INTRODUCTION

Titanium dioxide (TiO2) has attracted the attention of many researchers due to its cheapness, availability, strong oxidizing power, non-toxicity, antibacterial properties and self-cleaning properties [1–3]. TiO2 can be deposited on the surface of various substrates, such as glass, ceramics, metals, cement, bricks to create a coating with self-sterilization and self-cleaning properties to destroy bacteria and kill them [4–6].

Due to the above properties, TiO2 is used in food industries, medical, automotive, tile, ceramic, solar cell [7–9]. However, the main problem is that, the lack of visible light prevents its practical application. To overcome this problem, many studies have recently used noble metal doping such as Ag and Cu to increase antibacterial activity in visible light [10–13]. In general, it is believed that, silver ions interact with proteins by reacting with the SH groups present in bacteria, leading to the inactivation of the proteins [14–16]. Moreover, silver ions can interact with DNA of bacteria preventing cell reproduction [17–21]. Both effects lead to the death of the bacterial cells [22]. Page et al [23] reported that preparation of Ag2O–TiO2 films on glass substrates by sol gel method and investigated their antimicrobial properties by irradiating UV light at 365 nm. They found that, Ag-doped TiO2 coatings were significantly more anti-microbially active than TiO2 coatings. Yu et al. [24] showed that, TiO2 composites and silver nanoparticles have antibacterial activity even in the absence of visible light, indicating that, silver nanoparticles are responsible for the antimicrobial effect of coatings in the dark. They also showed that, the higher antibacterial activity of composite films under UV light is due to the synergistic antibacterial effects of the photocatalytic reaction of TiO2 and Ag coatings. Sun et al. [25] deposited Ag–TiO2 thin films on glazed tile surface by liquid phase deposition method (LPD). The films were prepared from ammonium hexauororitanate(IV) and silver nitrate and annealed at 600°C which showed a high antibacterial activity versus Staphylococcus aureus and Escherichia coli.

Sol-gel process is a conventional method to prepare composite Ag–TiO2. In this study, TiO2 and Ag–TiO2 thin films deposited on floor tiles were prepared by a template sol-gel technique. Thin films with different contents of AgNO3 and TiO2 were deposited on floor tiles by dip coating at room temperature. Different characterization methods were used to analyze the resultant films. The antibacterial effect of the obtained thin films on Gram-negative and positive bacteria was tested. The hydrophilicity effect of films was obtained using water contact angle measurement.

EXPERIMENTAL

Materials

Propanol (CH3CH2CH2OH, 99% purity, Merck KGaA), titanium dioxide (TiO2, 99% purity, Sigma-Aldrich), tetraisopropyl orthotitanate (99% purity, Merck KGaA), silver nitrate (AgNO3, 99% purity, Merck KGaA) and bacteria of S. aureus ATCC 6538 and E. coli ATCC 25 922 (Microbiology Laboratory of Scientific Research Center, University of Tehran) were provided as initial materials. The strains were cultured in Muller Hinton broth and Standard Methods Agar (TSA (trypticase soy agar) using the appropriate times and temperatures of incubation. The substrates used were selected floor tiles from Golchin Co.

Preparation of TiO2 and Ag–TiO2 Films on the Floor Tile

The experiments were performed in the ceramic laboratory of Maybod Islamic Azad University. Samples were prepared by following procedures:

Preparation of TiO2 film on the floor tiles: 2 molar hydrochloric acid was obtained with 16.5 mL of concentrated hydrochloric acid (37% purity) and distilled water into 100-mL flask. To prepare the precursor solution for the production of TiO2 film, first 85 mL of propanol and 6 mL of titanium isopropoxide (Ti{OCH(CH3)2}4) were stirred at 40°C. Then, 2 molar hydrochloric acid was continuously added as catalyst to the solution until pH 3. The water in the solution caused a gentle hydrolysis of the solution. The solution was stirred at room temperature for 6 h to complete this reaction. In the next step, TiO+ particles were added to the solution with different weight ratios from 0.05 and 0.1 wt % and mixed for 15 h. The floor tiles were immersed in the solution at ambient temperature for 30 s to complete the immersion deposition process.

Preparation of Ag–TiO2 film on the floor tiles: For preparing the coating of Ag–TiO2 on the floor tiles, the steps are repeated as mentioned before. After adding of TiO2 particles, 0.1 and 0.2 wt % AgNO3 were added and the solution stirred for 6 h. The floor tiles were immersed in the solution at ambient temperature for 30 s to complete the immersion deposition process. The substrates dimensions of floor tiles were 30 × 15 × 3 mm. The substrates were cleaned in acetone and then an ultrasonic bath was used by ethanol.

Figure 1 shows a schematic of the preparation of nano Ag–TiO2 film. Primary tile samples were used as a control sample to evaluate the results. The codes of prepared samples with different weight ratios of TiO2 and AgNO3 were shown in Table 1. The prepared samples were studied by the following techniques. X-ray diffraction (XRD, Japan Shimadzu, CuKα 40 kV, 30 mA) through a D8-Advance Bruker instrument equipped with a CuKα radiation source with λ = 1.5406 Å, and configured in horizontal 2θ geometry. The surface morphology and grain size were measured by field-emission scanning electron microscope (FESEM) (Hitachi S-4100 SEM, Hitachi High-Tech, Minato-ku, Tokyo, Japan) with an EDS system (MIRA II, SAMX). For FESEM study, to prevent the charging effect, the samples were coated with a very thin layer of gold. The static contact angle of the distilled water drop with the surface of thin film was measured using a domestic made instrument with a × 1000 magnification lens and a digital camera (model canon-25 megapixel). Ten milliliters of distilled water was slowly released on the thin film surface. The image of the contact angle was analyzed by ImageJ software code and the method of low-bond axisymmetric drop shape analysis (LB-ADSA) [26]. To measure the antimicrobial activity, the killing time test was used.

Fig. 1.
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Schematic of the deposition steps.

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Table 1.   Prepared samples with different weight ratios of TiO2 and AgNO3
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RESULTS AND DISCUSSION

X-ray Diffraction Studies of TiO2 and Ag–TiO2 Films

The XRD analysis of samples with different weight ratios of TiO2 and AgNO3 on floor tiles was shown in Fig. 2. Crystal structure of samples was identified by JCPDS card which showed TiO2 and Ag2O3 phases, respectively. Specifications and preferential growth orientation of the created peaks by TiO2 and Ag2O3 phases are given in Table 2. As it was shown in Fig. 2 with addition of TiO2 and AgNO3, except TiO2 and Ag2O3 phases, mullite and quartz phases were detected due to the effect of the substrate. In all samples, the main diffraction angle at 2θ ≈ 27° was related to the rutile phase of TiO2 in (110) diffraction line. The diffraction lines related to Ag2O3 were 2θ = 37.05°, 42.70°, 60.45° that indicated the good dispersion of nanocrystalline silver particles in the matrix. The XRD pattern of the control sample (S) showed the amorphous structure and the formed peaks in the range of 2θ = 20°–30° were related to quartz and mullite phase with very wide peak. Fluctuations in the spectrum were owing to the amorphous nature of the tile. By increasing the amount of TiO2 from 0.05 to 0.1 wt % (S0.05T, 0.1A and S0.1T, 0.1A) and fixing the amount of AgNO3 (0.1 wt %) constant, the peak intensities was increased sharply, especially in 2θ = 27.9° for TiO2 and 2θ = 37.05° for Ag2O3. While with adding of 0.2 wt % of AgNO3 and increasing the amount of TiO2 from 0.05 to 0.1 wt % in S0.05T,0.2A and S0.1T,0.2A, the peak intensity 2θ = 27.9° of TiO2 was reduced. With comparing the X-ray diffraction pattern of the samples, it was seen that, with increasing of TiO2 and AgNO3, all of peaks were more intense.

Fig. 2.
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XRD patterns of films with different weight ratios of TiO2 and AgNO3 on floor tiles.

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Table 2.   Characteristic for coating the tiles with TiO2 and AgNO3 from XRD analysis
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Surface Morphology and Chemical Composition of TiO2 and Ag–TiO2 Films

Figure 3 shows the surface microstructures of the TiO2 and Ag–TiO2 films on the floor tile. The grain size of the FESEM results using Nanoscope software was given in Table 3.

Fig. 3.
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Surface FESEM images of films with different weight ratios of TiO2 and AgNO3 on floor tiles.

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Table 3.   Grains size obtained from FESEM analysis
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By coating of TiO2 film on the floor tile, the grain size of S0.05T and S0.1T samples were increased compared to control sample of S. However, with increasing of TiO2 from 0.05 wt % (S0.05T) to 0.1 wt % (S0.1T), the grain size was decreased. Moreover, by coating of Ag–TiO2 film on floor tile, the grain size and surface roughness were increased. By increasing of AgNO3 content from 0.1 wt % (S0.05T) to 0.2 wt % (S0.05T) with 0.05 wt % of TiO2 particle, the grain size was decreased. Also with adding of 0.1 wt % of TiO2 particle in all samples the grain sizes (S0.1T, 0.1A and S0.1T, 0.2A) were reduced. According to Table 3, with a constant value of 0.05 wt % of TiO2 and doping of AgNO3 from 0.1 to 0.2 wt %, the grain sizes were increased 2 and 1.4 times, respectively. Consistent with Table 3, with comparing of S0.1T, 0.1A, S0.1T,0.2A, S0.1T it was seen that, with a constant value of 0.1 wt % TiO2 and doping of AgNO3 from 0.1 to 0.2 wt % the grain size was increased 2.26 and 1.5 times, respectively. In addition, larger pores were produced by doping of AgNO3 which was resulted in increasing of porosity and surface roughness.

Figure 4 is shown the EDS spectra of the TiO2 and Ag–TiO2 films on the floor tile. The presented energy of each of the peaks in the spectra was assigned to a specific atom. Higher peaks were indicated the higher concentrations of the element in the sample. The EDS analysis was employed to verify the chemical composition of the film. In all samples in Fig. 3, it was seen that, the Si and Al elements were related to the substrate and the oxygen form the metal oxide had the highest percentage. In the samples of S0.05T, 0.1A, S0.1T, 0.1A, S0.05T, 0.2A, S0.1T, 0.2A, the peaks created by Ti and Ag element were very small and in the S0.05T and S0.1T samples, the Ti and O element peaks were increased significantly.

Fig. 4.
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EDS spectra of films with different weight ratios of TiO2 and Ag–TiO2 on floor tiles.

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Wettability Behaviors of TiO2 and Ag–TiO2 Films

TiO2 as a semiconductor has various applications that are inherently hydrophilic. The contact angle of a water drop on the solid surface is an important parameter in determining the hydrophobicity or hydrophobicity of the material.

The effect of doping of TiO2 and AgNO3 on wettability has been investigated using the contact angle analysis of deionized water droplets of 10 mg on surfaces.

The average contact angle of 5 droplets of water on the surface of the film is measured using ImageJ software to obtain data uncertainty (by precision of two digits). When the contact angle is greater than 90°, the surface is hydrophobic. However, when the contact angle changes is between 30° to 90°, the surface is hydrophilic and if the contact angle is less than 30°, the surface is super-hydrophilic. The wettability behavior of hydrophilic surfaces depends on the chemical composition and structure of the material surface.

Figure 5 shows the images of static contact angle of water drop on produced films on floor tiles. As it was shown in Fig. 4, the surface of the control sample (S) had a hydrophobic behavior with a contact angle of 98°. By adding TiO2 content, the contact angle of S0.05T and S0.1T samples was reduced and the hydrophobicity turned into hydrophilicity. As the TiO2 content increased from 0.05 to 0.1 wt %, the contact angle decreased from 88° to 78°. Also, by doping AgNO3 with TiO2 content, the contact angle was reduced. The static contact angle of the distilled water drop on surfaces of S0.05T, 0.1A and S0.1T, 0.1A samples were measured as 77° and 61°, respectively. The contact angle decreased with increasing of AgNO3 content from 0.1 to 0.2 wt %. S0.05T, 0.2A and S0.1T, 0.2A samples with contact angles of 65 and 20° were hydrophilic and super-hydrophilic, respectively. Silver nanoparticles reduced the contact angle and changed the behavior of the samples from hydrophilicity to super-hydrophilicity. In fact, silver had an important effect on wettability properties [27].

Fig. 5.
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The static contact angle of water drop with films with different weight ratios of TiO2 and AgNO3 on floor tiles.

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Antibacterial Activity Evaluation

The time-kill method was used to evaluate the antibacterial activity. The time-kill method is one of the best methods to obtain information about the dynamic relationship between antibacterial and bacterial species, which depends on the time and effective concentration of antibacterial substances. In this method, the percentage of dead cells relative to living cells in terms of CFU/mL is obtained using agar count and then compared with the growth control tube.

Bacterial testing was performed on two types of Gram-positive bacteria S. aureus ATCC 6538 and Gram-negative bacteria E. coli ATCC 25922 with an initial concentration of 1 × 105 CFU/mL.

Table 4 presented the experimental results for the qualitative evaluation. The results show that, all samples could prevent the growth of bacteria. This result was expected because of the driving force of silver ion diffusion on the surface which was larger for coatings with higher silver content. Figure 6 shows the reduction ratio of bacteria for different samples by two gram-positive bacteria, S. aureus ATCC 6538, and the Gram-negative bacterium E. coli ATCC 25 922 under visible light. Covering the floor tile with TiO2 film increased the antibacterial properties. With the increase of TiO2 content from 0.05 to 0.1 wt % (S0.05T and S0.1T) the destruction of bacteria for both bacterial species has increased so that, the percentage of reduction of Staphylococcus aureus ATCC 6538 was higher than Escherichia coli ATCC 259. However, in tiles coated with AgNO3 and TiO2 content (S0.05T,01.A and S0.1T,0.1A) caused to kill more bacteria. Although antibacterial properties in composite samples depend on the diffusion of silver ions, therefore the composite with higher silver content had a greater antibacterial effect. Antibacterial properties increased with increasing of silver nitrate from 0.1 to 0.2 wt % (S0.05T,0.2A, S0.1T,0.2A, S0.1T,0.2A) and the amount of S. aureus ATCC 6538 and E. coli ATCC 259 were reduced by 99 and 95%, respectively. With comparing all the coated samples according to Table 4 and Fig. 5, it could be expected that, the percentage of reduction of S. aureus ATCC 6538 was higher than that of E. coli ATCC 259.

Table 4.   Percentage of bacterial reduction of different samples on the floor tile by two different bacteria
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Fig. 6.
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Killing ratio two species of bacteria on the surfaces of films with different weight ratios of TiO2 and AgNO3.

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CONCLUSIONS

TiO2 and Ag–TiO2 films were prepared by the sol-gel dip coating method on floor tiles. X-ray diffraction patterns for all samples with different percentages of TiO2 and AgNO3 on the floor tile substrates showed that all of the samples had crystal structure. With increasing the weight percentage of TiO2 from 0.05 to 0.1, the intensity of the peaks, especially related to TiO2 phase was increased, while with increasing the weight percentage of AgNO3, the X-ray diffraction pattern did not change clearly. The results of FESEM showed that, the grain size was decreased with increasing of TiO2 content. Also, by adding of AgNO3 content, the grain size and surface roughness were increased. Prototype surfaces with floor tile substrate had a hydrophobic behavior with a contact angle of 98°. Adding of TiO2 content reduced the contact angle and turned the hydrophobic behavior into hydrophilicity. As the percentage of TiO2 content was increased from 0.05 to 0.1, the contact angle was decreased from 88° to 78°. Also, silver nanoparticles reduced the contact angle and changed the behavior of the samples from hydrophilicity to super-hydrophilicity.

The inactivation of Gram-negative and positive bacteria (S. aureus ATCC 6538 and E. coli ATCC 25922) under visible illumination of all films was compared and the films showed the antibacterial activity and better wettability effect with increasing of the silver content. Antibacterial properties increased with increasing of AgNO3 content from 0.1 to 0.2 wt %. In S0.1T,0.2A sample, the amount of S. aureus ATCC 6538 and E. coli ATCC 259 were reduced by 99 and 95%, respectively. With comparing all the coated samples, it was observed that , the percentage reduction of S. aureus ATCC 6538 was higher than that of E. coli ATCC 259.