Abstract
Structures, morphological control, and antibacterial activity of silver-titanium dioxide (Ag/TiO2) micro-nanocomposite materials against Staphylococcus aureus are investigated in this study. Horizontal vapor phase growth (HVPG) technique was used to synthesize the Ag/TiO2 micro-nanomaterials, with parameters of growth temperature and baking time. The materials were characterized by using scanning electron microscopy (SEM), energy dispersive X-ray (EDX) spectroscopy, and atomic force microscope (AFM). The result indicated that the HVPG technique is able to synthesize Ag/TiO2 with many shapes in micro- and nanoscale such as nanoparticles, nanorods, triangular nanomaterials, and nanotubes. The results showed that the shape of micro- and nanocomposites material could be arranged by adjusting the parameters. The results revealed that the nanorods structure were obtained at 1000°C growth temperature and that 8 hours of baking time was ideal for antibacterial application. Treating the S. aureus stock with Ag/TiO2 nanocomposites is able to reduce bacterial growth with a significant result.
1. Introduction
Recent forays into novel developments of medical research have involved nanomaterials as useful tools to combat cancer or bacteria, viruses, and other microbial pathogens [1–4]. Nanomaterials based on silver, titanium dioxide, carbon, and graphene have been studied for food packaging process, especially for medical applications such as antipathogenic activity or tissue regeneration [5–8]. There are eight main applications for nanotechnology in medicine: pathogen detection, protein detection, DNA structure penetration, tissue engineering, tumor eradication, pathogenic cell or molecule eradication, magnetic resonance imaging, and phagokinetic studies [9–11].
Synthetic silver or Ag/TiO2 nanomaterials have been developed and successfully manufactured by several methods due to the wide applications that are found tremendously in different fields. The synthetic methods include chemical reduction [12], chemical vapor deposition [13], electrochemical [14], green synthesis [15], HVPG [16–18], microwave [19], photochemical [20], radiation [21], and sonochemical [22].
In the case of synthesized silver-titanium nanocomposite, the research of this material applied in medical and health applications had attracted many researchers. Table 1 shows the use of silver-titanium dioxide nanocomposite material for antibacterial applications that has been performed in the previous study.
The previous study shows nanocomposite materials successfully synthesized by using HVPG technique. This technique offers certain advantages as follows: a large amount of nanocomposites material can be fabricated from the limited amount of powder-form source material, the synthesis occurred at the vacuum condition that minimizes contaminants, and by adjusting baking time and growth temperature, variations of the nanostructures can be produced. Moreover, statistics analysis of synthesis nanomaterials by using the HVPG technique reported that it was capable of creating nanomaterials with various shapes such as nanoparticles, nanorods, triangular nanomaterials, and nanotubes. [17, 18, 29, 30]. Table 2 shows the previous work that has been done by researchers regarding synthesized various nanomaterials by using the HVPG technique.
The goal of this study is to investigate the structures and morphological behavior of Ag/TiO2 micro-nanocomposites materials synthesized by using the HVPG technique. The study also evaluated the antibacterial effect of Ag/TiO2 nanocomposite materials against the gram-positive bacterium S. aureus, one of the species that is commonly infectious to humans [35–37] with bacterial colonies quantified through the pour plate technique. The micro-nanostructures synthesized from the HVPG technique were evaluated by using SEM and EDX to determine the shape of the Ag/TiO2 micro-nanocomposite. The AFM is used to determine the 3D surface roughness of the nanocomposite to explain the geometrical effect of nanocomposite that can eradicate bacteria. The antibacterial testing was conducted by comparing 2 different types of tubes with and without Ag/TiO2. By synthesizing the above material with HVPG technique and investigating with SEM, EDX, AFM, and pour plate technique, it is a step forward in the development of manufacturing of silver-titanium dioxide nanomaterials for antibacterial purposes with a simple, noncontaminant technique, which shapes a variety of nanomaterials.
2. Materials and Methods
The starting material was a mixture of 17.5 mg silver (Ag) powder from Aldrich Corporation and 17.5 mg titanium dioxide (TiO2) powder from Degussa P25. JEOL JSM-5310 scanning electron microscopy (SEM) was used to image and measure the synthesized nanomaterials found at all three zones of the tube. Energy dispersive X-ray spectroscopy (EDX) was used to determine the composition of the material in terms of silver or titanium dioxide. The 3D surface roughness of nanocomposite material was characterized by using Park System XE-100 atomic force microscopy (AFM).
The silica quartz tubes with an outer diameter of 11 mm and an inner diameter of 8.5 mm are used as the vessels for synthesis. The quartz tubes containing silver and titanium dioxide powder then were evacuated using the thermionic high vacuum system to create vacuum conditions with a pressure of approximately 10−6 Torr and then sealed. This effectively sterilizes the tubes and removes possible contaminating substances.
Figure 1 shows the nanocomposite materials after sealing and then placed half inside the furnaces and divided into 3 zones. The tubes were placed horizontally, with variable baking times of 4, 6, and 8 hours and variable temperatures growth of 800°C, 1000°C, and 1200°C. The tubes were only halfway inserted into the furnace, creating a temperature gradient between the hotter region inside the furnace and the cooler region outside the furnace. Figure 2 schematically depicts the vaporization of the source material located in the furnace and subsequent deposition outside the furnace.
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To quantify bacterial colonies, McFarland standard 0.5 was used, with serial dilutions to dilution factors 10−3 to 10−6. About 1.5 × 10−8 CFU/mL of inoculum of test organism, which is S. aureus, is spread onto the agar plate. The plate was placed on the inoculated plates and then incubated at 37°C for 16–24 hours. The pour plate technique was used to grow the colonies under three different setups: negative control of only the bacterial suspension and two setups with the nanocomposites of 17.5 gram Ag and 17.5 gram TiO2 [38–40].
A vortex mixer was used to homogenize the S. aureus suspension with the Ag/TiO2 nanocomposites. This was inoculated into Petri dishes and incubated at room temperature for 18 hours before colony counting. Figure 3 shows a flowchart of the experiment conducted in the study.
3. Results and Discussion
3.1. Material Characterization via SEM and EDX
SEM revealed the external morphology of the nanocomposites, while EDX determines its relative composition. Figure 4 shows the SEM image of Ag/TiO2 material before it was mixed. The results show that most of the materials are macro in size and in bulk form. In this study, the nanomaterials deposited at different zones of the quartz tube were separately investigated by using SEM.
Figure 5 shows the SEM images of the as-prepared hybrid nanocomposite materials Ag/TiO2 with a baking time of 4 hours. As shown, the materials are not fully transformed to its nanosized form and visible in zone 3 and partially shown in zone 2 both at 800°C of growth temperature. The results indicate that the baking time is not enough to evaporate silver and titanium dioxide macromaterials to nanoscale especially at 800°C growth temperature. Some amount of Ag and TiO2 bulk materials source material still had not changed into nanomaterials. In contrast, nanomaterials exhibit in zone 2 and zone 3 at 1000°C and 1200°C growth temperature. The nanoparticle size ranges from 700 to 1416 nm.
Figure 6 shows that the majority of silver nanomaterials were detected after Ag/TiO2 evaporated in zone 1, zone 2, and zone 3. The particles were exhibited on the inner surface of the silica tubes. It also indicates that the majority of the Ag/TiO2 deposit appeared at zone 2 of the tube. EDX spectroscopy in Figure 7 shows the majority of the material consisting silver exhibits in zone 2 of the tube at 1200°C growth temperature and 6 hours baking time. This phenomenon occurred due to inadequate growth temperature for the evaporation of TiO2.
Figure 8 shows the SEM images of the Ag/TiO2 nanocomposite materials. The results indicate that the majority of materials appear to be nanoparticles and nanorods. The diameter of the nanocomposites varies and has different shapes with different baking time. The results show that the smallest diameter of nanocomposites in all combinations occurred at 8 hours baking time and 1000°C growth temperature with a range of 361 nm–382 nm. In this combination, especially at zone 2, the silver nanorods are extended with sharp end and covered by nanocotton-like titanium dioxide.
The average diameters of the micro- and nanocomposites concerning the parameters of the study which are baking time and growth temperature are shown in Table 3. The material measurements based on SEM image and EDX results were evaluated from 3 different points as shown in Figure 9(a). The measurement method was performed by manual calculation. The results show the diameter of nanocomposites can be predicted statistically [17]. The results show that the smallest average diameter of micro-nanocomposite occurred at the 1000°C growth temperature and 8 hours baking time.
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Size, shape, and surface of the nanocomposites are the several critical aspects in determining their interaction behavior with cells. The study proved that the smaller the nanocomposites size, the higher the antimicrobial efficiency of the nanocomposites due to their direct interaction with the interface of the bacteria (membrane). Furthermore, the shape of the nanocomposites could directly influence the available contact area required to facilitate the interactions of nanocomposites with the bacterial membrane [41]. Silver nanoplates and silver nanorods showed higher antimicrobial activity towards bacteria compared to other silver nanoshapes [10]. The silver nanorods were capable of inhibiting all viable S. aureus cells within 4 hours by using 5 ppm silver nanorods solution and 3.5 hours by using 10 pp solution, respectively [42]. Moreover, based on Table 3 and images from SEM, 8 hours of baking time followed by 1000°C growth temperature could be an effective way to achieve high antimicrobial efficacy. The results show the nanocomposites diameter is from 300 nm to 600 nm with the majority of nanomaterials form as nanorods.
Figure 9 shows Ag/TiO2 nanorods with diameters around 300 nm to 400 nm (Figure 9(a)). Other forms of the synthesized nanocomposite include strand-like or cotton-like structures with bigger diameters that addressed to Ag/TiO2 micro-nanocomposites (Figure 9(b)). EDX analysis (Figure 9(c)) revealed appropriate amounts of Ag, Ti, and O in the nanocomposites along with Si likely from the quartz tube and Au from the coating material.
Figure 9 shows the typical Ag/TiO2 nanorods synthesized via HVPG technique. It is shown that silver nanomaterials are grown with the titanium dioxide covered on the surface. By using EDX spectroscopy, the nanorods come from silver nanomaterials, and cotton-like nanomaterials are from titanium dioxide nanomaterials. Furthermore, the results show that combination of silver and titanium dioxide can be fabricated with a controllable diameter as explained in the previous study by Muflikhun et al. [17]. To ensure Ag/TiO2 is capable of eradicating bacteria, the antibacterial test was conducted by using a combination tube with 8 hours baking time and 1000°C growth temperature.
3.2. Investigation of Antibacterial Property
The antibacterial property of the Ag/TiO2 nanocomposites were tested against S. aureus colonies. The standard analysis was conducted through the 0.5 McFarland standard (approximately 1.5 × 108 CFU/mL). Serial dilution was performed with dilution factors from 10−3 to 10−6. Figures 10 and 11 show the colonies grown at dilution factors 10−5 and 10−6, with and without the nanocomposites.
Figures 10 and 11 show that treatment with the Ag/TiO2 nanocomposites resulted in curbing the growth of S. aureus colonies. The result and images are presented as the significant capability of Ag/TiO2 nanocomposite to eradicate the S. aureus in terms of the amount of S. aureus with the nanocomposites. The antibacterial efficacy of the nanocomposite treatment is computed, and the complete data collected from counting bacterial colony growth in the different setups are compiled in Table 4.
Table 4 shows the bacterial test by using the Ag/TiO2 nanocomposite material (nanorods). The results show the nanocomposite materials are successfully capable of reducing bacterial growth with approximately 75% efficiency. It can be noted that the test is based on the dilution factor by using a 0.5 McFarland standard. The solution that contains bacterial colony was placed inside the tube that consists of nanomaterials. The tube that did not contain nanomaterials was used as the control plate. The tubes were rotated and shaken for around 5 minutes by using a Vertex machine to mix the bacterial colony with nanocomposite materials. The bacterial colony attached Ag/TiO2 nanorods when the rotation occurred. Since there is no standard for shaking and rotating the tubes by using the Vertex machine, the efficiency of the treatment can be improved by increasing the amount of nanocomposite material and the rotating time. Presumably, the more the nanorods formed and the longer the shaking time, the faster the bacteria eradication.
3.3. Nanocomposite Growth Mechanism
The synthesis of nanomaterials by using the HVPG technique is based on the thermal method where the material is changed at different stages as shown in Figure 12. The conversion depends on the temperature wherein, at low temperature, the material becomes solid. At high temperature, the material becomes liquid (at melting point) and gas (at boiling point) before it condenses in lower temperature and becomes solid again. In the case of Ag/TiO2 nanocomposite material, the shape of the nanocomposite depends on the growth temperature and baking time. The higher temperature will fasten the material conversion from liquid to gas and move from zone 1 (inside the furnace) to zone 2 and zone 3. Longer baking time will speed up the material deposit mostly in the bigger size (micro and nano) at zone 2 and zone 3, and lower baking time will create the smaller size of nanocomposite materials in zone 2 and zone 3.
3.4. Antibacterial Mechanism
Modification of silver nanomaterials with specific shape combined with titanium dioxide nanomaterials gives significant results in effectiveness against S. aureus. To understand the mechanism of how nanorods can eradicate the bacteria, some relevant papers discussed the nanomaterials contacted to bacteria should be considered. The previous studies conducted by Hajipour et al. [35] and Salata [9] show that nanomaterials have higher efficiency in eradicating bacteria. Bacteria are a robust organism that its cell wall is designed to provide strength and can protect its cell from osmotic rupture and physical damages. As a result, there are two common ways to eradicate bacteria, by the toxicity mechanism and by breaking the membrane of bacteria by the mechanical process. By toxicity, nanoparticles can attach to the membrane of bacteria by electrostatic interaction and disturb the integrity of it. For example, TiO2 nanomaterials are toxic to the bacterial only under ultraviolet (UV) illumination. The effectiveness of TiO2 under UV light was able to eradicate the bacteria in 1 hour. The mechanism of TiO2 to eradicate the bacteria can increase peroxidation of the polyunsaturated phospholipid component in the lipid membrane. This condition can disturb cell respiration. The Ag nanomaterials can eradicate bacteria by penetrating their membrane and increasing the toxicity in the presence of external magnetic field, electrostatic interaction, and physical damage of the bacteria [9, 35].
In this study, the nanocomposite materials are reported with the unique ability of Ag/TiO2 to eradicate bacteria caused by physical damage due to its shape. The present study shows that silver nanomaterials can be fabricated with a different shape depending on the baking time and growth temperature. Among the various shapes of nanomaterials, nanorods are the one candidate that can be used to eradicate bacteria by using its geometry. The shape of nanorods was sharp at the top of it, and this shape can break the membrane of the bacteria. The S. aureus membrane size has been investigated by Touhami et al. [43], and Lee et al. [44] show that the bacterial cell wall and plasma membrane are around 10–20 nm thickness. By using the nanorods that have sharp end on the top of it, the bacterial membrane can be broken, and it caused physical damage to the bacteria. The schematic of bacterial eradication (not to scale) and AFM test are shown in Figure 13.
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4. Conclusion
The HVPG technique was successfully used to synthesize silver-titanium dioxide micro-nanocomposites, with the desired optimal nanorods structures fabricated at for 8 hours. The SEM and EDX analyses were used to characterize the material. The pour plate technique was used to grow S. aureus colonies to determine the antibacterial activity.
The SEM and EDX analyses revealed that the micro- and nanoparticles, nanorods, triangular nanomaterials, and nanotubes were successfully produced in high proportions. Antibacterial treatment with the Ag/TiO2 nanocomposites was able to reduce bacterial growth by approximately 75%. This result indicated that the bacteria were successfully eradicated with Ag/TiO2 nanocomposites. In general, the effect of adding Ag/TiO2 will give more significant effect on the performance of nanocomposite materials to eradicate S. aureus. The result is strongly dependent on the growth temperature and baking time to produce and increase the amount of nanorods that have been proven more effective to eradicate bacteria. Furthermore, the study was capable of controlling the structures and morphology of micro- and nanomaterials based on growth temperature and baking time. In the future, the effect of Ag/TiO2 nanocomposites will be studied for their antibacterial property with different types of bacteria and will be tested against fungi as well as bacteria.
Data Availability
No data were used to support this study.
Conflicts of Interest
The authors declare that there are no conflicts of interest in this study.
Acknowledgments
The study was supported by the Solid State Physics Lab, Mechanical Engineering Department of De La Salle University, and AUN SEED NET Project JICA Funding. The authors would also thank Siwat Manomaisantiphap for reviewing the manuscript and Gwen Castillon for assisting during the experiment.