3.1. FTIR analysis
FTIR spectra were collected and shown in Figures 1-3. Figure 1 shows the FTIR spectra of pure TiO2 and Ag-doped TiO2 (5,10,15%) nanoparticles synthesized by the sol-gel method and calcined at 450°C. Figures 2 and 3 show the FTIR spectrum of pure TiO2 and Ag-TiO2 nanoparticles synthesized by the coprecipitation method and calcined at 450°C.
Due to the need for anatase crystalline forms, all samples were calcined at 450°C for 1 hour. A broad band observed between 3700-3000 cm-1 relates to the O-H stretching mode of the hydroxyl group, indicating the presence of moisture in the sample. The peaks between 3000-2800 cm-1 are assigned to C-H stretching vibrations of alkane groups. The observed peak in the region 1636 cm-1 can be associated with the asymmetric stretching mode of titanium carboxylate. The Alkane and carboxylate groups are the precursors TTIP and 2-propanol used in the synthesis process. The strong absorption band observed between 800 and 450 cm-1 can be related to the vibrational modes of TiO2 [34]. (or bond stretching vibrations (Ti-O-Ti) [35]. On the other hand, all the IR spectra of Ag-TiO2 nanoparticle samples have a peak in the region of 1385 cm-1, which is not observed in undoped TiO2, and its intensity increases by increasing the amount of Ag in precursor )AgNO3) in the samples. The peak in the region of 1385 cm-1 is empirically attributed to the mutual effects between TiO2 and Ag particles [34].In Figure 1, peaks in the regions 3417.39 cm-1 (graph a), 3412.13 cm-1(graph b), 3424.76 cm-1 (graph c), and 3425.12 cm-1 (graph d) are related to the O-H stretching state of the hydroxyl group, the observed peaks in the regions 1621.09 cm-1 (graph a), 1620.32 cm-1 (graph b), 1629.28 cm-1 (graph c), and 1627.43 cm-1 are assigned to the asymmetric stretching mode of titanium carboxylate and the peaks in the regions 752.69 cm-1 (graph a), 462.19 cm-1(graph b), 663.62 cm-1, 808.70 cm-1 (graph c), and 750.22 cm-1 (graph d) are attributed to the vibrational modes of TiO2, the peaks in the regions 2879.25 cm-1 (graph b), 2847.83 cm-1 (graph c), and 2882.86 cm-1 graph (d) are assigned to C-H stretching vibrations of alkane groups. The peaks in the regions 1344.45 cm-1 (graph b),1313.36 cm-1 (graph c), and 1342.42 cm-1 (graph d) to the mutual effects between TiO2 and Ag particles are assigned.
In Figures 2 and 3, the peaks in the areas of 1620.94 cm-1 (graph 2a), 1617.05 cm-1 (graph 2b), 1647.75 cm-1 (graph 2c), 1616.08 cm-1 (graph 3a),1630 cm-1 (graph 3b) are related to the asymmetric stretching state of titanium carboxylate, the peaks in the regions 449.55 cm-1 (graph 2a), 454.16 cm-1 (graph 2b), 470.01 cm-1 (graph 2c), 439.13 cm-1 (graph 3a), 665.80 cm-1, 843.41 cm-1 (graph 3b) are attributed to the vibrational modes of TiO2 and the peaks in the regions 1344.42 cm-1 (graph 2b), 1316.07 cm-1 (graph 2c), 1313.17 cm-1 (graph 3a), 1313.08 cm-1 (graph 2c ) are attributed to the mutual effects between TiO2 and Ag particles, which were not observed for the undoped TiO2 (graph 2a). The intensity of these peaks increases by increasing the silver precursor (AgNO3) in the samples.
3.2. XRD analysis
The XRD measurements were carried out to investigate the crystal structure of nanoparticles, determine the crystalline phases, and estimate the crystallite size of the prepared nanoparticles shown in Figures 4-7. In Figure 4, we have two samples: one by coprecipitation method and another by sol-gel method were Synthesized and baked at 450°C for one hour. The XRD pattern for the sample synthesized by the coprecipitation method shows the presence of peaks at the angles of 2θ=25.209°, 36.746°, 37.527°, 38.404°, 47.874°, 53.533°, 54.864°, 62.365°, 68.298°, 70.030°, and 74.626° corresponding to the lattice planes of 101, 103, 004, 112, 200, 105, 211, 204, 116, 220, and 215, respectively, which are related to the given phase-pure TiO2 nanoparticles in anatase. It corresponds to the ICDD card number (01-071-1168) (tetragonal crystal structure). The sample synthesized by sol-gel method shows the presence of peaks at the angles of 2θ=25.156°, 36.637°, 37.385°, 38.314°, 47.782°, 53.344°, 54.752°, 62.191°, 68.052°, 69.884°, and 74.397° corresponding to the lattice planes of 101, 103, 004, 112, 200, 105, 211, 204, 116, 220, 215, respectively, which are related to the given phase-pure TiO2 nanoparticles in anatase. It corresponds to the ICDD card (01-071-1169) (tetragonalcrystal structure). The XRD results show that the synthesized TiO2 powder is mainly crystallized in the anatase phase.
Figure 5 shows the XRD pattern of TiO2 particles for the sample synthesized by sol-gel method, calcined at 700°C, and compared with 450°C. At the calcination temperature of 700°C, the rutile phase was noticed as the dominant phase. Diffraction patterns of calcined TiO2 show rutile peak 2θ=27.434°, 36.076o, 39.188°, 41.236o, 44.040°, 54.316o, 56.622o, 62.752°, 64.043°, 68.999°, 69.794°, and 74.394° corresponding to lattice planes of 110, 101, 200, 111, 210, 211, 220, 002, 310, 301,112, and 320, respectively, are which is consistent with the ICDD card number (01-072-1148) and in this model phase is rutile and crystalline structure is tetragonal. While in the calcined sample at a temperature of 450° anatase phase is formed, and the crystalline structure is tetragonal.
Figure 6 shows the XRD spectrum of Ag-TiO2 nanoparticles (5% Ag) synthesized by the coprecipitation method and annealed at 450°C for an hour. The XRD pattern shows the presence of peaks at the angles of 2θ= 25.293°, 36.910°, 37.731°, 38.543°, 48.023°, 53.810°, 55.042°, 62.631°, 68.659°, 70.266°, and 74.971°, which are related to the pure anatase phase of TiO2 with the ICDD card number (01-071-1167), which were assigned to the lattice planes 101, 103, 004, 112, 200, 105, 211, 204, 116, 220, and 215, respectively, and have a tetragonal crystal structure. Also, the normal diffraction peaks 200, 112, and 310 are located at the angles of 2θ=32.035°, 50.033°, and 55.984°, respectively. The phase is AgO, corresponding to the ICDD card number (00-043-1038), and has a tetragonal crystal structure, and the normal diffraction peaks 003, 200, and 204 are located at the angles of 2θ=34.253°,44.386°, and 65.661°, respectively. The phase is Ag2O corresponding to the ICDD card number (00-042-0874), and has a hexagonal crystal structure, so the anatase phase was identified as the dominant phase.
Figure 7 shows the XRD spectra of pure and Ag-doped (15,20%) TiO2 NPs at 550°C. Diffraction peaks positioned at 2θ=5.293°, 36.910°, 37.731°, 38.543°, 48.023°, 53.810°, 55.042°, 62.631°, 68.659°, 70.266°, and 74.971° corresponds to the pure anatase phase of TiO2 with the ICDD card number (01-071-1167) and has a tetragonal crystal structure. Also, the normal diffraction peaks 200,112,310 are located at the angles of 2θ=32.035°, 50.033°, and 55.984°, respectively, corresponding to the AgO phase with the ICDD card number (01-043-1038) and have a tetragonal crystal structure and the normal diffraction peaks 003, 200, 204 are located at the angles of2θ=34.253°, 44.386°, and 65.661°, respectively corresponds to the Ag2O phase with the ICDD card number (00-042-0874) and has a Hexagonal crystal structure.
Moreover, the typical diffraction peaks (110), (202), positioned at 27.470° and 76.586° correspond to the rutile phase of TiO2 with the ICDD card number (01-021-1276), so the anatase phase was identified as the dominant phase.
The calcined samples at a temperature of (450, 700, and 550°C) are shown in Figures 4-7. As shown in the figure, in the calcinated samples at a temperature of 450°C, the crystalline phase of titanium oxide, the anatase phase, is formed in the presence of Ag. Also, increasing the calcination temperature from 450 to 550°C accompanies the anatase phase, i.e., the rutile phase is formed next to Ag. With further increase in temperature up to 700°C, all the anatase phase is converted to the rutile phase, and the Ag crystals also become larger. These spectra show that the phase transformation from anatase to rutile is in the temperature range of 450-700 °C. Therefore, 450 °C is considered optimal for the anatase phase formation.
3-3- Antibacterial test
The antibacterial activity of pure TiO2 and Ag-TiO2 nanoparticles was investigated using the agar disk diffusion method, as shown in Figures 8-12.
Figure 8-11 shows that the pure sample of TiO2 and Ag-TiO2 (5% Ag) synthesized with coprecipitation and sol-gel methods show the presence of bacterial colonies. In contrast, Ag-TiO2 (20%) such bacterial colonies reduce significantly in the sample. A clear zone of inhibition is shown around the disks loaded with these nanoparticles. The formation of the zone of inhibition has confirmed the antimicrobial effectiveness of Ag-TiO2 (20%). Excellent antibacterial performance was achieved using TiO2 by increasing Ag concentration compared to pure TiO2.
Figure 12 indicates a clear zone of inhibition around the disks loaded with Ag-TiO2 nanoparticles (15% Ag) synthesized by the sol-gel method in an alkaline environment (PH=8) in the presence of both Staphylococcus aureus and E. Coli bacteria. Also, the disk loaded with Ag-TiO2 nanoparticles (15% Ag) synthesized by the coprecipitation method in an alkaline environment (PH=8) in the presence of E. Coli bacteria shows the formation of the zone of inhibition. However, no zone of inhibition is observed in the presence of Staphylococcus aureus bacteria. During the incubation, the Ag ions from the doped nanomaterials gradually leave the disks and enter the granular agar. The Ag ions bind to the cell membrane, interact with protein thiol groups, and inactivate respiratory enzymes, producing reactive oxygen species [36-37].
Table 1 shows the effect of Ag-TiO2 against Gram-positive bacteria S. aureus and Gram-negative bacteria E. coli. It should be noted that the tetracycline antibiotic was used as a positive control, and the diameter of the zone of inhibition for this antibiotic was 26 mm in the case of Staphylococcus aureus and 18 mm in the case of Escherichia coli.
Therefore, our observations showed that in plates impregnated with Ag-TiO2 (20% Ag) synthesized by coprecipitation (for both bacteria) and Ag-TiO2 (15% Ag) synthesized by sol-gel method (for both bacteria) and Ag-TiO2 (15% Ag) synthesized by coprecipitation method (for E. Coli bacteria in alkaline media at PH=8) zone of inhibition was formed. While in plates impregnated with the other nanoparticles and Ag-TiO2 (15% Ag) synthesized by the coprecipitation method )except E. Coli bacteria in alkaline media), a zone of inhibition was not formed, and numerous bacterial colonies were observed.
Table 1. The diameter of the zone of inhibition (cm) of S. aureus and E. coli bacteria by TiO2 and Ag- TiO2 nanoparticles prepared through coprecipitation and sol-gel methods.
Diameter of containment area (cm)
|
Diameter of containment area (cm)
|
|
|
|
|
|
Staphylococcus aureus
|
Escherichia coli
|
pH
|
Type of nanoparticles
|
Precipitation method
|
Ag (%)
|
No.
|
0mm
|
0mm
|
|
Pure TiO2
|
|
0
|
1
|
0mm
|
0mm
|
|
Ag- TiO2
|
Sol-gel
|
5%
|
2
|
0mm
|
0mm
|
|
Ag- TiO2
|
Sol-gel
|
10%
|
3
|
0mm
|
0mm
|
|
Ag- TiO2
|
Co-Precipitation
|
5%
|
4
|
0mm
|
0mm
|
12
|
Ag- TiO2
|
Co-Precipitation
|
12%
|
5
|
0mm
|
0mm
|
4.5
|
Ag- TiO2
|
Co-Precipitation
|
15%
|
6
|
0mm
|
15mm
|
8
|
Ag- TiO2
|
Co-Precipitation
|
15%
|
7
|
10mm
|
14mm
|
8
|
Ag- TiO2
|
Sol-gel
|
15%
|
8
|
12mm
|
13mm
|
|
Ag- TiO2
|
Co-Precipitation
|
20%
|
9
|
26mm
|
18mm
|
|
Tetracycline
|
|
|
10
|
These tests show that the antibacterial activity depends on the percentage of doping, PH, the reaction environment, and the synthesis method. The higher the percentage of doping, the higher the antibacterial activity. Also, the antibacterial activity in alkaline pH (8-8.5) is more than in acidic pH (4.5). Using TiO2 nanoparticles together with ultraviolet radiation can increase the antibacterial properties, and since we did not have ultraviolet radiation here, no antibacterial properties were seen from TiO2 nanoparticles. And as mentioned before, to have this property in visible light, doping with Ag is one way to modify it.
The antibacterial test against Staphylococcus bacteria and especially E. Coli showed a significant increase in the antibacterial properties of Ag-TiO2 nanoparticles, which is due to the action of doped Ag as an electron trap and, as a result, preventing the recombination of produced electrons and holes and increasing the antibacterial activity. It becomes bacterial.
This observation can also indicate the higher resistance of Gram-positive bacteria (ATCC 33591) S. aureus against synthesized nanomaterials compared to Gram-negative bacteria (ATCC 35218) E. coli. It is consistent with reports showing that S. aureus is more resistant to Ag nanoparticles than E. coli [38]. Our results showed that E.coli is more sensitive than S. aureus at the same concentration of tested nanomaterials. This can be attributed to structural differences in the outer cell wall between Gram-positive and Gram-negative bacteria, which determine cell permeability. It should be noted that Jayesh P. Ruparelia et al. [39] reported that some E. Coli strains, such as MTCC739 and MTCC1687, are more resistant than S. aureus strains. Therefore, the antibacterial effect of nanoparticles does not depend only on the structure of the bacterial membrane.
4.3.SEM images
In order to investigate the surface morphology of Ag-TiO2 nanoparticles, SEM studies were conducted. SEM images of TiO2 and Ag-TiO2 samples are shown in Figures 13-15.
Figure 13 shows an SEM image of TiO2 prepared by coprecipitation and sol-gel method with irregular porous particles whose average size is 291 and 381 nm, respectively. In Figure 13 a, we can see that in the coprecipitation method, the first sample contains agglomerates (dense masses) and also a large amount of fiber caused by Titanium tetra-isopropoxide (Ti (OC3H7)4) (TTIP) precursor. On the other hand, the second sample is very different. In Figure 13b sol-gel method, there are no fibers, and Titanium tetrabutoxide Ti(OC4H9)4 (TTB) precursor is used.
In Figure 14, Ag-TiO2 nanoparticles show relatively compact surface morphology with less porosity than pure TiO2 samples. In Figure 14a, SEM images show samples of TiO2 nanoparticles with lower density and high porosity. In contrast, In Figure 14b, SEM images show Ag-TiO2 nanoparticle powders with higher density and lower porosity.
In Figures 15 and 16, the TiO2 sample at 450°C at pH = 4.5 shows higher accumulation than at pH = 12. The aggregation of particles leads to an increase in particle size. The effect of pH also contributes to changes in particle size. It was observed that the morphology of TiO2 largely depends on the pH values. The higher the pH value, the smaller and denser the particle. The size of Ag-TiO2 particles prepared in Figure 15a with pH = 12 is 113.86 nm, and Figure 15b with pH = 4.5 is 246.64 nm.
The morphology of TiO2 was observed to be largely similar to that of Ag-TiO2 nanoparticles, forming irregular aggregates. These irregular aggregates are formed due to the addition of H+ ions to the solution when titanium alkoxides are subjected to hydrolysis. Adding Ag nitrate lowers the pH level, increasing the presence of H+ ions. The positive ion breaks down the broken particles into smaller sizes and prevents initial crystallization, which will interact with each other to form an amorphous mass. However, after drying, the absence of the H+ ion and its replacement with Ag makes the crystallization of TiO2 happen earlier [40].