Synthesis of Silver Doped Titanium Dioxide by Wet-Ball Milling Sol–Gel Method for Antibacterial Application

Titanium dioxide (TiO2) has been extensively studied as photo-catalyst for water treatment, air purification and antibacterial applications due to its challenging properties such as chemical stability, environmental friendly and strong photocatalytic activity. However, the limitation of TiO2 on its dependent to ultraviolet radiation for photocatalytic activity is still aroused. In this study, silver doped titanium dioxide (Ag-TiO2) was synthesized by wet-ball milling sol–gel method (WBMS). Ag-TiO2 molar ratio was varied from 0% to 10% to study the effect of silver content on the synthesized Ag-TiO2 characteristics and the ability to apply on antibacterial applications. The objective of this work was to find an optimal concentration of Ag in Ag-TiO2. Characterization of the particle size, morphology, and surface area of synthesized Ag-TiO2 were discussed by techniques of transmission electron microscopy (TEM) and Brunauer-Emmett-Teller (BET). Photocatalytic activity was investigated from degradation of methylene blue. Antibacterial activity was conducted by finding minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests performed on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) under dark condition and under visible light. The results demonstrated that the doping of Ag inhibited crystal growth of Ag-TiO2. The smallest particle size and the highest surface area were obtained from 5% Ag-TiO2. Also, it was found that methylene blue degradation rate increased to the highest number of 1.62x10-3 min-1 when Ag concentration reached 5%, and methylene blue degradation rate reduced when Ag concentration was higher than 5%. The antibacterial activity of Ag-TiO2 was better than TiO2. The optimal concentration of 3-5% Ag-TiO2 was observed from the


Introduction
Titanium dioxide (TiO2) nanoparticle is photocatalyst has been widely researched in waste treatment applications due to its strong oxidizing property, non-toxicity and long-term photo stability (Zaleska, 2008). It has been also in the research interest for the antibacterial applications. However, the limitation of using TiO2 upon its dependency on UV light is prominent. To overcome this limitation of TiO2 nanoparticles, as well as enhancing the effectiveness of its photocatalytic and antibacterial activity, silver doped TiO2 (Ag-TiO2) nanoparticle is proposed. In this study, Ag-TiO2 was synthesized by wet ball milling sol-gel method (Phromma et al., 2017). Effect of silver concentration on the photocatalytic activity and antibacterial activity was investigated. Characterization of the particle size and morphology of synthesized Ag-TiO2 were determined.

Ag-TiO2 Preparation
Ag-TiO2 was synthesized by wet-ball milling sol-gel method, modified from the previous report (Phromma et al., 2017). The substitution reaction of Titanium (IV) isopropoxide 97%wt (TTIP) with methanol (MeOH) were carried out with a molar ratio of 1:15. The solution pH was adjusted to 2 by adding 2 ml of HNO3. The various amount of Ag was added to the TiO2 solution in concentration of 3%, 5%, 6% and 10% by mole. The yellow colloid of Ag-TiO2 was obtained. Citric acid was used to reduce Ag + into Ag 0 . The Ag-TiO2 gel was then observed. Washing Ag-TiO2 gel with DI water by centrifuged at 10,000 rpm for 15 min, then the yellow gel was dried at 110°C for 24 hours. The yellow powder, then, appeared. The yellow powder was dry ground with 10 mm-ball milling at 300 rpm for 20 min and consequently wet ground by 2 mm-ball milling at 500 rpm for 3 hours in IPA. The Ag-TiO2 solution was dried again at 110°C for 24 hours and was calcined at 400°C for 4 hours with a heating rate of 5°C/min. After all the processes mentioned, Ag-TiO2 nanoparticle powder was synthesized. The nomenclature 3% Ag-TiO2, 5% Ag-TiO2, 6% Ag-TiO2 and 10% Ag-TiO2 are used to represent the various concentration of silver doped.

Characterization of synthesized Ag-TiO2
The morphology and particle size distribution of Ag-TiO2 were studied by high resolution transmission electron microscopy (HRTEM, JEOL JEM-2100) operated at 200 kV. Brunauer-Emmett-Teller (BET) was used to determine surface area of Ag-TiO2 particles. The degradation of methylene blue (MB), (Ajax Finechem), under UV radiation was used as a model system to evaluate the photocatalytic activity of Ag-TiO2. The photocatalytic reaction was carried out in the in-house photocatalytic chamber. The initial concentration of MB (C0) was prepared at 5 ppm. A 10-mg of Ag-TiO2 was mixed with 40 ml of MB solution and then stirred in the photocatalytic chamber. The mixture was kept in the dark for 1 h to ensure the saturation of MB on the surface of the catalysts. The MB solution was irradiated by 0.25 UV light Wm -2 (λ=351 nm). The sample was collected at every hour for 24 hour. The concentration of MB in suspension was analyzed using a UV-Vis spectrophotometer at wavelength of 635 nm. The kinetic constants of reaction rate were determined according to the pseudo-first-order kinetic model as follows: where t is the irradiation time k is kinetic constant (min -1 ), and C is the concentration of the MB (mg/L).
The antibacterial activity of the synthesized Ag-TiO2 was investigated using the standard microdilution method. Minimum inhibitory concentration (MIC) that inhibited the growth of bacterial strain was determined on 96-well microdilution plates. Initial concentration of microorganisms (Staphylococcus aureus; S. aureus 8739 as a gram positive or Escherichia coli; E. coli 6538P as a gram negative bacteria) was 105 cfu/ml. Preparation of Ag-TiO2 solution by dissolved 10 mg Ag-TiO2 in 10% dimethyl sulfoxide (DMSO) and make a serial dilution of Ag-TiO2 powder with Mueller-Hinton Broth (MHB). End point were determined when no turbidity in the 96-well was observed after 24 hours under dark and UV light condition of incubation at 37°C and 95% relative humidity. Minimum bacterial concentration (MBC) was also determined by counting colony on agar plates after incubated at 37°C for 24 hours.

Results and Discussions
From TEM images, as shown in Figure 1, silver distribution was observed as black particles scattering on white powder. The scattered silver particles came close to each other more when the silver concentration was higher. The agregation of particles seemed to be more obvious with higher silver amount added. As demonstrated in Table 1, particle size of Ag-TiO2 increased from 16.4±5.2 nm, when there was no Ag added, to 34.0±9.1 nm when it was 3% Ag-TiO2. Higher concentration of silver, however, did not cause further increasing of the particle size. It stayed in a range of 21 -28 nm. This pattern might be well-explained owning to the larger size of Ag+(ca.126 pm) when compared to the size of Ti4+(ca.68 pm). With small amount of silver added, the possibility of silver doped in the lattice of TiO2 is greater, and expand the particle size of the Ag-TiO2. When more silver was added, however, silver ion rather stayed on the outer surface of the TiO2 than doped into the structure. The Ag+ dispersed on surface of TiO2 effect the density of surface increased defect at the surface of anatase grain which favors the rutile nucleation (Chao et al., 2003). Then, surface area of 3% Ag-TiO2 decreased from 18.62 to 13.97 m2 /g as shown in Table1 due to large particle size of 3% AgTiO2. Increasing of molar ratio of Ag from 5% to 6%, the particle size decreased from 23.0±7.4 nm to 21.8±7.2 nm. Since the Ag doping in optimum molar ratio usually hinders the growth of particle size (Ahmad et al., 2007). The highest surface area was obtained at 5% AgTiO2. Moreover, when molar ratio of Ag increased from 6% to 10%, the particle size was increased to 28.5±9.6 nm relative with decreasing of surface area. Then, 10% Ag-TiO2 showed agglomeration of nanoparticle since it presented high surface energy of the particle which the particle size of TiO2 is not limit of growth.  Figure 2 showed photocatalytic activity of TiO2 and Ag-TiO2 with the degradation of MB solution under UV light and calculated the kinetic constant rate (k) as shown in Table 2. It was shown that the photocatalytic activity of Ag-TiO2 were higher than TiO2 powder with kinetic constant rate increased from 1.03x10 -3 min -1 to 1.62x10 -3 min -1 The highest photocatalytic activity with the kinetic constant of 1.62x10 -3 min 1 was obtained with 5% Ag-TiO2. This result was compliant to the highest surface area of 5% Ag-TiO2 as discussed earlier. When Ag-TiO2 was increased from 6% to 10%, the photocatalytic activity decreased as can be seen by the decreasing of kinetic constant to 1.53x10 -3 min -1 . Since a large silver nanoparticle hindered UV absorption onto TiO2, in consequence, the degradation of MB was slower.   Table 3 show MIC of TiO2 and Ag-TiO2 with different concentrations under dark and UV light condition. The results showed that neither MIC nor MBC of microorganism growth could not be found in case of using TiO2 under dark condition. This is obvious that the antibacterial applications using TiO2 solely cannot be acheived due to its dependance to UV light. Considering MIC obtained when using different concentrations of Ag-TiO2, it was clearly seen that the bacteria growth can be inhibited even in the dark condition when silver is doped to TiO2. MIC of E. coli and S. aureus were the same when 3% Ag-TiO2, 5% Ag-TiO2, and 6% Ag-TiO2 were used with both dark and UV-light conditions. However, the MIC was decreased to almost half when 10% Ag-TiO2 was applied. It might be able to explain that 10% Ag-TiO2 showed the highest percentage of silver nanoparticles which affect directly to bacteria cell and cause its death. The MBC is smaller when Ag-TiO2 was used in UVlight condition, indicating that the synegistic effect of bacteria degradation by titanium and silver was presented. The MBC of E. coli and S. aureus under UV-light condition of TiO2 showed the highest amount of 10 mg/ml to kill bacteria. On the other hand, Ag-TiO2 gave lower amount of concentration to kill bacteria. Therefore, Ag-TiO2 showed the higher antibacterial activity than TiO2. MBC of E. coli of 3% Ag-TiO2 under UV light was at 0.625 mg/ml which is twice lesser than that of the dark condition at 1.250 mg/ml. As MBC of S. aureus under dark and UV light conditions were the same at 2.500 mg/ml. It suggested that S. aureus is more resistant than E. coli. As can be seen when 5% Ag-TiO2 was used, MBC of E. coli was less than that os S. aureus. When increasing silver concentration to 6% Ag-TiO2 -10% Ag-TiO2, however, MBC of E. coli and S. aureus were the same. The lowest MBC of E. coli is showed when using 3% Ag-TiO2 -5% Ag-TiO2 and MBC of S. aureus also the same value at 2.500 mg/ml. It was proved that the antibacterial activity of Ag-TiO2 is higher than that of TiO2. Looking back to the images shown in Figure 1, it can be seen that Ag nanoparticle tends to deposit on TiO2 surface. The deposited Ag can be oxidized with oxygen to generate Ag+ ion. Ag+ ion can attack the cell wall of bacteria and diffuse into the cytoplasm leading to the cell death. Moreover, TiO2 makes photocatalytic reaction happened and generate reactive oxygen species that can kill bacteria. Furthermore, from the results we obtained the higher value MBC of S. aureus than E. coli is because the difference in cell wall structure between gram positive and gram negative bacteria. Because gram negative consists of a thin peptidoglycan layer (PG) So, gram negative bacteria can be attacked easier than gram positive.

Conclusion
Doping silver to TiO2 nanoparticles by WBMS method are successful. It results in modifying the lattice structure of TiO2 nanoparticle and cause the different size and surface area depending on the concentration of silver doping. The synthesized Ag-TiO2 nanoparticle shows higher performance on antibacterial applications when compared to TiO2. The optimal amount of Ag in TiO2 is 5%by mole. It shows the highest photocatalytic activity with kinetic constant rate 1.62x10 -3 min -1 . Antibacterial activities of 3% Ag-TiO2 and 5% Ag-TiO2 for E. coli and S. aureus are the highest and higher than those of TiO2 as well.