The synthesis of silver, zinc oxide and titanium dioxide nanoparticles and their antimicrobial activity

Three different types of nanoparticles were synthesised in this study, viz silver (Ag), zinc oxide (ZnO) and titanium dioxide (TiO 2 ) using different chemical methods. These materials were then characterised using Transmission Electron Microscopy (TEM), Fourier Transform Infra-Red Spectroscopy (FTIR), Ultraviolet Visible Spectroscopy (UV-Vis) and Thermal Gravimetric Analysis (TGA). The materials were also tested for anti-bacterial activity. TEM showed that the particles were in the nano-size range (1 – 100 nm). FTIR and UV-Vis Spectroscopy showed the different absorption bands of the synthesised nanoparticles, respectively. Silver nanoparticles showed greater antibacterial activity against several bacteria than titanium dioxide and zinc oxide nanoparticles. The highest inhibition was observed for Klebsiella pneumoniae . The results showed that antimicrobial activity of nanoparticles increases with increasing concentration of the nanoparticles. Copyright © 2017 VBRI Press.


Introduction
The re-emergence of infectious diseases (e.g. Ebola haemorrhagic fever, meningitis and tuberculosis) and increasing rates of resistant bacterial strains remains a big threat to health worldwide [1]. Bacteria develop resistance because they adapt to different antibiotics. Some of the pathogenic microorganisms that cause infections and fatal disease are from the Enterococcus, Staphylococcus, Bacillus and Streptococcus genera [2].
Antibiotics have been used as major treatment for such infections. Microorganisms, however have developed resistance towards these antibiotics. Due to emerging resistant bacterial strains, nanoparticles are now being used as antibacterial agents to fill the gaps where antibiotics fail. There are several methods that are used to test antimicrobial activity of nanoparticles including disc diffusion, broth dilution, agar dilution and minimum inhibitory concentration. Disc diffusion involves the use of antimicrobial discs which are placed on agar media. After incubation, the zone of inhibition is measured. Dilution methods are used to measure the antimicrobial concentration that can kill organisms [3].
Metal oxides nanoparticles have two pathways of attacking and destroying bacteria. Firstly, the disruption of the bacterial membrane. Secondly, they produce reactive oxygen species (ROS). The nanoparticles bind electrostatically to the bacterial cell wall and destroy the inner components causing the cell to die. Alternatively, they cause lysis by disturbing the respiratory chain of the bacteria. The main aim is to prevent bacterial growth [1].
Silver oxide (AgO) is an inorganic metal that possesses electrical, thermal conductivity and antimicrobial activity. Ag nanoparticles are frequently prepared via chemical reduction method. There are several reducing agents used for the synthesis of Ag nanoparticles including sodium borohydride, tri-sodium citrate and hydrogen peroxide. Silver nanoparticles antibacterial activity is dependent on their size, the smaller the nuclei the higher the antibacterial activity. Silver is commonly used in the medicinal sector to treat infections, burns and wounds. In the food industries it is used to control food spoilage. Environmentally it acts as a water purifying agent. Maiti et al. [4] reported that bacteria are less prone to develop resistance against Ag nanoparticles than against conventional antibiotics. Ag nanoparticles as antimicrobial agents are active against pathogens such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa [5].
Titanium oxide (TiO2) is a white powder. In nature it occurs as rutile, brookite and anatase mineral. In the study our focus was to produce anatase TiO2 nanoparticles. Anatase nanoparticles have large surface volume and high photocatalytic activity as compared to other phases. TiO2 is nontoxic to humans. It has a wide range of applications from purifying agents, antimicrobial agents and as coating agents in food industries. Zinc oxide (ZnO) is a well-studied metal oxide. It is insoluble in water, and it is used as a coating agent in the food, plastic, glass and paint industries. ZnO nanoparticles exhibit high activity towards Escherichia coli and Staphylococcus aureus. Their activity can be increased by capping these nanoparticles with other antibacterial agents [6].
This study aims to synthesise and characterise silver (Ag), titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles and to determine their antimicrobial activity.

Materials/ chemicals details
All chemicals were purchased from Sigma-Aldrich, South Africa. The biomolecules were purchased from Anatech, South Africa.

Material synthesis / reactions
Silver nanoparticles A 0.1 M silver nitrate (AgNO3) solution was prepared by dissolving 0.849 g (AgNO3) in 50 ml distilled water in a 100 ml beaker. Sodium borohydride 0.1 M (NaBH4) was prepared by dissolving 0.0567 g NaBH4 in 50 ml distilled water in a 100 ml beaker. An ice bath was prepared and placed on a magnetic stirrer. 15 ml of NaBH4 was measured and poured in a 100 ml beaker in the ice bath. 15 ml of AgNO3 was added drop-wise in the beaker with constant stirring. Polyvinylpyrrolidone (PVP) of 0.2 g was added to the solution. Mole ratios of NaBH4 and AgNO3 were varied (1:1, 1:2 & 2:1). The solution was stirred for 45 minutes in the ice bath. The solution was then centrifuged at 2000 rpm for 20 minutes. To remove the excess, the precipitate was washed three times with 40 ml distilled water. The precipitate was dried at 80 0 C in an oven [7].

Zinc oxide nanoparticles
Ethanol solution (0.5 M) was prepared by measuring 14.652 ml of pure (99.9%) ethanol and diluting with distilled water to make a 500 ml solution. A solution of Zinc nitrate (Zn (NO3)2.4H2O) was prepared by dissolving 2.62 g (Zn (NO3)2.4H2O) salt in 10 ml of 0.5 M ethanol with constant stirring using a magnetic stirrer for 1 hour. A solution of sodium hydroxide (NaOH) was prepared by dissolving 0.0399 g (NaOH) in 10 ml of 0.5 M ethanol with constant stirring using a magnetic stirrer for 1 hour. After, NaOH solution was added drop-wise into a solution of Zn(NO3)2 with constant stirring at a higher speed for 45 min.
Polyvinylpyrrolidone of 0.2 g was added to the solution. The reaction was allowed to proceed for 2 hours after complete addition of NaOH. After 2 hours, the beaker was sealed and allowed to settle overnight. The mole ratio of Zn(NO3)2 was varied (1:1, 1:2 & 1:3). The solution was centrifuged at 2000 rpm for 20 minutes. The precipitate was washed three times with 40 ml deionized water to remove the excess. The precipitate was dried in a muffle furnace at 500 0 C for 2 hours [8].

Titanium dioxide nanoparticles
A 25 ml solution of acetic acid (AcOH) was prepared by measuring 0.43 ml of concentrated acetic acid and diluted with distilled water in a 50 ml beaker. Acetic acid solution was placed on a magnetic stirrer. 2.58 ml of titanium tetrabutoxide (TBT) was added drop-wise into 25 ml acetic acid with constant stirring for 45 minutes. Polyvinylpyrrolidone of 0.2 g was added to the solution. After 45 minutes, the mixed solution of TBT and AcOH was allowed to stand overnight at room temperature. The mole ratio of AcOH and TBT were varied (1:1, 1:2 & 1:3). The solution was centrifuged at 2000 rpm for 20 minutes. The precipitate was washed with 40 ml deionized water and finally dried in an oven at 80 0 C. To obtain pure anatase TiO2 phase, the prepared powder was calcined in a muffle furnace at 400 0 C for 2 hours [9].

Maintenance and growth of bacterial strains
All the bacterial strains were collected from the Vaal University of Technology Biotechnology laboratory. The strains were grown and maintained on nutrient agar at 37 0 C for 24 hours and subcultured weekly. Stock cultures were stored at 4 0 C. Below (Table 1) is a list of the bacteria that were used for the antimicrobial studies.    Peaks between 1100 and 1700 cm -1 are characteristics of hydroxyl group and the absorbance of water [16]. Sample (c) shows a different pattern with two bands at 775.21 and 950.63 cm -1 . The latter band corresponds to the formation of O-Ti-O bonds. There were no peaks observed at 2900 cm -1 for all the prepared nanoparticles, which means that all organic compounds were removed during calcination [16].   Fig. 3a shows the TGA results for silver nanoparticles. Two weight loss stages were observed (figure 3a) between 100 0 C and 400 0 C for (1:2 and 2:1). In the first stage weight was lost from 100 0 C to 300 0 C. This was due to the evaporation of moisture [13; 20]. The second drop was from 300 0 C to 400 0 C. This was due to the degrading of some un-reacted materials [20].
There was no weight loss observed from 400 0 C to 700 0 C. This shows the stability of the pure synthesized silver nanoparticles [21]. Still in figure 3a, the 1:1 (mole ratio) nanoparticles exhibit a drastic weight loss from room temperature to 700 0 C. This is an indication of unstable nanoparticles. Hence, they cannot be used for application studies. This can be due to the imbalance of chemicals. Fig. 3b shows TGA curves of titanium dioxide nanoparticles of different mole ratios. In figure 3b, samples at all mole ratios show single continuous weight loss from room temperature to 700 0 C, at percentage losses of 2.28% (1:1), 5.378% (1:.2) and 5.07% (1:3). The weight loss in the 100 o C region was attributed to the release of water and the subsequent loss was due to the breakdown of the PVP in the sample [22]. At (1:2) mole ratio, a single weight loss stage which range from room temperature to 300 0 C was recorded. From 300 0 C the sample did not show any weight loss till 700 0 C (It remained constant). This shows the stability of the nanoparticles according to Bagheri et al. [15]. The sample of 1:3 mole ratio, shows two weight loss stages. The first stage ranges from room temperature to 300 o C; this is due to a combined process of removal of water (100 o C) and unreacted materials (300 o C). The second stage ranges from 300 0 C to 700 0 C. This is due to the decomposition of PVP from the sample [22]. Fig. 3c shows TGA curves for zinc oxide synthesized nanoparticles with different ratios. In figure 3, the 1:1 mole ratio sample shows that a small amount of weight loss has accrued (about 1%) partly caused by evaporation of water from the sample at 100 o C. The 1:2 mole ration sample shows three weight loss stages. The first stage from 100 to 200 0 C is related to the removal of moisture from the sample. Second stage from 540 to 670 0 C is due to decomposition of PVP [19]. The 1:3 mole ratio sample shows two weight loss stages. The first stage is between 100 to 360 0 C and is attributed to the evaporation of moisture and decomposition of un-reacted material. The second stage ranges from 400 to 650 0 C due to the decomposition of PVP from the sample.  . 4a) shows mixed shapes Ag nanoparticles of mole ratio 1:2. The particle size distribution of the single nanoparticle ranges from 0.5 to 3 nm in diameter. This confirms that the particles are in the nanometer range. The image shows aggregated nanoparticles, this means that PVP was limiting. PVP is used to stabilize and prevent agglomeration of the nanoparticles [23]. Fig. 4b shows the micrograph of TiO2 of mole ratio 1:2. The image shows mixed in shape nanoparticles with particle size between 2 to 12 nm in diameter. It is clear from the image that particles are not agglomerated as compared to Ag nanoparticles micrograph. The morphology and ZnO nanoparticles and size distribution is shown on figure 4c. The histogram shows that the particle size ranges from 2 to 12 nm in diameter. The micrograph shows mixed in shape ZnO nanoparticles. There is much less agglomeration as compared to Ag nanoparticles.  Table 2 shows disc diffusion results after adjusting the concentration of the nanoparticles to 200 mg/ml. Six organisms were used throughout the test. The Ag, TiO2 and ZnO nanoparticles at concentration 25, 50 & 100 mg/ml showed no bacterial inhibition as compared to 200 mg/ml. At a concentration of 200 mg/ml Ag nanoparticles showed antibacterial activity against selected bacteria including Escherichia coli (7 mm), Bacillus cereus (9 mm), Bacillus subtilis (6 mm) and Klebsiella pneumoniae was the most sensitive bacteria with 11 mm zone of inhibition (  [4] showed that inhibition depends upon silver nanoparticle concentration, so when silver concentration is increased the bacterial concentration was found to decrease. Ghosh & Ramamoorthy [26] also reported that antibacterial activity of silver is dependent on size and concentration. The antibacterial activity is due to their size and large surface area volume. Ag particles managed to penetrate through the membrane of selected gram negative and positive bacteria. It appears that Ag nanoparticles are bactericidal at high concentration.  [27] reported that the activity of both nanoparticles is limited and dependent on size, morphology of nanoparticles and the characteristics of the organism being tested. Small nanoparticles are more effective than large nanoparticles. Padmavathy & Vijayaraghavan (2008) showed that ZnO nanoparticles of 12 nm are more effective than suspension with larger particle size [29]. These nanoparticles are active against gram negative bacteria rather than to gram positive. Gram positive bacteria have a thicker 50% peptidoglycan layer than gram negative [30]. This thick layer makes it difficult for the nanoparticle to penetrate or disrupt the cell membrane. Zones of inhibition were observed where streptomycin (positive control) was used with a large zone of inhibition of 23 mm for Bacillus cereus. The tested bacteria were inhibited by this antibiotic. No inhibition was observed on the negative controls. Resazurin dye was used as a growth indicator. Active bacterial cells reduce the Resazurin dye to produce pink colour. Blue colour indicates inhibition of the bacteria. Tests were done in triplicate. Ag nanoparticles showed inhibition for several bacteria such as Bacillus cereus, Klebsiella pneumoniae, Escherichia coli and Bacillus subtilis for the initial concentration 33.34 mg/ml only (Fig. 5). Ag nanoparticles were less effective against Pseudomonas aeruginosa and Staphylococcus aureus. These results are comparable to disc diffusion. Titanium dioxide and ZnO nanoparticles were less effective against all the organisms at all concentrations as shown by the appearance of pink colour. These extracts were unable to kill the organisms. It is reported in the literature that the activity of both nanoparticles is limited and dependent on size, morphology of nanoparticles and the characteristics of the organism being tested [30]. Small nanoparticles are more effective than large nanoparticles. They also active against gram negative bacteria because they have less peptidoglycan layer than gram positive [30]. This thick layer makes it difficult for the nanoparticle to penetrate or disrupt the cell membrane. No colour changes were observed where streptomycin (antibiotic) was used. Negative control showed growth of the tested bacteria. The antibiotic (positive control) managed to inhibit the bacterial growth even at low concentration of 0.026 mg/ml. These controls were used to make sure that the solvent doesn't have any inhibitory effects and to make sure the test is working.

Conclusion
This study showed that chemical reduction and precipitation methods can be used for the synthesis of silver, titanium dioxide and zinc oxide nanoparticles due to its purity and cost-effectiveness. The formation of these nanoparticles was confirmed by UV-Vis spectroscopy, FTIR, TGA and TEM. TEM analysis confirmed that the synthesized nanoparticles were in the nanometer range. The chemical reduction and precipitation methods produced mixed shaped nanoparticles between the ranges of 0.5 to 12 nm. Based on the results obtained after characterization, second mole ratios of Ag, TiO2 and ZnO nanoparticles were used for application studies. Silver nanoparticles inhibited both gram positive and negative bacteria. Titanium dioxide and zinc oxide nanoparticles didn't show any antibacterial activity. The antibacterial activity of Ag nanoparticles increases with increasing concentration of nanoparticles.