Preparation and antibacterial properties of titanium-doped ZnO from different zinc salts
© Sun et al.; licensee Springer. 2014
Received: 19 December 2013
Accepted: 7 February 2014
Published: 27 February 2014
To research the relationship of micro-structures and antibacterial properties of the titanium-doped ZnO powders and probe their antibacterial mechanism, titanium-doped ZnO powders with different shapes and sizes were prepared from different zinc salts by alcohothermal method. The ZnO powders were characterized by X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV-vis), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and selected area electron diffraction (SAED), and the antibacterial activities of titanium-doped ZnO powders on Escherichia coli and Staphylococcus aureus were evaluated. Furthermore, the tested strains were characterized by SEM, and the electrical conductance variation trend of the bacterial suspension was characterized. The results indicate that the morphologies of the powders are different due to preparation from different zinc salts. The XRD results manifest that the samples synthesized from zinc acetate, zinc nitrate, and zinc chloride are zincite ZnO, and the sample synthesized from zinc sulfate is the mixture of ZnO, ZnTiO3, and ZnSO4 · 3Zn (OH)2 crystal. UV-vis spectra show that the absorption edges of the titanium-doped ZnO powders are red shifted to more than 400 nm which are prepared from zinc acetate, zinc nitrate, and zinc chloride. The antibacterial activity of titanium-doped ZnO powders synthesized from zinc chloride is optimal, and its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are lower than 0.25 g L−1. Likewise, when the bacteria are treated by ZnO powders synthesized from zinc chloride, the bacterial cells are damaged most seriously, and the electrical conductance increment of bacterial suspension is slightly high. It can be inferred that the antibacterial properties of the titanium-doped ZnO powders are relevant to the microstructure, particle size, and the crystal. The powders can damage the cell walls; thus, the electrolyte is leaked from cells.
KeywordsAntibacterial Microstructure Alcohothermal method Zinc oxide Titanium-doped
Antibacterial agents are applied to many fields, such as food [1, 2], care , packaging , synthetic textiles , environmental , and so on. Chemical synthesis antibacterial agent is divided into two categories: organic and inorganic antibacterial agent [7–9]. Organic antibacterial agent has many disadvantages, including the toxicity hazard to the human body and instability in high temperature and pressure . By comparison, inorganic antibacterial agent has the properties of heat resistance, long life, and chemical stability . Nowadays, metallic simple substances and their compounds are used widely in antimicrobial application research, such as Ag [12–16], Fe2O3, TiO2, CuO [19, 20], MgO , Mg (OH)2, and ZnO [23, 24]. Among metal oxide antibacterial agents, ZnO has aroused concern due to its good antibacterial activities on a broad spectrum of bacteria [24–26]. The antibacterial properties of ZnO have been studied broadly with pathogenic and nonpathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas, etc. [26, 27]. Zinc oxide is an interesting material due to its extensive applications in various areas, such as antibacterial, optical, piezoelectric, magnetic, and gas sensing properties [24, 26–31]. Therefore, many of the synthetic approaches such as sol-gel method , co-precipitation , hydrothermal method , microwave synthesis [23, 26], and thermal evaporation method  have been used for the preparation of ZnO powders. Hydrothermal method is an important technology in synthetic material. Using this method, the crystal grain can develop completely and the particle size is uniform.
In this work, in order to research the influence of the microstructure and crystal on the antibacterial properties of titanium-doped ZnO powders, the powders were synthesized by alcohothermal method from different zinc salts, and the antibacterial activities against E. coli and S. aureus were evaluated. Moreover, the antibacterial mechanism of titanium-doped ZnO powders was deduced.
Materials and methods
The reagents (e.g., two hydrated zinc acetate, zinc vitriol, zinc nitrate, zinc chloride, lithium hydroxide monohydrate, absolute ethyl alcohol, tetrabutyl titanate, glutaraldehyde, disodium hydrogen phosphate 12-hydrate, monopotassium phosphate) used in this study were analytically pure chemicals. Biological reagents (e.g., nutrient broth, nutrient agar medium) were used as received. De-ionized water and aquae sterilisata with conductivity lower than 0.5 μS/cm were used to prepare all the solutions. E. coli (ATCC44104) and S. aureus (CMCC26001) bacterial strains were obtained from Beijing Assay Institute of Biological Products. Phosphate-buffered saline (PBS; pH = 7.4) was prepared with disodium hydrogen phosphate 12-hydrate and monopotassium phosphate.
Synthesis of titanium-doped ZnO powders
Under magnetic stirring condition, 0.1 mol/L zinc salts and 0.14 mol/L lithium hydroxide alcoholic solution were prepared. Meanwhile, 0.01 mol/L tetrabutyl titanate alcoholic solution was prepared. Then, 80 mL of lithium hydroxide alcoholic solution was dropwise added to 160 mL zinc salt alcoholic solution during the stirring process. After that, 80 mL of tetrabutyl titanate alcoholic solution was added to it drop by drop. Subsequently, 8 mL of deionized water was added into the mixed solution, and then the mixed solution was treated by ultrasound for 1 h. The mixed solution was shifted into the hydrothermal reactors with 70% filling, and then the reactors were sealed and heated for 24 h at 140°C. After the reactors were cooled naturally to room temperature, the precipitates were collected and washed several times using distilled water and then were dried at 40°C. After grinding, the titanium-doped ZnO powders were prepared.
Evaluation of antibacterial activity
Bacterial strains (E. coli and S. aureus) were cultured overnight in nutrient broth medium at 37°C before being used. The strains were diluted to 105 to 106 colony forming units (CFUs) per milliliter with PBS. Twenty milliliters of dilute bacterial suspension was taken in each of the iodine number flask, respectively. The powders of 0.25 to 2.5 g/L were added into each flask. The bacterial suspension without powders was used as positive control. All the iodine number flasks were put on a shaker bed at 150 rpm and incubated at 37°C for 24 h. Both the treated and control bacterial suspensions were diluted by a series of twofold dilutions in PBS solution. The dilute solutions with appropriate dilution ratio were then plated on nutrient agar plates to assay the colony forming ability. Plates were incubated at 37°C for 48 h, and the colonies were counted. All experiments were performed in triplicate, and the averages were obtained.
Characterization of titanium-doped ZnO powders
The crystalline phases of the powders were characterized by X-ray powder diffraction (XRD) using D/MAX-RB X-ray diffractometer (Rigaku, Tokyo, Japan) with Cu K radiation in the 2θ range of 10° to 70° at a scan rate of 8°/min. Fourier transform infrared spectra (FT-IR) of the powders were characterized using Scimitar 2000 Near FT-IR spectrometer (Thermo Electron, Madison, WI, USA), and the spectra were recorded in the range of 4,000 to 400 cm−1. The UV-visible diffuse reflectance spectra of the powders were recorded with a model Shimadzu UV2550 spectrophotometer (Shimadzu, Nakagyo-ku, Kyoto, Japan). The morphologies of the powders were examined by field emission scanning electron microscopy (FESEM; S-4800, Hitachi, Ltd., Chiyoda, Tokyo, Japan) and field emission transmission electron microscopy (FETEM; JEM-2100 F, JEOL Ltd., Akishima, Tokyo, Japan). Meanwhile, the crystalline characters of the powders were examined.
Characterization of cells' morphology
Fresh bacterial culture was treated with titanium-doped ZnO powders at 37°C for 18 h, and then the bacterial suspension of control and treatment were fixed with 2.5% (v/v) glutaraldehyde for 2.5 h. After being centrifuged at 2,500 rpm for 5 min, the liquid supernatant of bacterial suspension was discarded. After being added aquae sterilisata and centrifuged for three times, a drop of bacterial suspension was placed on the cover slip and dried under vacuum. The bacterial cells' morphologies were examined using FESEM (S-4800, Hitachi).
Measurement of the electrical conductance of bacterial suspension
The E. coli and S. aureus in logarithmic phase were washed with aquae sterilisata for three times, and then the concentration of the bacterial suspension was adjusted to 107 CFU/mL. One hundred milligrams of titanium-doped ZnO powders were added to 50 mL 107 CFU/mL of bacterial suspension. The bacterial suspension without powders was used as control. The electrical conductance of the bacterial suspension was measured with 10-min interval. All experiments were performed for three times, and averages were obtained.
Results and discussion
XRD characterization of titanium-doped ZnO powders
FT-IR spectra of titanium-doped ZnO powders
UV-visible spectra of titanium-doped ZnO powders
SEM characterization of titanium-doped ZnO powders
TEM characterization of titanium-doped ZnO powders
The EDS spectrums (Figure 5(a1, a2)) of the samples synthesized from zinc acetate show that titanium is almost undetected in the rods, yet the fine particles next to the rods contain a certain amount of titanium. It indicates that the titanium is not doped in the ZnO and there is amorphous substance in the samples. This is why the titanium is not detected in the XRD. Figure 5(b1) shows that a large number of titanium is in the agglomerate substance of the samples synthesized from zinc sulfate. When the samples are prepared from zinc nitrate, EDS results (Figure 5(c1, c2)) show that the sheets contain more titanium than the rods. Similarly, (Figure 5(d1, d2)) shows that the fine particles contain more titanium than the formed particles which is synthesized from zinc chloride. Meanwhile, the atomic percentage content of titanium in the tooth shape particles is 12.14%; it is almost consistent with the experimental process in which the molar ratio of titanium and zinc is 1 to 10. It manifests that titanium is almost utterly doped in the ZnO.
The crystalline characters of the samples are checked by selected area electron diffraction. Figure 5(a3) shows that samples synthesized from zinc acetate have certain crystalline state, and the crystalline grain size is slightly larger. The (101), (102), and (112) crystal faces are detected. This is consistent with the XRD. When the raw material is zinc sulfate, the diffraction pattern displays the ( 10) lattice plane of Zn (SO4)2 · 3Zn (OH)2 and (101), (102), and (201) lattice planes of ZnO (Figure 5(b2)). The result is consistent with the XRD. When the raw material is zinc nitrate, (101), (102), and (201) crystal planes of ZnO are detected, and the diffraction rings are obscure (Figure 5(c3)). It demonstrates that the samples are composed of amorphous and crystalline forms. The SAED pattern of the samples prepared from zinc chloride displays the (002), (101), (102), (110), (103), (200), and (201) crystal planes of ZnO (Figure 5(d3)). It indicates that the samples are hexagonal phase. Besides, there are some scattered bright spots in the diffraction pattern. It demonstrates that the grain size is slightly larger.
Antibacterial properties of titanium-doped ZnO powders
Colony count of E. coli after antibacterial activities by titanium-doped ZnO powders
Powder concentration (g/L)
1.25 × 108
2.1 × 107
1.95 × 107
1.75 × 107
1.2 × 107
3.85 × 106
2.9 × 103
1.65 × 103
1.1 × 107
9.75 × 106
5.3 × 106
2.95 × 105
5.6 × 104
1.6 × 104
7.65 × 103
2.15 × 107
1.9 × 107
1.65 × 107
1.6 × 107
3.35 × 105
2.8 × 103
3.05 × 104
6.55 × 103
3.9 × 103
2.5 × 103
2.3 × 103
2.0 × 103
Colony count of S. aureus after antibacterial activities by titanium-doped ZnO powders
Powder concentration (g/L)
1.95 × 108
5.25 × 107
5.2 × 107
4.0 × 107
3.4 × 107
3.0 × 107
4.15 × 105
2.1 × 103
8.85 × 107
8.3 × 107
7.55 × 107
4.35 × 107
4.0 × 107
6.25 × 106
2.0 × 105
9.65 × 107
9.15 × 107
8.9 × 107
8.3 × 107
1.01 × 107
2.6 × 105
6.0 × 102
7.35 × 104
5.6 × 104
2.0 × 104
3.5 × 103
1.9 × 103
1.7 × 102
SEM characterization of E. coli and S. aureus cells
From what is mentioned above, we can reach the conclusion that the extent of damage to E. coli and S. aureus cells is positively related to the antibacterial properties of titanium-doped ZnO powders (Tables 1 and 2). Moreover, many powders are attached to the bacterial cells' surfaces, and the energy-dispersive spectrometer results (Additional file 1) demonstrate that they are titanium-doped ZnO particles (yellow circles in Figures 6 and 7 correspond to the EDS spectra in Additional file 1 in sequence).
The electrical conductivity of bacterial suspension before and after treatment
The bacterial cell wall can provide strength, rigidity, and shape for the cells and can protect the cells from osmotic rupture and mechanical damage. The bacterial cells can be divided into Gram-positive cells and Gram-negative cells according to their cell wall structure. Besides, the wall of Gram-positive cells contains a thick layer of peptidoglycan (PG) of 20 to 80 nm, which is attached to teichoic acids. By contrast, Gram-negative cell walls are more complex, both structurally and chemically. The wall of Gram-negative cell contains a thin PG layer of 2 to 3 nm and an outer membrane of 8 to 10 nm, which covers the surface membrane .
Comprehensive analysis reveals that the antibacterial properties of titanium-doped ZnO powders are affected not only by the size but by the crystallinity. When the powders are attached to the bacterial surface, titanium-doped ZnO crystals reacted with PG, teichoic acids, and lipoteichoic acids, and then the structure of bacterial cell wall is damaged. The titanium-doped ZnO powders are crystalline nanorods synthesized from zinc acetate, and its antibacterial activities are lower than the others. Meanwhile, the bacterial cell wall is damaged slightly, and the electrical conductance of bacterial suspension is increased; it indicates that the destroy capacity of the powders to bacterial cell wall and cell membrane is feeblish. This could be because of the weak doping level of titanium in ZnO crystal, although the particle size is smaller than the others. When the titanium-doped ZnO powders are prepared from zinc nitrate, the particles are six prismatic crystals with big size. The bacterial cell wall is damaged seriously, and the electrical conductance of bacterial suspension is increased; it proves that the powders' damage capability to the bacterial cell wall and cell membrane is great. It could be due to good doping level of titanium in ZnO crystal and high dissolving ability of metal ion from the crystals. The titanium-doped ZnO powders are spherical and tooth shape nanoparticles, which are synthesized from zinc chloride. After treatment with them, the bacterial cell wall and cell membrane are damaged seriously, and the increase of electrical conductance of the bacterial suspension is greater than the others. It indicates that the capability of the powders to the cell wall is high and makes the penetrability of cell membrane increased. This is due to high doping level of titanium and small size of particles. When the bacterial suspension is treated by the powders prepared from zinc sulfate, the antibacterial activity is weak and the damage degree of bacterial cell wall is slight. It demonstrates that the antibacterial activities of ZnTiO3 and ZnSO4 · 3Zn (OH)2 crystal are weaker than ZnO.
Furthermore, when the E. coli cell walls are damaged by titanium-doped ZnO powders, the holes appeared on the cells; this may be because the thin cell wall and outer membrane are easy to break. When the S. aureus cell walls are damaged by the powders, the cell walls become crinkly or honeycomb; this could be due to the thick layer of PG and the PG chemical network structure.
On the basis of the above analysis, it is inferred that the antibacterial properties of the titanium-doped ZnO powders are relevant to the particle size and the crystallinity.
The titanium-doped ZnO powders with different shapes and sizes were synthesized from different zinc salts. Antibacterial property results show that the titanium-doped ZnO powders have different antimicrobial activities. The antibacterial properties of the powders prepared from zinc chloride are optimal, and its MIC and MBC are lower than 0.25 g L−1. Moreover, the antibacterial action of the powders toward E. coli is stronger than that towards S. aureus.
This study was supported by the grant from the National Natural Science Foundation of China (No. 31371858), the National Key Technologies R & D Program of China during the 12th Five-Year Plan Period (No. 2012BAD29B06), and the Open Project of Food Safety Key Laboratory of Liaoning Province (LNSAKF2011022).
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