurin dye solution was added to the well plates and incubated at 37˚C for an additional 2 h. The MIC was qualitatively determined by the smallest concentration of nanoparticle at which the blue colored solution remained unchanged [19] [20].

To determine the minimum bactericidal concentration (MBC) [21], from each nanoparticle concentration for which absolute inhibition was observed, 1 µL of suspension was used to inoculate selective agar plates and incubate at 37˚C for 24 h. The MBC was determined as the nanoparticle concentration at which no bacterial growth was observed on the agar plate. The selective agars used were MacConkey Agar for Escherichia coli (E. Coli), SS Agar for Salmonella typhimurium (Salmonella), Baird Parker Agar for Staphylococcus aureus (S. aureus), and Sabdex Agar for Candida albicans (C. albicans).

In order to establish the optimal temperature parameters, Cu, Ag and AgCu nanoparticles were synthesized at different temperatures ranging from 155˚C to 180˚C. Following the synthesis of the nanoparticles, UV-Vis analyses were carried out to confirm the presence of Ag and Cu metals in the nanoparticles. The UV-Vis absorption spectrum recorded for the silver nanoparticles (shown in Figure 1(b)), synthesized at temperatures 155˚ to 180˚ exhibited a maximum absorption peak at 406 nm attributed to the plasmon resonance characteristic of silver nanoparticles. UV-Vis analyses showed broader and red-shifted peaks at higher synthesis temperatures suggesting particle agglomeration and greater size distributions [22] [23]. The XRD patterns shown in Figure 1(a) demonstrated the formation of the face-centered cubic (FCC) silver crystalline structure in accordance with PDF No. 98-000-0398. The crystallite size of the AgNP, for each synthesis temperature, was calculated using the Debye-Scherrer relationship at the most intense peak by MDI/JADE software, which corresponds to the (111) plane (Table 1) [24] [25] [26] [27]. The obtained values demonstrated an increment in the nanoparticle’s crystallite size as synthesis temperatures increased.

CuNPs were also synthesized at temperatures ranging from 155˚C to 180˚C. However, only the syntheses performed at temperatures above 175˚C were successful at reducing Cu²⁺, evidenced by a change of color in the solution from blue to the red-wine [22] [28]. Since the copper standard potential reduction from Cu²⁺ to Cu⁰ is +0.34 V, whereas for silver reduction from Ag⁺ to Ag⁰ is +0.80 V, more energy is required to reduce copper nanoparticles [29]. UV-Vis absorption spectra, presented in Figure 2(b), shows a characteristic maximum absorption peak at 653 nm attributed to copper nanoparticle’s plasmon resonance [30]. The X-ray diffraction patterns of Figure 2(a) confirmed that the synthesized nanoparticles exhibited a copper FCC crystalline structure corresponding to PDF No 98-000-0172. The average crystallite size for CuNP was estimated at 14 nm for nanoparticles synthesized at both temperatures, as is shown in Table 2.

† These values were obtained using the Debye-Scherrer relationship by MDI/JADE software.

† These values were obtained using the Debye-Scherrer relationship by MDI/Jade software.

The previously discussed results from the synthesis of AgNP and CuNP indicate that the smallest nanoparticle size is obtained at a temperature of 175˚C. Consequently, the synthesis of AgCu bimetallic nanoparticles was performed at 175˚C in a single 2-minute step. The crystalline structure of the nanoparticles was confirmed by XRD measurements which evidences the presence of both Ag and Cu structures (Figure 3(a)). In the UV-Vis absorption spectrum (Figure 3(b)) a redshift in the absorption peak at 443 nm can be observed, which suggests the formation of an AgCu bimetallic type nanoparticle [31]. The average crystallite size was estimated at 12.89 ± 2.00 nm considering the peak at 2θ = 38.521 of Ag and 2θ = 44.761 of Cu, as it is shown in Table 3.

The UV-Vis spectroscopy technique has been widely used to characterize metallic nanoparticles due to the surface plasmon resonance produced by the collective oscillations of the free conductive electrons at the nanoparticle’s surface. The electron’s vibration mode is related to the size distribution and shape of nanoparticles that produce an optical absorption in the UV-Vis region characteristic of the metal [32] [33] [34] [35] [36] As a result, the stability in the suspension of AgNP, CuNP, and AgCuNP over time, was evaluated by collecting their UV-Vis spectra for 12 days (aging time). UV-Vis measurements of the synthesized nanoparticle solution were done before the washing step (BW), in which nanoparticles are coagulated using 2-propanol. Also, after the washing step (AW), when the previously washed nanoparticles are resuspended in ethylene glycol, and every 24 hours for the following 12 days. Figure 4(a) shows the overlaid UV-Vis spectra of AgNP for the evaluated period, the absorbance peak intensity at 410 nm stays constant while its width becomes slightly wider over time, as shown in Figure 4(b). The absorbance peak wavelength redshifts from 410 to 432 nm suggests an increase in nanoparticle size distribution. In the case of CuNP, the UV-Vis spectra exhibit a characteristic peak at 583 nm (Figure 5(a)), this peak red-shifts to 628 nm at the end of the 12 days and its plasmon peak intensity decreases (Figure 5(b)). This suggests the decrease of the NP size and the amount of Cu⁰ by the Ostwald Ripening effect. It also suggests NP surface oxidation, and aggregation-precipitation of CuNP with aging time [37] [38]. The UV-Vis spectral overlay for AgCuNP (Figure 6(a)) shows peaks at 391 nm and 573 nm characteristics of the plasmon resonance of AgNP and CuNP, respectively. There were no changes observed in the shape of the spectra and wavelength peaks positions through time, evidencing the stability of the nanoparticles in suspension over these 12 days period (Figure 6(b)).

† These values were obtained using the Debye-Scherrer relationship by MDI/Jade software.

HRTEM images of the synthesized AgNP are shown in Figure 7 and Figure 8, at 20 nm and 10 nm scale bar, respectively. The images reveal the spherical shape of the nanoparticles with an average diameter of 9.7 ± 4.5 nm and a narrow particle size distribution as evidenced by the histogram presented in Figure 7(b). The particle size distribution analyses were done using ImageJ software. The ED pattern in Figure 8(b) exhibits the FCC crystalline structure corresponding to Ag, indexed to the (111), (200), (220) and (222) planes. These results confirm the XRD pattern results obtained from the synthesized AgNP. Figure 9(a) and Figure 10(a) correspond to HRTEM images showing spherical crystalline CuNP with an average 7.3 ± 2.2 nm diameter. The size distribution analysis, shown in Figure 9(b), reveals that synthesized CuNP were smaller than the synthesized AgNP. The formation of CuNP was demonstrated by the corresponding ED pattern indexed to (111), (200), (220), and (311) planes related to the FCC crystalline structure of Cu, consistent with the XRD analysis. Comparatively, AgCuNP consisted of irregular-shaped nanoparticles (Figure 11(a) and Figure 12(a)) with an average size of 11.7 ± 3.8 nm (Figure 11(b)). Figure 12(b) shows the ED pattern results obtained after a mapping control using only carbon film. The measurements of the ring’s diameter were 0.238 nm corresponding to miller index (111) from 2θ = 38.114 degree for Ag, and 0.203 nm corresponding to miller index (111) from 2θ = 43.317 degree for Cu. The above results fully confirm the successful synthesis of nanoparticles with Ag and Cu metal composition.

To corroborate the chemical composition and particle distribution of Ag and Cu in the synthesized nanoparticles, EDX analysis was performed. Figure 13 shows the elemental mapping of AgCuNP (Figure 13(b)), which revels the homogeneous distribution of Ag (Figure 13(a)) and Cu (Figure 13(c)). The EDX elemental analysis demonstrates the presence of Ag and Cu in a single particle with an atomic percent composition of 51.74% of Cu and 48.26% of Ag with an Ag:Cu atomic ratio around 1:1, which is consistent with the molar ratio used in the synthesis. These results reaffirm the successful formation of an AgCuNP structure through a microwave synthesis time as short as two minutes (Figure 14).

The minimum inhibitory concentration (MIC) is the lowest concentration at which an antimicrobial agent is able to inhibit microbial population growth, and the minimum bactericidal concentration (MBC) is the lowest concentration of an antimicrobial agent required to reduce the population at ≥99.9% from the initial inoculum population. Both concentrations are complementary, that is, if a MIC shows inhibition, plating the bacteria onto agar might still result in organism proliferation because the antimicrobial did not cause death, unlike the MBC that it does cause death [20] [39]. The MIC and MBC of AgNP, CuNP and AgCuNP were determined against Gram negative bacteria E. coli and Salmonella, Gram positive S. aureus, and a yeast C. albicans. The AgCuNP showed greater activity against E. coli (Figure 15(a)) with a MIC = 60 µg/mL and MBC = 300 µg/mL. Salmonella (Figure 15(c)) showed more sensitivity in presence of AgCuNP (MIC = 100 µg/mL and MBC = 100 µg/mL) and AgNP (MIC = 200 µg/mL

and MBC = 200 µg/mL), where AgCuNP was the best. As same as in the case of Gram-negative bacteria, AgCuNP exhibited the best antibacterial performance against S Aureus (Figure 15(b)), with MIC = 200 µg/mL and MBC = 200 µg/mL. In the case of C. albicans, CuNP showed the best results with MIC = 100 µg/mL and MBC = 1900 µg/mL. Whereas, for Ag and AgCu NP the MBC values could not be determined as bacterial growth was observed even at the maximum evaluated nanoparticle concentration (2200 µg/mL). The above results indicate that AgCuNP are more effective in inhibiting the growth of most of the studied microorganisms. It was evident that Salmonella showed an enhanced vulnerability to these nanoparticles. Differences in the composition and structure of the cell wall of bacteria and yeasts could explain the resistance of the yeast to the effect of nanoparticles. The bacterial cell wall structure is formed by lipopolysaccharides and peptidoglycan layer mainly, the Gram-positive bacteria has a thicker peptidoglycan layer than the Gram negative [40]. In a different way, the yeast cell wall is formed by an inner wall of β, 1-6 glucan, β, 1-3 glucan and chitin and an outer protein wall [41]. It has been reported that metallic nanoparticles are able to attach more easily to DNA and proteins [8] [13], which explains why the evaluated yeast was more resistant to the effect of AgNP and AgCuNP, which are more stable than CuNP, hence it would be easier for them to cross the yeast cell wall in an ionic state rather than its metallic state [42]. The MBC/MIC ratio has been denominated Tolerance by May, et al. 2006 [43] and Das, et al. 2017 [5]. When a tolerance value for bacteria is ≥16, the antimicrobial agent is considered bacteriostatic, and when the tolerance value is ≤4 the agent is considered bactericidal. Accordingly, a bacteriostatic agent just causes an inhibition of bacterial growth while a bactericidal agent kills bacteria [44] [45]. The tolerance values for AgNP, CuNP and AgCuNP were calculated and are shown at the right scale of Figures 15(a)-(c). From the calculated values, AgNP and AgCuNP demonstrated a bactericidal effect against all bacteria, whereas CuNP demonstrated a bactericidal effect against E. coli and S. aureus and a bacteriostatic effect against Salmonella. The bacteriostatic effect of CuNP can be explained by its antimicrobial mechanism, which has been previously reported in different studies [46]. Cu NP’s antibacterial effect is mainly triggered by the release of copper ions in aqueous solution which lead to the disruption of the bacteria’s cell membrane causing cell death.