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nanoparticles, which is of great importance to assess the nature of the stabilizing agent (capping) [22]. The presence of the band positioned at 230 cm−1 can be attributed to the Ag-N stretching mode, which may be originated from the fungal extract, providing the nitrogen atoms that will finally take part of the capping of the silver nanoparticle. Even more, bands positioned at the fingerprint region can be Maotlterciubleust2e0d20,to25,t2h0e23presence of L-valine and L-alanine, which indicate that proteins from the f6uonfg1a4l extract are part of the capping, or are near the nanoparticle metallic surface [22]. Molecules 2020, 25, x FOR PEER REVIEW 7 of 15 a decrease in intensity at the Surface Plasmon Resonance (SPR) band, as well as a shift in the visible spectrum towards the infrared. Although PchNPs were stable for most of the evaluated conditions, a decrease at the band corresponding to the SPR band, as well as a slight broadening of the peak, were observed at 100 and 500 mM NaCl and pH 3. These results could be attributed to the neutralization of surface charges of PchNPs, which presented negative net charge at pH 6, resulting Figure 5. Raman spectrum for PchNPs with superimposed Raman spectra of l-valine and l-alanine. in their aggregation. Figure 5. Raman spectrum for PchNPs with superimposed Raman spectra of L-valine and L-alanine. 2.4. Colloidal Stability Assays The colloidal stability of PchNPs over pH 3–9 and under conditions of high ionic strength ([NaCl] = 10–500 mM) were evaluated (Figure 6a,b respectively). Aggregation of the nanoparticles generates Figure 6. Stability of PchNPs (a) from pH 3 to 9, and (b) at different ionic strength, [NaCl] 0–500 nM Figure 6. Stability of PchNPs (a) from pH 3 to 9, and (b) at different ionic strength, [NaCl] 0–500 nM 2.5. Antimicrobial Activity of PchNPs against E. coli 2.5. Antimicrobial Activity of PchNPs against E. coli 2.5.1. Antibacterial Activity against E. coli 2.5.1. Antibacterial Activity against E. coli In order to determine the antibacterial activity of PchNPs, a bacterial inoculum of E. coli in LB In order to determine the antibacterial activity of PchNPs, a bacterial inoculum of E. coli in LB media was supplemented with different concentrations of PchNPs and incubated for 24 h. Consistent media was supplemented with different concentrations of PchNPs and incubated for 24 h. Consistent with results reported by other authors using biogenic nanoparticles [23], in this work the Resazurin cell with results reported by other authors using biogenic nanoparticles [23], in this work the Resazurin viability assays showed minimum inhibitory concentration (MIC) of PchNP of 0.25 nM for E coli cells. cell viability assays showed minimum inhibitory concentration (MIC) of PchNP of 0.25 nM for E coli 2c.e5l.l2s.. ESEM and TEM of E. coli Cells Exposed to PchNPs Based on the results of antibacterial assays, we opted to use electron microscopy techniques 2.5.2. ESEM and TEM of E. coli Cells Exposed to PchNPs (TEM and ESEM) to evaluate the interaction of the silver nanoparticles (at 0.12 and 0.25 nM) with Based on the results of antibacterial assays, we opted to use electron microscopy techniques E. coli. ESEM imaging shows that when E. coli cells are exposed to PchNPs (Figure 7c–f), their cell (TEM and ESEM) to evaluate the interaction of the silver nanoparticles (at 0.12 and 0.25 nM) with E. wall shows loss of integrity when compared to the control (Figure 7a,b). At lower, sub-MIC, PchNPs coli. ESEM imaging shows that when E. coli cells are exposed to PchNPs (Figure 7c–f), their cell wall concentrations (Figure 7c,d), bacterial cells appear to be losing part of their cytoplasmic components shows loss of integrity when compared to the control (Figure 7a,b). At lower, sub-MIC, PchNPs (indicated by arrows in Figure 7d) and, with the increment of the concentration (Figure 7e,f), the concentrations (Figure 7c,d), bacterial cells appear to be losing part of their cytoplasmic components damage of the cell membrane is clearly evidenced (indicated by arrows in Figure 7f). Meanwhile TEM (indicated by arrows in Figure 7d) and, with the increment of the concentration (Figure 7e,f), the images demonstrate that at the lowest PchNP concentration of 0.12 nM (Figure 8c,d), bacterial cells damage of the cell membrane is clearly evidenced (indicated by arrows in Figure 7f). Meanwhile TEM possess large conglomerates of silver nanoparticles on their surface (indicated by arrows) and show images demonstrate that at the lowest PchNP concentration of 0.12 nM (Figure 8c,d), bacterial cells distinctive morphological changes when compared to control (Figure 8a,b). possess large conglomerates of silver nanoparticles on their surface (indicated by arrows) and show distinctive morphological changes when compared to control (Figure 8a,b).PDF Image | Biofilm Eradication Using Biogenic Silver Nanoparticles
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