Synthesis of ZnTiO3 and Ag/ZnTiO3 and Their Antibacterial Performances
2018 Volume 59 Issue 7 Pages 1112-1116
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2018 Volume 59 Issue 7 Pages 1112-1116
Antibacterial materials, namely ZnTiO3 and Ag/ZnTiO3, were prepared by a sol–gel method from tetra-n-butyl orthotitanate, zinc nitrate, and ethylenediaminetetraacetic acid (EDTA) as a complexing agent. The effects of the synthetic conditions on the properties of the obtained ZnTiO3 samples, i.e., the Zn2:Ti4:EDTA ratios and the calcination temperature, and the effects of different Ag:Ti4+ ratios on the properties of ZnTiO3 doped with Ag were investigated. These materials were characterized by powder X-ray diffraction, scanning electron microscopy, and transmission electron microscopy, and their antibacterial activities against Staphylococcus aureus were also evaluated. The results showed that the optimum conditions for ZnTiO3 synthesis were Zn2+:Ti4+:EDTA ratios of 1:1:1 and calcination at 650°C for 2 h. Spherical Ag/ZnTiO3 nanoparticles of average diameter 30–50 nm showed effective antibacterial properties with and without exposure to sunlight. Ag/ZnTiO3 (Ag:Ti4+ = 1:20) at a concentration of 10 mg/mL killed over 99.86% of S. aureus bacteria within 4 h.
Fig. 9 Effect of exposure to sunlight on antibacterial activity of Ag/ZnTiO3 (Ag/Ti4+ = 1:20) off concentration 10 mg/mL.
Antibacterial compounds either locally inhibit bacterial growth or kill bacteria without releasing toxic by-products to the surrounding environment.1) Ag nanoparticles show excellent antibacterial activity.2–5) Cho et al. reported strong antibacterial activity of Ag nanoparticles against Staphylococcus aureus and Escherichia coli, and complete inhibition of S. aureus growth was achieved with 100 ppm of Ag nanoparticles.6) Reduction of the cost of Ag nanoparticles by doping with other stable nanomaterials needs to be considered.7)
Recently, the synthesis of antibacterial materials from metal oxides such as titanium and zinc oxides has been reported.8) The ZnO–TiO2 system can generate several compounds such as ZnTiO3 (ZTO), Zn2TiO4, and Zn2Ti3O8, which can be used for various applications, e.g., as catalysts, pigments, and antibacterial materials.9) Among these compounds, ZTO has attracted much attention in recent years because of its perovskite structure and antibacterial properties, which are effective with or without sunlight exposure.10) Sotyanova et al. synthesized ZTO via a sol–gel method, with Ti(OEt)4 and Zn(CH3COO)2 as the main precursors.11) Ruffolo et al. reported the use of ZTO nanopowders as antimicrobial coatings for stone. ZTO showed high biocidal effectiveness against Aspergillus niger.12)
Doping of ZTO nanostructures with Ag to improve their properties has been investigated in several studies. For example, Ag-doped ZTO showed improved photocatalytic activity in the degradation of organic pollutants under solar irradiation and increased antibacterial activity.13) A Ag/TiO2/ZnO nanopowder (AZTO), prepared by a facile one-pot hydrothermal process, showed good antibacterial and photocatalytic properties.14) AZTO nanopowder therefore has potential as an antibacterial material.
ZTO with a combination of the antibacterial activities of TiO2 under sunlight and of ZnO without sunlight has not been previously reported. The synthesis of ZTO by a sol–gel method with use of a complexing has not been reported. Perovskite materials with small and uniform particles can be produced by a sol–gel method and a low calcination temperature. Moreover, Ag metal can be doped into ZTO during the preparation process to enhance the antibacterial activity at the nanometer scale.
In this study, we synthesized ZTO with the perovskite structure by a sol–gel method with use of a complexing agent. The effects of Ag metal on the ZTO (AZTO) perovskite structure were studied. The structures and morphologies of ZTO and AZTO were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and powder X-ray diffraction (XRD). The antibacterial activities of ZTO and AZTO against S. aureus were also studied.
Zn(NO3)2·6H2O (99 mass%), AgNO3 (99.8 mass%), and tetra-n-butyl orthotitanate [(C4H9O)4Ti, 99.8 mass%], purchased from Merck, were used as the precursors. Ethylenediaminetetraacetic acid (EDTA, 99.8 mass%) and ethylene glycol (EG, 99.8 mass%) were used as a chelating agent and dispersing agent, respectively. All the chemicals were analytical grade and used without further purification.
2.2 Synthesis of ZnTiO3 and Ag/ZnTiO3A ZnTiO3 nanocrystalline powder was prepared by a sol–gel method with use of a complexing agent and a low calcination temperature. The first reactant mixture, containing Zn(NO3)2 and EDTA, was prepared as an aqueous solution at 80°C. The pH of the mixture was adjusted to 4.5 ± 0.5 with NH4OH aqueous solution under stirring at room temperature. The second mixture, which was prepared by dissolving (C4H9O)4Ti in EG, was slowly added to the first mixture under continuous stirring. The mixture was then dried at 150°C for 5 h and the precursor, which was a black powder, was collected. The precursor was then calcined at various temperatures for 2 h, and then ground with a ball mill for 5 h.
The ZTO structure was doped with Ag metal by adding AgNO3 solution to the reaction mixture after addition of the solution of (C4H9O)4Ti in EG. A similar process was used for AZTO preparation.
2.3 CharacterizationXRD patterns of ZTO and AZTO were recorded with a D8 Advance X-ray diffractometer (Japan). The instrument was operated at 40 kV and 40 mA, with Ni-filtered Cu Kα radiation (λ = 0.15418 nm), in the 2θ range 10° to 70°. The morphologies of AZTO and ZTO were examined using a FE-SEM instrument (Hitachi S-4800), operated at 10 kV. TEM micrographs of the samples were obtained with a JEM-1400 instrument, which was operated at 80 kV. The specific surface areas of the samples were determined by the Brunauer–Emmett–Teller method from N2 adsorption–desorption isotherms at 77 K.
2.4 Antibacterial activityThe antibacterial activities of the samples were determined by the colony count method and from the minimum inhibitory concentration (MIC). S. aureus (ATCC 29523) with an initial cell density of 9.8 × 104 colony-forming units per milliliter (CFU/mL) was used. Samples of bacteria (105 CFU/mL) were treated with various concentrations of ZTO and AZTO powders (5–20 mg/mL) at 37°C for 24 h. The viable cells were counted. The removal efficiency was defined as
| \begin{equation*} \eta (\%) = (N_{1} - N_{2}) \times 100\%/N_{1} \end{equation*} |
The MIC was considered to be the lowest nanopowder concentration that gave no visible growth of S. aureus after incubation.
The effect of the Zn2+:Ti4+:EDTA ratios on the ZTO morphology was investigated. Precursors with different Zn2+:Ti4+:EDTA ratios were calcined at 650°C for 2 h. Figure 1 shows that mainly ZnTiO3 was formed when ratios of 1:1:1 were used. When the ratios of Zn2+:Ti4+:EDTA were changed to 2:1:6, Zn2TiO4 was the main product. A trace of ZnO and a small amount of ZnTiO3 were found in the mixture. These results show that increasing the concentrations of Zn2+ and EDTA in the reactant mixture increased the proportion of Zn2TiO4 and decreased that of ZnTiO3 in the product. Zn2+:Ti4+:EDTA ratios of 1:1:1 were therefore chosen for the synthesis of ZnTiO3.
XRD patterns of samples synthesized with Zn2+:Ti4+:EDTA ratios of 1:1:1 and 2:1:6.
The effect of the calcination temperature on the microstructure of ZnTiO3 is shown in Fig. 2. The precursor, which was prepared with the optimum Zn2+:Ti4+:EDTA ratios, was calcined for 2 h at 500, 650, and 750°C. The XRD patterns clearly show that increasing the calcination temperature improved the ZnTiO3 crystallinity. The crystalline phase of ZnTiO3 was first formed at 500°C. Some low-intensity peaks from ZnTiO3 were present in the XRD pattern. When the calcination temperature was increased to 650°C, all the XRD peaks matched those in the ZnTiO3 standard pattern [JCPDS 022380]. On further increasing the calcination temperature to 750°C, the peaks became more intense, which shows that the particles were highly crystalline; however, heat could cause particle aggregation. Smaller nanopowder particles have better antibacterial properties because the higher surface area leads to more interactions between the material and the bacteria. A calcination temperature of 650°C was therefore used in subsequent experiments.
Effect of calcination temperature on crystal structure of ZnTiO3.
A Ag/ZnTiO3 composite was synthesized with Ag:Ti4+:Zn2+:EDTA ratios of 0.1:1:1:1 (corresponding to a Ag:Ti4+ ratio of 1:10). The structure of the Ag-doped ZnTiO3 was determined and the presence of Ag metal in the product was confirmed. The XRD patterns of Ag-doped ZnTiO3 and ZnTiO3 are shown in Fig. 3; the Ag standard pattern [JCPDS 040783] is shown for comparison. The results show formation of a composite of Ag and ZnTiO3, with peaks at 2θ 38.116°, 44.277°, and 64.426°. In addition to incorporation into the perovskite structure, Ag+ in Ag2O, which was formed during calcination, was present on the surface of the material.13)
XRD patterns of ZnTiO3 and Ag/ZnTiO3 samples.
Figure 4 shows that when the Ag:Ti4+ ratio was increased from 1:100 to 1:20 and 1:10, the specific peaks from Ag became sharper and more intense. The amount of Ag+ incorporated into ZTO also increased with increasing Ag:Ti4+ ratio in the initial reactant mixture.13)
XRD patterns of Ag/ZnTiO3 samples with different Ag/Ti4+ ratios.
SEM and TEM images of Ag/ZnTiO3 are shown in Figs. 5 and 6, respectively. The images show some agglomeration regions with average particle sizes below 50 nm. The specific surface area of the Ag/ZnTiO3 sample was 13.79 m2/g; this is low because of particle agglomeration. However, this value is twice that of the specific surface area of Ag/ZnTiO3 (7 m2/g) reported by Dutta et al.15)
SEM image of Ag/ZnTiO3.
TEM image of Ag/ZnTiO3.
The effect of the proportion of Ag incorporated into ZnTiO3 on the antibacterial activity of the nanomaterial was investigated. Samples with Ag:Ti4+ molar ratios of 1:100; 1:20; and 1:10 were prepared and their antibacterial activities against S. aureus were determined by the MIC method. As shown in Fig. 7, the antibacterial efficiency of the material increased with increasing Ag:Ti4+ ratio.
Effect of Ag/Ti4+ ratio in Ag/ZnTiO3 on efficiency of S. aureus removal.
The maximum MIC for ZnTiO3 was 25 mg/mL; the value for Ag-doped ZnTiO3 was lower. At Ag:Ti4+ ratios of 1:100 and 1:20, the MIC value for AZTO was 5 mg/mL and the value further decreased to 2.5 mg/mL when the Ag:Ti4+ ratio was changed to 1:10. The results indicate that ZnTiO3 has antibacterial properties and the antibacterial activity can be enhanced by incorporating Ag into the ZnTiO3 structure.
3.3.2 Effect of Ag/ZnTiO3 concentrationThe effect of the Ag/ZnTiO3 (Ag:Ti4+ 1:20) concentration on the antibacterial activity was studied. A colony of S. aureus with an initial cell concentration of 9.8 × 104 CFU/mL was incubated with various concentrations of AZTO. As shown in Fig. 8, all the S. aureus bacteria were killed by incubation with 5 mg/mL of Ag/ZnTiO3 for 12 h. When the AZTO concentration was increased to 20 mg/mL, the S. aureus colony was effectively killed in 2 h. This indicates that a higher concentration of antibacterial material led to a shorter bactericidal time. At a Ag/ZnTiO3 concentration of 10 mg/mL, over 99.86% of S. aureus bacteria were killed within 4 h. These results are comparable to those reported by Stoyanova et al.,13) which showed that 100 mg of Ag:TiO2:ZnO (with molar ratios of 1:2:3) in 100 mL of 105 CFU/mL E. coli killed 99% of bacteria after 60 min. Pant et al.13) reported that under UV irradiation of intensity 20% Ag:TiO2:ZnO of concentration 0.4 mg/mL decreased the concentration of E. coli from 107 CFU/mL by about 99% in 80 min.
Effect of various concentrations of Ag/ZnTiO3 (Ag/Ti4+ = 1:20) on efficiency of S. aureus removal.
The results in Fig. 9 show that Ag/ZnTiO3 (Ag:Ti4+ = 1:20) showed excellent antibacterial activity against S. aureus under both sunlight and dark conditions. However, the antibacterial activity of AZTO was slightly more effective under exposure to sunlight. This is because sunlight has a phototoxic effect, and desorption of loosely bound oxygen from the surface of the material assists the generation of reactive oxygen species such as O2−, H2O2, and OH−. The generated active species can penetrate the cell membrane. When these toxic species are internalized into bacterial cells, they can destroy cellular components such as lipids, DNA, and proteins; this results in inhibition of bacterial activity and killing of microorganisms.16,17)
Effect of exposure to sunlight on antibacterial activity of Ag/ZnTiO3 (Ag/Ti4+ = 1:20) off concentration 10 mg/mL.
In this study, nanoscale ZnTiO3 and Ag/ZnTiO3 samples were successfully synthesized by a sol–gel method with use of EDTA as a complexing agent. Either ZnTiO3 or Zn2TiO4 was formed, depending on the ratios of the reactants. When the reactant ratios were 1:1:1, ZnTiO3 was the dominant product. Ag doping of ZnTiO3 significantly increased its antibacterial activity. The antibacterial activity of AZTO was improved by increasing the Ag:Ti4+ ratio. Ag/ZnTiO3 had highly effective antibacterial activity without exposure to sunlight.
We would like to thank the Department of Science and Technology, Ho Chi Minh City, Vietnam for financial support for this research through contract No. 87/2016/HĐ-SKHCN.
