Photocatalytic Antibacterial Effectiveness of Cu-Doped TiO₂ Thin Film Prepared via the Peroxo Sol-Gel Method

Abstract

Cu-doped titanium dioxide thin films (Cu/TiO₂) were prepared on glass substrate via peroxo sol-gel method and dip-coating process with no subsequent calcination process for the degradation of organic dye and use as an antibacterial agent. The as-prepared materials were characterised using transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). For photocatalytic degradation of methylene blue in water, the samples were subjected to Ultraviolet C (UVC) and visible light irradiation. Degraded methylene blue concentration was measured using UV-Vis spectrophotometer. The antibacterial activities of the samples were tested against the gram-negative bacteria Escherichia coli (ATCC25922). Copper species were present in the form of CuO on the surface of modified TiO₂ particles, which was confirmed using TEM and XPS. The optimal observed Cu/TiO₂ weight ratio of 0.5 represents the highest photocatalytic activities under both UVC and visible light irradiation. Moreover, the same composition remarkably exhibited high antibacterial effectiveness against E. coli after illumination with ultraviolet A. The presence of CuO on TiO₂ significantly enhanced photocatalytic activities. Therefore, active Cu-doped TiO₂ can be used as a multipurpose coating material.

1. Introduction

Photocatalysis has garnered plenty of attention from the scientific community in recent decades, resulting in various commercialized products having photocatalytic functions. Among photocatalysts, titanium dioxide (TiO₂) has received the greatest interest because of its remarkable stability and non-toxicity. Modification of TiO₂ has been extensively studied to improve its physical and chemical properties and to overcome the limitation of TiO₂ in photocatalytic processes. Modified TiO₂ has been deployed both environmentally and hygienically, including in the photocatalytic decomposition of organic pollutants [1,2,3,4,5], in self-cleaning materials [6,7,8,9], and as an antibacterial agent of photo-induced photocatalytic reactions [10,11,12,13].

The activity of TiO₂ nanoparticles is due to the oxidative stress and/or the production of reactive oxygen species (ROS), including hydroxyl radical (OH^•) and hydrogen peroxide (H₂O₂) under UV light irradiation; therefore, TiO₂ is used as an antimicrobial agent. The produced ROS can cause cell membrane damage, cell cycle cessation, DNA damage and lipid peroxidation in microorganisms via direct contact between cells and nanoparticles, thereby resulting in cell death [14,15,16].

Several transition metals are toxic to various microbial pathogens. In addition to considerable commercialization in this field, this finding has led to widespread research on the use of such materials as practical antimicrobial agents. Among several transition metals, copper has gained considerable attention, both as dispersions and, in the case of elemental copper and as alloys [10,17,18]. For use as antibacterial agents, copper and its compounds achieve antibacterial activity by the accumulation of copper ions within cells, eventually causing degradation of cell membranes [19,20,21].

Using titanium tetrachloride (TiCl₄) as a precursor and H₂O₂ as a peptizing agent, previous studies have proposed a peroxo sol-gel method for the synthesis of neutral TiO₂ sol [22,23,24,25]. The advantages of this method include not needing a calcination process to obtain the anatase structure of TiO₂, as well as the ability to use H₂O₂ as an oxidizing agent. It results in the formation of TiO₂ nanoparticles dispersed in neutral, stable, and transparent sol.

The purpose of this study was to prepare Cu-doped TiO₂ thin films using the peroxo sol-gel method, thus determining how the addition of Cu to TiO₂ influenced its antibacterial effectiveness, as well as the photocatalytic degradation of methylene blue (MB) aqueous solution under either UVC or visible light irradiation.

2. Results and Discussion

2.1. Characteristic of Cu-Doped TiO₂ Particles

The peroxo sol-gel method was used to prepare Cu-doped TiO₂ sol by direct addition of the precursor of copper (Cu(NO₃)₂·3H₂O) during the heating of TiO₂ sol. To obtain powder nanoparticles, the as-prepared sols were further dried at 70 °C for several days. The X-ray diffraction (XRD) patterns of samples are shown in Figure 1. The diffraction peaks of the TiO₂ and a series of Cu-modified TiO₂ were located at the same positions and showed a similar pattern. The diffraction peaks located at 2θ = 25.31°, 37.80°, 48.05°, 53.89°, 55.06°, 62.69°, 68.76°, and 75.03° corresponded to the anatase phase of (101), (004), (200), (105), (211), (204), (116), and (215), respectively (JCPDS 21-1272). Furthermore, no additional peaks of copper oxide or other forms were found, implying that copper oxides were either highly dispersed with little TiO₂ particles or the amount of Cu dopant was below the detection level of the technique. The size of the crystallite was calculated by the Scherrer equation [26,27]:

$$L=\frac{0.9\lambda }{\beta \cos \theta }$$

(1)

where λ is the X-ray wavelength (0.1540 nm), β is the full width at half maximum (FWHM), θ is the diffraction angle, and L is the average crystallite size. The results are listed in

Table 1. Crystallite sizes of TiO₂ and a series of Cu-doped TiO₂.

Sample	Weight Ratio of Cu:TiO2	Crystallite Size (nm)
TiO2	0:100	14.96
0.1Cu/TiO2	0.1:100	9.12
0.3Cu/TiO2	0.3:100	9.40
0.5Cu/TiO2	0.5:100	9.21
0.7Cu/TiO2	0.7:100	9.60
1Cu/TiO2	1:100	10.16

. The presence of copper slightly decreased the crystallite size of TiO₂, which was attributed to the inhibition of titania condensation and the crystallization in the Cu-doped system [21].

Morphology of the as-prepared samples was investigated using TEM and HRTEM. Figure 2 depicts TEM images of TiO₂ and 0.5Cu/TiO₂ particles. The morphology of the TiO₂ particles prepared via the peroxo sol-gel method is best described as an elliptical shape with particle size of 40–60 nm and 15–30 nm for long and short axes, respectively [3,7,12,23,24,25]. In Figure 2b, HRTEM image displayed the lattice fringe of TiO₂ of 0.327 nm, corresponding to the anatase (101) plane. Figure 2c shows 0.5Cu/TiO₂ particles having the particle size ranging from 20 to 30 nm and 5 to 10 nm for long and short axes, respectively. In addition, magnified view clearly identified some small copper nanoparticles (≤4nm) deposited on the surface of the TiO₂ nanoparticles (see Figure 2c). Therefore, elliptical anatase TiO₂ nanoparticles can be synthesised via the peroxo sol-gel method without a subsequent annealing process, and the presence of copper could decrease the particles size and crystallite size of TiO₂, attributed to phase deterioration of the TiO₂ anatase [21].

2.2. Characterization of Cu-Doped TiO₂ Thin Film

XPS was performed to investigate the electronic state of each element in TiO₂ and Cu-doped TiO₂ thin films. Figure 3a–c shows the XPS spectra of Ti 2p, O 1s and Cu 2p in the TiO₂ and 0.5Cu/TiO₂ thin films. The peaks of Ti 2p_1/2 and Ti 2p_3/2 in pure TiO₂ were located at 464.6 and 458.9 eV, respectively, corresponding to the tetravalent state (Ti⁴⁺) [28]. The characteristic peaks of Ti 2p did not change in the presence of copper, indicating that the cations in the Cu-doped TiO₂ film are all in the Ti⁴⁺ state. Figure 3b shows the XPS spectra of the O 1s region for TiO₂ and 0.5Cu/TiO₂ films. The binding state of O 1s region of TiO₂ was deconvoluted into two peaks centred at 530.7 eV and 531.7 eV, which were ascribed to lattice oxide ions in TiO₂ and hydroxyl groups on the surface, respectively (see Figure 3b and

Table 2. O1s XPS data and the fraction of total area of TiO₂ and 0.5Cu/TiO₂ thin films.

Sample	Lattice O2−		Ti-OH
	BE (eV)	Fraction (%)	BE (eV)	Fraction (%)
TiO2	530.7	81.48	531.7	18.52
0.5Cu/TiO2	530.6	73.72	531.6	26.28

) [22]. The characteristic Cu 2p_3/2 and Cu 2p_1/2 peaks were observed at 934.9 eV and 954.9 eV, respectively, which were consistent with those of the Cu²⁺ cations [29]. The presence of Cu ions could capture the photogenerated carriers to accelerate the separation of charge carriers, subsequently transferring them to the surface of TiO₂ thin film, resulting in the improvement in the photocatalytic activity of the Cu-doped TiO₂ [30].

Wettability measurements were performed using a customized in-house contact angle meter. To measure the water contact angle (WCA), a 5 μL DI water drop was dripped on the films. The TiO₂ film prepared via the peroxo sol-gel method showed hydrophilicity with an average WCA of 6.4°. After doping with copper, the average WCA of the TiO₂ dramatically increased from 6.4° to 35.9° (see Figure 4). In general, elemental copper exhibits super hydrophilicity, whereas copper oxide (e.g., CuO and Cu₂O) shows hydrophobicity. This finding confirms the presence of CuO (Cu²⁺) in the Cu-doped TiO₂ films, which was in accordance with the XPS [31,32].

2.3. Photocatalytic Degradation of MB Aqueous Solution

Prior to the photocatalytic activity, all samples were immersed into the set-up reactor and kept in the dark for 1 h to attain equilibrium adsorption of MB. The photocatalytic degradation of MB in water under UVC and visible light irradiation is shown in Figure 5. The highest photocatalytic activity under both UVC and visible irradiation was shown for the modified TiO₂ with the weight ratio Cu:TiO₂ = 0.5:100 (0.5Cu/TiO₂). The amount of copper beyond a certain loading decrease the photocatalytic activity of TiO₂ due to the Cu light absorption [33]. Furthermore, larger doping of copper resulted in CuO acting as a recombination centre [34].

The photodegradation of MB was fitted to the Langmuir–Hinshelwood model. The slope of ln (C₀/C) plotted versus irradiation time (h) indicates the reaction rate constant of the sample. Under UVC and visible light irradiation, the photocatalytic activity was the highest for 0.5Cu/TiO₂, with a rate constant of 0.737 h⁻¹ and 0.160 h⁻¹, respectively (

Table 3. Rate constant of the reaction from pseudo-first order kinetics under visible light irradiation.

Samples	Rate Constant (k, h−1)
	Under UV light	Under Visible Light
TiO2	0.449	0.070
0.1Cu/TiO2	0.687	0.066
0.3Cu/TiO2	0.546	0.098
0.5Cu/TiO2	0.737	0.160
0.7Cu/TiO2	0.524	0.146
1Cu/TiO2	0.317	0.076

). Higher photocatalytic activity was attributed to the photoelectron transfer of the conduction band (CB) of TiO₂ to CuO, leaving the hole on TiO₂ to take part in oxidation reaction. In other words, under UV irradiation, the presence of CuO can slow down the recombination of electron/hole pairs in TiO₂, as shown in the following equations [34,35].

$${\mathrm{Cu}}^{2+}+{\mathrm{e}}^{-}\mathrm{CB}\to {\mathrm{Cu}}^{+}$$

(2)

$${\mathrm{Cu}}^{+}+\left({\mathrm{O}}_2,{\mathrm{H}}_2{\mathrm{O}}_2\mathrm{or}\mathrm{other}\mathrm{ROS}\right)\to {\mathrm{Cu}}^{2+}+{\mathrm{e}}^{-}$$

(3)

CuO nanoparticles deposited on the TiO₂ surface received the photogenerated electrons from the CB of TiO₂ to form Cu⁺ ion as shown in Equation (2), and Cu⁺ ions could be re-oxidised to Cu²⁺ by the ROS species present in the surrounding media (3).

2.4. Photocatalytic Antibacterial Effectiveness of Cu-doped TiO₂ Thin Film

Under irradiation with UVA light, the antibacterial activity of the samples was tested against Escherichia coli (ATCC25922). E. coli is present as a normal intestinal flora and is commonly found in contaminated drinking water. After a UVA radiation of 3 h at a low UVA intensity of 33 µW/cm², the 0.5Cu/TiO₂ coating showed high antibacterial effectiveness of >99% when tested against E. coli. In contrast, TiO₂ displayed an average effectiveness of 61.20% when tested against E. coli (see Figure 6). Antibacterial activities of Cu-doped TiO₂ could be attributed to the production of ROS species (e.g., O₂⁻, OH^•, and H₂O₂) using TiO₂, as well as CuO nanoparticles that trigger oxidative stress and cell damage in bacteria [36]. In addition, released Cu ions (Cu²⁺) increased intracellular ROS in bacteria using the following pathway [17,21]:

$${\mathrm{Cu}}^{+}+{\mathrm{H}}_2{\mathrm{O}}_2\to {\mathrm{Cu}}^{2+}+{\mathrm{OH}}^{-}+\mathrm{OH}•$$

(4)

H₂O₂ is a byproduct of normal metabolism of oxygen in bacterial cells. The accumulation of ROS dramatically increased, eventually causing cell death [10,17,18,19,20,21,36]. Therefore, Cu-doped TiO₂ showed antibacterial effectiveness, even though a low-intensity of UVA light source was applied.

3. Materials and Methods

3.1. Materials

TiCl₄ (purity > 99.9%) and H₂O₂ (30% in water) were purchased from Showa Chemicals Industry, Ltd. (Tokyo, Japan). NH₄OH was purchased from Merck Co. (Kenilworth, NJ, USA). Copper (II) nitrate trihydrate (Cu(NO₃)₂·3H₂O) was purchased from Sigma-Aldrich (St. Louis., MO, USA). Distilled water was used throughout the experiments.

3.2. Preparation of TiO₂ and Cu-Doped TiO₂ Sol

The typical procedure of preparing TiO₂ and Cu/TiO₂ sols went as follows: Three milliliters of TiCl₄ were added dropwise into 150 mL 1 M HCl aqueous solution (under magnetic stirring) and kept in an ice bath so as to maintain the temperature at 0 °C for 30 min. An aqueous solution of 1 M NH₄OH was then added dropwise to form the white hydrated titanium oxide gel Ti(OH)₄. The pH of the solution was adjusted to 8.0 by adding the required amount of ammonia solution. After aging and stirring for 30 min, a Ti(OH)₄ cake was filtered and washed with distilled water until no chloride ions were detected (QUANTOFIX^®). An amount of the as-prepared gel was re-dispersed in distilled water under magnetic stirring to form a milky solution. An aqueous solution of H₂O₂ was added dropwise to the solution under vigorous stirring for 1 h. The resultant solution was heated at 95 °C for 4 h under magnetic stirring.

For preparation of Cu-doped TiO₂ sol, the copper precursor was added into the TiO₂ sol during heating at 95 °C. The solid content of the TiO₂ in the solution was 1.0 wt %; the molar ratio of H₂O₂:TiO₂ was 4:1; and the weight ratio of Cu:TiO₂ was: 0.1:100, 0.3:100, 0.5:100, 0.7:100, and 1:100. These ratios were denoted as 0.1Cu/TiO₂, 0.3Cu/TiO₂, 0.5Cu/TiO₂, 0.7Cu/TiO₂, and 1Cu/TiO₂, respectively.

3.3. Preparation of Films

The sols were aged at room temperature for 8 h before deposition on glass. The films were prepared using the dip-coating method. Soda lime glass was used as the substrate. The total coating surface area of each glass substrate was 40 cm². The glass substrate was cleaned using a commercial dishwashing detergent. Subsequently, it was ultrasonicated for 30 min in 1 M NH₄OH solution, thoroughly rinsed with distilled water and oven-dried at 60 °C. The glass substrate was vertically dipped into the as-prepared sol with a withdrawal speed of 30 cm/min for 7 times. The thickness of the films was 250–300 nm, as measured by SEM.

3.4. Characterization of Cu-Doped TiO₂ Particles and Thin Films

All samples were air dried at 80 °C for 1 h before further characterisation. The XRD patterns of the samples were determined using a Siemens D500 powder diffractometer (Siemens, Westborough, MA, USA) with Cu Kα source (λ = 1.5405 Å). The morphology and structure of the samples were investigated using transmission electron microscopy (TEM) on a JEM-2000 EX II (JEOL, Tokyo, Japan) operated at an accelerating voltage of 120 kV and high resolution TEM (HRTEM) on a JEOL JEM-2010 (JEOL, Tokyo, Japan) operated at 200 kV. The lattice spacing of the samples was measured using Gatan Digital Micrograph software. The chemical composition and chemical state of the samples were determined using X-Ray photoelectron spectroscopy (XPS) with a Thermo VG Scientific Sigma Prob spectrometer (Thermo Fisher Scientific, Logan, UT, USA). The XPS spectra were collected using Al K_α radiation at a voltage of 20 kV and current of 30 mA. The binding energy (BE) was calibrated using contaminant carbon (C_1S = 284.6 eV). The peaks of each spectrum were organised using XPSPEAK software (Thermo Fisher Scientific, Logan, UT, USA); Shirley type background and 30:70 Lorentzian/Gaussian peak shape were adopted during the deconvolution. The thickness of the films was measured using scanning electron microscopy (SEM) (Hitachi-3000, Tokyo, Japan).

3.5. Photocatalytic Degradation of MB Aqueous Solution

The photocatalytic activity of the samples was determined by inducing the decomposition of MB under irradiation with either UVC or visible light. An aqueous solution of MB (40 mL) with a concentration of 10 mg/L was loaded onto a quartz glass plate. The samples were horizontally immersed into the MB solution. Before the photocatalytic activity was measured, the reactor was kept in the dark under magnetic stirring for 1 h to achieve the saturation adsorption of MB on the coatings. The catalysts were irradiated by either two 9 W UVC lamps (wavelength = 254 nm, TUV PL-L 18W/4P 1CT/25, Philips) or two 18 W compact fluorescent lamps (1200 lumen, Philips (Pro UV Lamps Ltd., Bucks, UK)) equipped with UV cut-off filters with a cut-off wavelength of 410 nm. The distance from the lamp to the surface of the solution was 15 cm, and the concentration of the aqueous solution of MB was determined at intervals of 1 h using UV-vis spectrophotometer (JASCO V-630 (Japan Spectroscopic Company, Tsukuba, Japan)). The wavelength selected for the measurements was 663 nm, which is the characteristic maximum absorption wavelength of MB.

3.6. Study of Antibacterial Activity

The test method of the coatings against E. coli’s (ATCC25922) antibacterial activity was modified from the certificate “JIS Z 2801: 2000 (E)—Antimicrobial products-Test for antimicrobial activity and efficacy” and “TN-050—Standard on nano anti-bacterial coating.” The strains were grown on tryptic soy agar and diluted to 5.5 × 10⁶–6.0 × 10⁶ cells/mL using distilled water. The bacterial concentrations were measured from the optical density reading at 600 nm (OD₆₀₀). The as-prepared sols were deposited on a 5 cm × 5 cm glass substrate using the dip-coating technique and a catalyst content of 0.2 mg/cm². Bare glass substrates were used as controls. Before testing, all samples were sterilized and activated using two 9 W UVC light (λ = 254 nm, TUV PL-L 18W/4P 1CT/25, Philips (Pro UV Lamps Ltd., Bucks, UK)) for 1 h. In order to quantitatively evaluate the antibacterial activity of the coatings, 0.4 mL of bacterial suspension was added onto each coating. Next, the test pieces were covered with 4 × 4 cm of adhesive film (with a transparency of >85% at 340–380 nm). Covered with the adhesive film, the test piece was placed into a Petri dish and exposed to two UVA lamps (λ = 365 nm, PL-L 36W/09/4P, Philips (Pro UV Lamps Ltd., Bucks, UK)) for either 1 h or 3 h. The irradiance of the UVA intensity was measured at 33 µW/cm², using a UV light meter (model UV-340A, Lutron (Lutron Electronic Enterprise Co., Taipei, Taiwan)). After UVA irradiation, the bacterial suspension on each coating was washed off and diluted with phosphate buffer saline (PBS). Bacterial colony-forming units (CFUs) were enumerated by plating serial dilutions (1:10–1:10⁵). The number of surviving bacterial colonies was counted (CFU/mL) after incubation at 37 °C for 24 h. The experiments were repeated three times for each sample type; therefore, three parallel CFU values were obtained for each type of sample.

The antibacterial effectiveness was calculated according to the following equation [12,25]:

$$C(\%)=\left(\frac{A-B}{A}\right)\times 100\%$$

(5)

where C represents antibacterial effectiveness, A is the average number of colonies formed in the blank control group (CFU/mL), and B is the average number of colonies formed in the experimental group (CFU/mL).

4. Conclusions

0.5Cu-doped TiO₂ nanoparticles can be successfully prepared via the peroxo sol-gel method without needing further calcination. The CuO nanoparticles, having a particle size of <4 nm, were deposited on the TiO₂ surface. The photocatalytic activity was the highest for 0.5Cu/TiO₂, with a rate constant of 0.737 h⁻¹ and 0.160 h⁻¹ under UVC and visible light irradiation, respectively. Moreover, the 0.5Cu/TiO₂ coating showed high antibacterial effectiveness of >99% against E. coli after illumination with 33 µW/cm² UVA radiation for 3 h. Therefore, the presence of CuO significantly enhanced the photocatalytic activity as well as antibacterial effect of TiO₂. Therefore, the Cu-coped TiO₂ materials prepared via the peroxo sol-gel method can be an alternative and promising solution to increasing environmental contamination.