UV-Vis Spectroscopy and Z-potential
Figure 1 (A) shows the UV-vis absorption spectra of the synthesized nanomaterials. The absorbances have been normalized for the maximum localized surface plasmon resonance (LSPR) corresponding to each nanoparticle system.
Au@AgNps absorption spectrum in Figure 1 has a single band centered at 474 nm, which almost covers the entire region of the experiment. The wavelength of the LSPR for Au@AgNps is located between the AgNPs LSPR (445 nm) and the AuNPs LSPR (544 nm). The absence of a gold-like absorption peak on Au@AgNPs suggests that obtained nanomaterials by sequential synthesis are core@shell structures. It is not possible to detect any absorption band associated with Au belongs to the nucleus. Some authors assume that for core@shell systems, the absorption spectra are composed of two bands associated with each of the metals for shell thicknesses between 3 and 4 nm. The absorption associated with metalcore disappears for higher thicknesses, obtaining a single absorption band where the maximum location depends on the thickness/core size ratio of the bimetallic particle [49, 50].
Samal et al. [51] synthesized core @ shell nanoparticles (Au@Ag) by controlling the nuclei sizes and adding different thicknesses shells. In particular, our UV-vis result for Au@AgNPs coincides with that reported by Samal et al. for 32 nm gold cores and a silver thickness greater than 15 nm, where spectra are characterized by a single absorption band (~ 450 nm), and the suppression of Au surface plasmons are observed.
Additionally, in Figure 1A, absorption centered at 280 nm (region highlighted in blue) can be observed, corresponding to molecules from the Rumex hymenosepalus extract used as a reducing agent in our nanoparticle synthesis. Figure S1 corresponds to Rumex hymenosepalus aqueous solution absorption spectrum. A characteristic band centered at 278nm is observed, associated with the electronic transitions of the aromatic rings conjugated with the carbonyl groups of polyphenolic compounds [52]. This absorption band in the nanoparticles UV-Vis spectra indicates that final products contain extract molecules that remain in them.
Figures 1B, 1C, and 1D show the average curves of Z potentials corresponding to AuNPs, AgNPs, and Au@AgNPs. The graphs show a Gaussian behavior with peaks centered at -49.7mV, -47.2mV, and -51.3mV, respectively. These highly negative Z potential values indicate that nanoparticles experience repulsive interactions between them that prevent their aggregation and allow the long-term stability of metal colloids [53–55]. By correlating the UV-vis spectroscopy results with the obtained Z potential values, we can establish that the highly negative values may be due to the complexing of polyphenolic molecules of the extract onto the nanoparticle surface [56].
HAADF-STEM
Figure 2A corresponds to a representative bright-field STEM micrograph of the Au@AgNPs system at low magnification (100 nm scale bar). A set of nanoparticles without agglomeration and with mostly quasi-spherical geometry can be seen. The same region is shown in dark field (HAADF) in Figure 2B, and the core@shell structure can be observed, where can we distinguish Au-core looks more intense than Ag-shell, due to the difference in atomic number. Figures 2C and 2D correspond to STEM higher magnification micrograph (scale bare 20nm) of a nanoparticles group of system core@shell in a bright and dark field, respectively. Can be appreciated with clarity brilliant Au core and Ag shell lightly contrasted. These images show that the thickness of Ag-shell varies between 3 and 5 nm. Figure S2 corresponds to an individual images gallery where can be observed uniformity of Ag-shell.
TEM, HRTEM, and EDS
Figures 3A, 3C, and 3E correspond to micrograph TEM of representative nanoparticles systems AuNPs, AgNPs, and Au@AgNPs, respectively. In all cases, nanoparticles have sphere-like morphology and are shown well separated from each other. This can be explained by the extract molecules onto nanoparticle surfaces, acting as spacers between them. Figure 3B, 3D, and 3F are shown histograms correspond to size distribution obtained by TEM and performed with 500 nanoparticles. The histogram presents Gaussian distribution with a mean size of 24.23 ± 3.78nm (AuNPs), 13.20 ± 2.81nm (AgNPs), and 36.19 ± 11.09nm (Au@AgNPs).
For biological applications, it is valuable to obtain nanoparticles population with monodisperse sizes[57], so that dispersity was calculated from TEM data as reported by Tiunov et al. [58]. Polydispersity values obtained are 0.156, 0.212, and 0.306 for AuNPs, AgNPs, and Au@AgNPs, respectively, which correspond to the nanoparticles population of highly homogeneous sizes [59].
Figure 4 corresponds to the Au@AgNPs HAADF-STEM micrographics. A single nanoparticle is shown in figure 4A with a gold nucleus and silver cover perfectly delimited. The red square region is amplified to obtain an HRTEM micrography of the shell portion (Figure 4B), then to verify the crystalline shell structure, the nanoparticle periphery region was analyzed (discontinued square) with the Digital Micrograph 3.0 software (Gatan). Fast Fourier Transform (FFT) image of the selected area was obtained (Figure 4C). Using the Inverse Fast Fourier Transform was possible to estimate interplanar distances of 2.3 Å, 2.0 Å, and 1.4 Å in Figure 4D. These distances can be assigned respectively to the crystalline planes (111), (200), and (220) of face-centered cubic (fcc) silver according to Powder Diffraction File Card 00-004-0783 [60]. A similar analysis of crystal structure by HRTEM was carried out for monometallic nanoparticles as illustrated in Figures S4 (for AuNPs) and S5 (AgNPs). In both cases, crystal structure corresponds to face-centered cubic (fcc).
EDS
EDS chemical analysis shows the presence of both metals for a group of bimetallic Au@AgNPs observed by TEM (Figure 5A) in proportions of the atomic weight percent 77% of Ag (shell) and 23% of Au (cores) (Figure 5B). In comparison, a single bimetallic (Figure 5C) Au@AgNPs has proportions around 80% of Ag (shell) and 20% of Au (core) (Figure 5D).
These results are coincident with estimations effectuated (considering quasi-spherical NPs) for the Au and Ag content from the experimental results of size measurement by TEM for AuNPs and its corresponding Au@AgNPs; our calculations give an atomic content of 70.68 % for Ag (shell) and 29.32% (core).
XRD
Figure 6 corresponds to XRD patterns for AuNPs and AgNPs as well as bimetallic Au@AgNPs. All the synthesized products have fcc crystalline structure as previously reported in the characterization by electron microscopy. Peaks for Au@AgNPs are located at 2θ diffraction angles of 38.25 °, 44.4 °, 64.9 °, 77.85 °, and 81.25 °. As can be appreciated in the figure, the AgNPs and AuNPs diffraction peaks are found in the same positions mentioned with a difference of ± 0.5 °. This is because Au and Ag have very similar lattice constants, so their diffraction patterns for fcc crystal structure are almost identical (REF: Green synthesis and applications of Au–Ag bimetallic nanoparticles). In this way, the diffraction peaks in figure 6 are assigned, respectively, to the crystalline planes (111), (200), (220), (311) and (222) of the gold and silver fcc structure in accordance with ICSD #180868 and ICSD #604631.
Antimicrobial Activity
Monometallic (AgNPs, AuNPs) and bimetallic (Au@AgNPs) materials were tested at four different concentrations: 1, 10, 50, and 100 μg/mL. Selected microorganisms to evaluate antimicrobial activity were yeast Candida albicans, Gram positive bacteria S.aureus, and Gram negative bacteria E. coli. Growth kinetics curves in a time-lapse of 24 h are shown in Figure 7.
C. albicans
AuNPs show no effect on growth kinetics until 10 h (Figure 7A), varying in a dose-dependent manner the absorbance reached at 24 h. Interestingly, with 50 µg/mL or more, the growth kinetic shows a steep negative slope from 10 h until reaching a 45% reduction at 24 h, which suggests an antifungal effect of these materials. This can be attributed to the ability of gold nanoparticles to interact with relevant proteins present in fungus such as H+-ATPase, affecting proton pump activity. This atrophying the ability of yeast to incorporate nutrients causing its death [61]. In Figures 7B and 7C was observed that AgNPs and Au@AgNPs inhibit the growth of the yeast Candida albicans from 10µg/mL. The determination of the MIC50 concentration for both materials was estimated from the dose-response curve shown in Figure S5. MIC50 is defined as the concentration of nanoparticles that produces a 50% decrease in absorbance concerning the control (yeast without treatment). For AgNPs and Au@AgNPs, MIC50 were 2.21µg/mL and 2.37µg/mL, respectively.
However, according to the EDS results (Figure 7B), the silver content in Au@AgNPs is 64.85% mass. Thus, the concentration of silver in Au@AgNPs for MIC50 is 1.53µg/mL, 30% lower than in the case of AgNPs.
E coli
In Figure 7D, AuNPs do not show significant inhibition (<15%) or affect the growth kinetics of E.coli. For AgNPs (Figure 7E) at low concentrations, the Lag phase remains unchanged, but there is a marked decrease in growth ratio indicated by the slope decrement. At 50 µg/mL Lag phase lasts up to 16 h and viability reaches a maximum of 20% at 24 h. For 100 µg/mL, an apparent detachment of the growth phase of the microorganism is not observed In Au@AgNPs (Figure 7F), the first two concentrations do not show changes in their growth phase, but a phase delay of up to 2 h is observed compared to the control. It is interesting to note that the lag phase lasts up to 21 h for the 50 µg/mL concentration, finally there is no explicit growth behavior for the 100 µg/mL concentration.
S. aureus
A comparative analysis of lag phase regrowth occurred after 12 h for Au (Figure 7G), Ag (Figure 7H), and Au@AgNPs (Figure 7I) in the case of S. aureus at 50 µg/mL. For the highest concentration at 100 µg/mL, there is no growth of the bacteria. Additionally, we observe changes in the slope of respect control for Au, Ag, and Au@AgNPs at 1 and 10 µg/mL.
AuNPs interaction with these Gram-positive bacteria could be due to the charged surface that causes an electrostatic interaction, destabilizing membrane structure. Similar results, but with higher NPs concentrations are reported for AuNPs synthesized using Ananas comosus fruit extract as reducing agent [62] and blue-green alga Spirulina platensis protein [63]. Yang et al. show MIC > 500 µg/mL for S. aureus (CMCC(B)26003), our AuNPs has shown inhibition with 10 times less concentration, in this case, a critical synergy exists with polyphenols molecules on coating and stabilizing the surface of nanoparticle [64]. ROS is generated of less to higher intensity [65] by AuNPs, polyphenols (plant extracts), and AgNPs, so AuNPs in synergy with resveratrol and EGCG promotes antibacterial response over S. aureus [66] had the most feasible mechanism in this case. Penders et al. reported 250 and 500 µg/mL of AuNPs like antibacterial agents over S. aureus increases in bacterial growth lag time and antibacterial effect [62, 63, 67].
We believe that inhibition is caused by AgNPs accumulation and diffusion on bacteria related to NPs surface charges that promote electrostatic interactions [68] with the bacteria´s membrane leading to higher penetration and damage. We think this is a similar mechanism described for interactions between E. coli biofilms and AgNps [69].
For Au@AgNPs, the obtained results are comparable to those reported by other workgroups [64, 65]; however, different authors suggest that the inhibition of the growth of the microorganisms is directly related to the thickness of the shell [64, 65]. Core-shell NPs showed low cytotoxicity when tested in NIH-3T3 fibroblasts cells (normal mammalian cells) [62]. A lower proportion of silver in the shell of the Au@AgNPs shows similar results to AgNPs [64], Au core potentialize antibacterial effect, and minimize the cytotoxicity.
Curves Growth analyzed by the modified Gompertz model
To know how the growth ratios (µ) and Lag phase (λ) are quantitatively modified, the growth curves of microorganisms exposed to different concentrations of nanomaterials (Figure 7) were adjusted by the Gompertz model (Eq. 1).
In Figure 8A, it can be seen that all nanomaterials produce a decrease in the replication rate of S. aureus populations when the concentration of nanoparticles increases. This effect results in slightly higher sensitivity for AuNPs. At a concentration of 50 µg/mL, the growth ratio is only 30% concerning control (Table S1-S24); at a concentration of 100 µg/mL, all materials inhibit the growth of the S. aureus population. The behavior of the adaptive phase for S. aureus with the different treatments is shown in Figure 8B. It is observed that there are no significant differences in the material used, and at 50 µg/mL, the Lag phase has increased by almost 5 times compared to the adaptive phase of S. aureus (Tables S1-S24). In general, we can establish that the different nanomaterials evaluated in S. aureus reduce the replication rate and postpone the adaptive phase in a dose-dependent manner until its inhibition at 100 µg/mL.
Figure 8C clearly shows that AuNPs do not affect the growth ratio µ of E. coli bacteria. Meanwhile, AgNPs produce a decrease over µ, reaching a minimum value corresponding to 19% to the control (µ for E.coli without treatment) for 50 µg/mL (see table S52). In contrast, Au@AgNPs completely inhibit the E. coli growth at 100 µg/mL. Analysis of the behavior of E. coli Lag phase exposed to different materials is shown in Figure 8D. In this case, unlike Figure 8B, each material has a characteristic response. Thus, AuNPs do not generate any modification in the adaptive phase of E. coli, while AgNPs and Au@AgNPs have a dose-dependent effect on the Lag phase, the latter material standing out. Thus, we can establish that AuNPs have no appreciable effect on E. coli bacteria, and Au@AgNPs can inhibit replication and, therefore, indefinitely postpone the Lag phase of E.coli. Interestingly, this effect is not achieved for AgNPs even though the net silver content is higher than in Au@AgNPs. This suggests that the core@shell presentation of both metals produces a synergy that favors antimicrobial activity.