Photocatalytic antimicrobial and photostability studies of TiO₂/ZnO thin films

2.1 Materials

Pure Zinc (Zn, 99.99%) and Titanium metal (Ti, 99.99%) as the target material for thin films development was obtained from Ultimate materials Technology Co., Ltd, Taiwan. Glass (FEA Microscope slides) with a 76 × 25 mm dimension was used as a substrate. Argon (Ar, 99.9999%, Xinguang Co., Taiwan) was used in the sputtering process as an inert gas. Acetone (CH₃COCH₃, 95%) and ethanol (C₂H₅OH, 95%) were supplied by Jingming Chemical Co.,Ltd, Taiwan. Methylene blue (MB) (Alfa Aesar; Thermo Fisher Scientific, UK) was used for photocatalytic analysis. The culture collection of Escherichia coli ATCC25922, Staphylococcus aureus ATCC10234, and Candida albicans ATCC10234 as pathogenic microbes was obtained from the Department of Biological Science and Technology, NPUST. Sodium chloride (NaCl), potassium chloride (KCl), sodium phosphate dibasic (Na₂HPO₄), and potassium phosphate monobasic (KH₂PO₄) as the chemicals for producing phosphate buffered saline (PBS) solution were purchased from Sigma-Aldrich, USA. Tryptic soy agar (TSB, DIFCO^TM), potato dextrose agar (PDA, DIFCO^TM), Mueller Hinton II agar (MH, BBL^TM) (from Becton, Dickinson, and Company, USA), yeast extract (Pronadisa, Lab. Conda, Spain), and meat peptone (STBIO Media Inc, Taiwan) were used as microbe growth media. Antibiotics as a positive control in antimicrobial analysis such as Kanamycin, Ampicillin, and Amphotericin-B were also obtained from Sigma-Aldrich, USA). The disk paper (Whatman, Schleicher & Schuell, US) with a 6 mm diameter was used for antibiotic-paper assay procedures. Deionized (DI) water generated by a PURELAB water purification system (Lane End, High Wycombe, UK) was used in all experiments and chemical solution preparation.

2.2 Synthesis of TiO₂/ZnO thin films

The TiO₂/ZnO thin films were developed on a glass substrate using a four-step process. Initially, Zn films were deposited on the glass substrate via magnetron sputtering. Before use, the glass substrates were ultrasonically washed for 30 min each in acetone, ethanol, and distilled water. Moreover, the Zn was then thermally oxidized to produce ZnO from the metallic Zn thin film layer. Furthermore, ZnO thin films were covered with a Ti layer by magnetron sputtering. Finally, thermal oxidation transformed Ti/ZnO thin films into TiO₂/ZnO. As comparative samples, TiO₂ and ZnO thin films were also produced.

The ZnO thin films were prepared by High Power Impulse Magnetron Sputtering (HiPiMS) through thermal oxidation, and their synthesis route has been described in our previous studies (Widyastuti et al., 2022). Direct-current (DC) magnetron sputtering methods with a pure Ti target were used to develop Ti films as an upper layer on ZnO films. The distance between the target and substrate was set at 100 mm. The base pressure was controlled at 1 × 10^-6 Torr, and the working pressure was 2 × 10^-3 Torr. The argon gas flow is controlled at 6 sccm by mass flow controllers. The substrate holder was rotated at 24 rpm (revolutions per minute). Ti-upper layer films were deposited on ZnO-bottom layer films at 25 °C. The DC power was fixed at 100 W with a 20 min deposition time. The Ti film upper layer thickness was controlled at about 100 nm.

The different oxidation temperatures at 400, 450, 500 and 550 °C for 3 h in the presence of air were designated “Ti/ZnO 400”, “Ti/ZnO 450”, “Ti/ZnO 500” and “ZnO 550“, respectively. For comparison, as references, the Zn and Ti single-layer thin films were oxidized at 400 °C and 500 °C, respectively.

2.3 Characterization of TiO₂/ZnO thin film

X-ray diffraction analyses were performed employing a Bruker D8 Advance model (Germany) with Cu Kα radiation (λ = 1.54). The instrument devices were operated at 40 kV and 40 mA, respectively. XRD analysis for 20 mins scans was continuously performed in a 10° ≤ 2θ ≤ 60° range, with a step size of 0.1° and a 5 s step size. The International Centre for Diffraction Data (ICDD) card is used for phase identification of crystalline phases in various films. The full width at half maximum (FWHM) value was used to estimate the crystalline size using Scherer’s formula. Moreover, the a and c lattice parameters and the total unit cell volume lattice were also calculated (Supplementary Material A).

Field-emission transmission electron microscopy (FE-TEM; FEI Tecnai G2 F20, FEI Company—, USA) was used to observe thin-film cross-section images. Sample preparation for the TEM was performed using a Focused Ion Beam (FIB) (Hitachi NX2000, Hitachi High-Tech, USA). Selected area electron diffraction (SAED) was analyzed using Gatan Software Digital Micrograph software version 3.7.4 (Gatan Inc., USA).

The optical characteristic of thin-film samples, including absorbance and transmittance analysis, were measured using a UV– vis spectrophotometer (U-3310, Hitachi Ltd., Japan) across the wavelength ranges of 200–1000 nm and 300–800 nm, respectively. The optical absorbance was used to measure the thin films band gap using Tauc’s equation (Eq. (1)): (Singh and Chakrabarti, 2013)

Where α is the absorption coefficient, hν is absorbed photon energy; A is a characteristic parameter independent of photon energy, E_g is the optical energy gap, n = 2 for permitted indirect transition, and n = 0.5 for permitted direct transition.

2.4 Assessment of photocatalytic degradation performance of TiO₂/ZnO thin films

A degradation methylene blue (MB) solution with an initial concentration (C₀) of 10 mg/L was used to evaluate the photocatalytic performance of the thin-film samples. Prior to degradation studies, MB solutions with concentrations ranging from 1 to 10 ppm were produced. The calibration equations were derived by linear regression using the intensity (664 nm) displayed for each concentration. Furthermore, the calibration equation was employed to determine the concentration of MB during photocatalytic studies. A 10 ml aqueous MB solution was used to soak thin-film samples, then held under dark conditions for 60 min to create MB absorption equilibrium on the photocatalysts (t = 0 min). Photo-irradiation was performed using five commons of UVA lamp tubes (Philips TUV-8-watt, 365 nm, Netherlands) and placed horizontally inside an artificial box. Thin films were retained at 9 cm from the UV light. The photocatalytic activity of thin-film samples was investigated by decomposition of Methylene Blue (MB) solution under UVA irradiation. The concentration of MB in the solution was measured every 30 min for 5 h (t = 30–300 min, C₃₀ to C₃₀₀), and the changes in the MB absorbance were analyzed using UV– vis spectrophotometer (U-3310, Hitachi Ltd., Japan) (Lee et al., 2021). The degradation performance was calculated using equation (Eq. (2)): (Lee et al., 2021)

Where C₀ is the initial concentration of MB and C_t is the absorbance after different time intervals. The detailed schematic diagram of photocatalytic set up was shown in Fig. 1.

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Fig. 1

Schematic presentation of photocatalytic set-up.

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The photostability analysis of thin films for five-cycle reusability was investigated. After the first photocatalytic treatment was finished, thin film samples were detached, rinsed with distilled water several times, air-dried, and placed in a new MB solution. The next step repeats as described previously. The degradation performance was deliberated by each cycle using Eq. (2).

2.5 Assessment of antimicrobial activity performance

Two different assays (qualitative and quantitative) were carried out to investigate thin-film samples' antibacterial and antifungal activities. In this study, Escherichia coli, Staphylococcus aureus, and Candida albicans were used as models for gram-negative, gram-positive, and pathogenic fungi (Table 1). The detailed microbial preparation was followed by a previous study (Widyastuti et al., 2022). Different media were used to enhance bacteria and fungi, which were then cultured aerobically on a rotary shaker for 18–24 h. The PBS solution was then used to collect, wash, and re-suspend the cells. Thin-film samples and glass were cut at 1 cm × 1 cm to evaluate the antimicrobial properties. Three types of antibiotics, Kanamycin, Ampicillin, and Amphotericin-B were used as positive control treatments for E. coli, S. aureus, and C. albicans, respectively.

2.6 Statistical analysis

The quantitative data was conducted in three replicates and analyzed using One-way analysis of variance (ANOVA) by Tukey HSD tests at a p < 0.05 significance level in SPSS Statistics version 16 software (SPSS for Windows; IBM Corp, USA).

3.1 Structural analysis of TiO₂/ZnO bilayer thin films

The X-ray diffraction patterns (XRD) analysis was employed to determine the crystal phase of ZnO, TiO_2, and TiO₂/ZnO bilayer thin films. The XRD pattern for Ti, Zn as a single layer, and Ti/ZnO bilayer thin films thermally oxidized at 400 °C are shown in Fig. 2a. The sputter-deposited single-layer Zn thin films were metallic and transparent when thermally oxidized at 400 °C with intensity diffraction peaks corresponding to hexagonal wurtzite zinc oxide (ZnO) (hexagonal, space group: P63mc, ICDD Card No. 36–1451). However, the pure Ti sample does not exhibit any peaks that only manifest the presence of an amorphous phase. Comparatively, the crystal phase of Ti/ZnO, when thermally oxidized at 400 °C, the TiO₂ rutile (tetragonal, space group = P42/mnm, ICDD Card No. 21–1276) and anatase phase (body-centered tetragonal; space group = I41/amd, ICDD Card No. 89–2809), Ti_0.895O_.77 (Face-centered cubic, space group: Fm-3 m, ICDD Card No. 89–3075) and ZnO phases appeared. The non-stoichiometry Ti_0.895O_.77 phases attributed to TiO₂ have insufficient oxygen during thermal oxidation, and this layer exists between the upper and the bottom layer. It is demonstrated that appearing a ZnO as the bottom layer could provide sufficient oxygen to the Ti upper layer during the thermal oxidation process. Lee et al. (Lee et al., 2021) reported that oxygen diffusion from the bottom layer significantly affects the film's phase structure evolution. Therefore, it is believed that the phase evolution effects on the Ti films oxidized using thermal oxidation depend strongly on the bottom layer. Previous studies demonstrated that the ZnO layer improved the crystallinity of the TiO₂ thin film (Hussin et al., 2019).

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Fig. 2

X-Ray Diffraction (XRD) Pattern of thermally oxidized Zn, Ti and Ti/ZnO thin films at (a) 400 and (b) 500 °C.

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A further increase in temperature to 500 °C resulted in increased diffraction intensity with a decrease in the amorphous background, indicating improved Ti film crystallinity as a single layer and TiO₂/ZnO bilayer thin film, as presented in Fig. 2b. Anatase and rutile phases were observed on both samples. This result demonstrated that increasing thermal oxidation temperature increased anatase and rutile peak intensity. It was reported that the temperature and time of heat treatment were found to enhance the crystallization degree and growth rate of ZnO and TiO₂ (Munguti and Dejene, 2020). In addition, the TiO₂ and ZnO thin films develop during the layer-by-layer sputtering process exhibit good contact, ensuring that the TiO₂/ZnO thin films possess a good heterojunction structure (Ton et al., 2020).

In order to determine the effect of oxidation temperature on the bilayer thin films, the Ti/ZnO thin films were thermally oxidized from 400 to 550 °C for 2 h (Fig. 3). Temperature conditions highly influenced the crystallinity during the thermal oxidation process. Increases oxidation temperature on TiO₂/ZnO thin films will transform Ti_0.895O_.77 to anatase/rutile TiO₂ phase. Moreover, the peak of (1 1 0), (1 0 1), (1 1 1), and (2 1 1) orientations of the rutile TiO₂ structure increases considerably during the thermal oxidation process. It is well known that the anatase is a meta-stable phase that may be transformed into a rutile phase during heat treatment (Zhou et al., 2013a). Interestingly, the anatase phase still possesses, although oxidation temperature increased to 550 °C. It demonstrates that surface modification with ZnO on bilayer thin films can effectively inhibit the phase transformation from anatase to rutile, which is favorable to improving anatase thermal stability. Similar results were reported by Jing et al. (Jing et al., 2009), which showed that modified Bi₂O₃ species on TiO₂ nanoparticles could improve the thermal stability of anatase crystallites and promote photoinduced charge separation. Further study of the microstructure using Transmission Electron Microscopy (TEM) will be performed to prove the phases existences.

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Fig. 3

X-Ray Diffraction (XRD) Pattern of Ti/ZnO bilayer thin films under different oxidation temperatures.

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The TiO₂ and ZnO thin films' crystallinity may be improved by reducing the diffraction peak's full width at half maximum (FWHM) as the oxidation temperature rises (Rusu et al., 2007). The result is in accordance with earlier studies of ZnO-TiO₂ composites prepared by different heat treatments such as calcination and annealing temperature (Munguti and Dejene, 2020). The Ti atoms in the films could absorb and/or absorb the O₂ in the air during thermal oxidation of the film. The atoms of Ti and O receive sufficient free energy simultaneously to assist in the nucleation, growth, and aggregation of crystal grains. (Zhou et al., 2013a). According to Perez-Gonzales et al.(Pérez-González et al., 2015), the introduction of ZnO to TiO₂ causes the removal of the anatase phase. Similarly, it has been discovered by other researchers that the anatase phase of TiO₂ may reduce by increasing the quantity of ZnO (Chen and Xu, 2014).

The crystallite size and lattice constants of the ZnO, TiO₂, and bilayer thin films are estimated using the Debye-Scherrer and Bragg’s equation, respectively (Supplementary Material A), from the (0 0 2) peak for ZnO and from the (1 1 0) peak for TiO₂. These peaks are chosen since they are relatively intense peaks in the recorded XRD spectrum. The calculated crystalline size (D), lattice parameter ZnO, TiO₂ as a single layer, and bilayer thin films are summarized in Table 2.

The average of crystallites ranged between 7.356 and 26.541 nm (Table 2). It was found that by employing thermal oxidation procedures, this research achieved TiO₂/ZnO bilayer thin films with crystallite sizes smaller than those previously reported by means of sol–gel (Lachom et al., 2017) and reactive-magnetron sputtering (Ton et al., 2020) that was earlier reported. The evidence clearly indicates that Ti/ZnO O₂, through thermal oxidation, can promote decreasing crystalline size. A photocatalyst with smaller crystal size and a high specific surface area has more specific reaction sites accessible to undertake photocatalytic reactions, which plays a fundamental role in increasing the photocatalytic performance (Pérez-González and Tomás, 2021).

Therefore, the crystallite size of ZnO and TiO₂ on TiO₂/ZnO bilayer thin film was also affected by increasing oxidation temperature. The higher mobility of atoms, which tend to migrate to the most stable in the lattice, may explain the increases in crystallite size with increasing oxidation temperature (Ayana et al., 2022) and small grains fused collectively to increase grain size (Ibrahim et al., 2013). However, the sample oxidized at 400 °C exhibited the smallest crystallite sizes, resulting from poor crystallization. A rise in oxidation temperature increased crystallite development up to 500 °C, where a decrease was observed. It could be seen that the grain size of the films oxidized 500 to 550 °C decreases from 16.251 to 11.514 nm and 26.541 to 24.159 nm for TiO₂ and ZnO, respectively. A similar trend was also reported by Munguti et al. (Munguti and Dejene, 2020), Mragui et al. (El Mragui et al., 2019), and Khan et al. (Khan et al., 2018). Wittawat et al. (2020) suggested that the crystal size of ZnO and TiO₂ decreased while the temperature increased from 400 °C to 600 °C due to an increasingly high nucleation rate (Wittawat et al., 2020). The results revealed that oxidation temperature is essential in crystal structure formation and crystallite size control.

The effect of oxidation temperature on the microstructure of TiO₂/ZnO bilayer thin films was confirmed using High-resolution TEM (HR-TEM). Fig. 4a shows the bright cross-sectional field of Ti/ZnO microstructures thermally oxidized at 400 °C. The thin film thickness was observed at around 121.500 and 132.280 nm for ZnO and TiO₂, respectively. Two sections zone (inset A and B) were selected to identify the crystalline phase of thin films' upper and middle areas. The circle area around 90 nm was used to observe the selected area electron diffraction (SAED). The interplanar distance of 0.351, 0.324, and 0.209 indicated in the image were ascribed to the (1 1 0) anatase, (1 1 0) rutile, and (2 0 0) Ti_0.895O_.77 microcrystalline phase (Fig. 4b). Fig. 4c displays the SAED pattern of section A, whereas the upper zone area of thin films showed a concentric diffraction ring revealing the polycrystalline of thin films. The (1 1 0) and (1 0 1) anatase, (1 1 0) rutile, and (1 1 1) and (2 0 0) Ti_0.895O_.77 microcrystalline phase were found in this area. Furthermore, in the middle layer of thin films coexisted (1 1 1) and (2 0 0) Ti_0.895O_.77 and (1 0 1) and (2 0 0) ZnO microcrystalline. This observation is consistent with the XRD results, as explained in Fig. 2a. Ti_0.895O_.77 phase existed in the middle layer, implying a lack of oxygen content in the film oxidized at 400 °C.

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Fig. 4

Cross-sectional Transmission Electron Microscopes (TEM) images of a thermally oxidized Ti/ZnO thin film sample at 400 °C: (a) bright field of the film with a thickness of 253.78 nm. The images (inset A and B) were selected for High-Resolution TEM and electron diffraction analysis: (b) HR-TEM image showing point “A” (c) Selected area diffraction (SAED) pattern of section “A” adjacent to anatase, rutile and Ti_0.895O_.77, (d) HR-TEM image showing point B, and (e) SAED pattern of section “B” adjacent to Ti_0.895O_.77 and ZnO.

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Furthermore, Fig. 5 shows HR-TEM micrographs and SAED patterns of Ti/ZnO bilayer thin film sample thermally oxidized at 500 °C. The bright cross-sectional field of the TiO₂/ZnO thin films sample shows a thin film thickness of approximately 125.560 and 173.170 nm for ZnO and TiO₂, respectively. This shows that the film has good crystallinity in all zones, as shown in Fig. 5b to e. According to HR-TEM and SAED pattern analysis, the crystalline grains belong to the upper layer's thin films' rutile and anatase phases (Fig. 5b and c). Instead of anatase and rutile, the ZnO phase also existed on the middle layer, as shown in Fig. 5d to e. Literature indicates that TiO₂ growth is triggered in the rutile and anatase mixture phase, and the rutile phase rises with increasing thermal oxidation temperature (Yeh et al., 2018, Lee et al., 2021, Zhou et al., 2013b). During thermal oxidation, increasing oxidation temperature will induce sufficient oxygen to alter Ti layer microstructure and phase development. It must be pointed out that anatase phase still existed at 500 °C, which was due to incorporated of ZnO bottom layer.

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Fig. 5

Cross-sectional Transmission Electron Microscopes (TEM) images of a thermally oxidized Ti/ZnO thin film sample thermally oxidized at 500 °C: (a) bright field of the film with a thickness of 299.73 nm. The images (inset A and B) were selected for High-Resolution TEM and electron diffraction analysis: (b) HR-TEM image showing point “A” (c) Selected area diffraction (SAED) pattern of section “A” adjacent to anatase and rutile (d) HR-TEM image showing point B, and (e) SAED pattern of section “B” adjacent to anatase, rutile and ZnO.

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3.2 Optical properties and band gap energy

To further determine the optical characteristic of the thin films under various oxidation temperatures, transmission and absorbance were measured using UV–VIS spectroscopy. Transmission spectra as a function of oxidation temperature are displayed in Fig. 6a. ZnO, TiO₂, and glass substrates were used as references. The average transmittance in the visible range is around 3.7–78.1 % for TiO₂/ZnO bilayer thin films. Increased visible transmittance is discovered to be a function of oxidation temperature. Enhanced transmittance is responsible primarily for the increased surface area's reaction to light emitted. The optical transmittance spectra of Ti/ZnO thin films thermally oxidized at 400 °C showed only 3.7 % in the visible region. However, oxidation temperature will increase film transmittance to 78.2 % at 500 °C, as seen in Fig. 6.

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Fig. 6

Optical properties of TiO₂/ZnO thin films. (a) Transmittance of the TiO₂/ZnO thin films under various oxidation temperatures. (b) Plot of ( $\alpha hv$ )^0.5 versus photon energy ( $\mathit{hv}$ ) of the TiO₂/ ZnO thin films under different oxidation temperature (inset: plot of ( $\alpha hv$ )² versus photon energy ( $\mathit{hv}$ ) of the ZnO thin films).

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Enhanced crystallinity of TiO₂ and reduced optical scattering owing to densification of film crystallites, thickness, and defects might contribute to an increase in optical transmittance (Salaken et al., 2013), (Kamaruddin et al., 2011). The lower transmittance in Ti/ZnO bilayer films with 400 °C oxidation temperature could be due to the presence of the (1 1 1) and (2 0 0) Ti_0.895O_.77, as shown on XRD patterns (Fig. 2a and Fig. 3), implying the lack of oxygen content in the film. Ti_0.895O_.77 exhibited higher reflective properties due to inadequate oxygen in films. Ting et al. (Ting et al., 2000) suggested that thin-film transmittance improved by raising the thermal oxidation temperature, which probably removed the residual stress, quantum confinement, and structure order improvement. Similarly, Sreedhar et al. (Sreedhar et al., 2017) demonstrated that transmission in the TiO₂/ZnO composite drops rapidly at about 400 nm, consistent with electron excitation from the valence to the conduction bands. It clearly demonstrates that TiO₂/ZnO films were sensitive to visible light owing to a red shift in the optical absorption edge, which lowered the band gap energy. Furthermore, the transmittance of TiO₂/ZnO bilayer thin films is lower than that of the single layer of ZnO or TiO₂ due to increasing the thickness of the film, leading to a decreasing transmittance.

The optical band gap of TiO₂/ZnO thin films was determined using an indirect band gap semiconductor by plotting the graph of the incident photon energy (hv) against (αhv)^0.5. The bandgap values in the range of 2.5–3.05 eV of TiO₂/ZnO bilayer film with oxidation temperature are shown in Fig. 6b, which is the crucial factor for carrier generation. This is in good agreement with the results reported earlier that showed TiO₂/ZnO films band gap under different TiO₂ thicknesses around 3.18–3.23 eV (Sreedhar et al., 2017). Moreover, the lower value energy band gap was observed in thin-film samples with a lower oxidation temperature. A possible reason is the higher density of donor states near the conduction band, which is governed by the oxygen vacancies on the thin films. This results in good agreement with XRD analysis that shows Ti/ZnO thin films thermally oxidized at 400 °C have a Ti_0.895O_.77 phase that implies a lack of oxygen content in the film.

TiO₂ shows an indirect bandgap while ZnO displays a direct bandgap, with corresponding Ε_g values of 3.14 eV and 3.26, respectively. The combination of ZnO and TiO₂ material reduced the energy band gap and could improve the minimum energy required for excitation electron. The electron will become quickly excited from the valence band to the conduction band (Firdaus et al., 2012). Hernandez et al. (Hernández et al., 2014) similarly observed band gap reduction of TiO₂/ZnO samples due to several possibilities, such as (i) functional band gap distinction among anatase TiO₂ (3.2 eV) and ZnO (3.37 eV), (ii) rising of TiO₂ content on the ZnO surface (Yan et al., 2012), (iii) interlayer Fermi level equalization arrangement (Kalanur et al., 2017) and (iv) The Burstein-Moss (BM) effect (Pérez-González et al., 2017).

This finding showed that the transmittance and energy band gap increased as a function of oxidation temperature. It is known that the thermal oxidation process of TiO₂/ZnO bilayer thin film is a controlled mechanism of oxygen diffusion. Increasing thermal oxidation temperature could be attributed to the reduction of oxygen vacancies in the films, which increases transmittance and energy band gap. Consequently, Alteration in oxidation temperature is emphasized here as a viable method for transmittance and band gap adjustment and collecting a greater proportion of incoming light. In addition, the increased film crystallinity is linked to a shift in the optical absorption edge towards the visible light range, improving charge carrier mobility concerning recombination rate.

3.3 Photocatalytic activity

The photocatalytic activity of thin-film samples was investigated by decomposition of MB solution under UVA irradiation. All thin films sample display increasing photocatalytic activity as a function of irradiation time, as shown in Fig. 7a. The MB degradation percentage for Ti/ZnO sample thermal oxidized at 400, 450, 500 and 550 °C were 54.641, 69.128, 85.021 and 87.009 %, respectively. It showed that the degradation efficiency of the Ti/ZnO thin films increases by increasing oxidation temperature. The highest degradation efficiency was found for Ti/ZnO thermal oxidized at 550 °C. The pure TiO₂ thin films sample shows only 53.510% of MB degradation percentage. TiO₂/ZnO thin films exhibit significantly higher photocatalytic performance than TiO₂ thin films. However, the degradation efficiency of ZnO single layer and TiO₂/ZnO bilayer are comparable, around 88.601 and 87.009%, which were the highest among the samples.

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Fig. 7

Photodegradation of Methylene Blue (MB) solution by photocatalytic reaction with TiO₂/ ZnO thin film under UV-light (λ = 365 nm) irradiation and (b) Kinetics curves (first-order reaction) of thin films samples with MB under various oxidation temperature. The experimental condition is: MB concentration (C₀) = 10 mg/L, T = 25 ± 2 °C.

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The possible reason is that ZnO is the primary source of photogenerated charge carriers, and its surface reactions proceed unobstructed. The inclusion of TiO₂ thin films on the ZnO layer could limit the wavelength of light absorbed due to the shift in the absorption edge to shorter wavelengths (Butalid et al., 2020). This phenomenon causes slightly lower degradation efficiencies when compared to ZnO single layer.

The kinetics of MB photodegradation on the thin film surface can be described by the Langmuir-Hinshelwood (LH) first-order reaction kinetics Equation (Eq. (4)): (Lee et al., 2021)

As illustrated in Fig. 7b, the ln (C_t/C₀) is the linear fit slope in the function of the time (t) plot shows the kinetic constant (k) of the photodegradation. The photocatalytic reaction process was matched with the pseudo-first-order reaction kinetics. The reaction constants (k) of the ZnO, TiO₂ and Ti/ZnO thin films oxidized at various temperatures are listed in Table 3. Linear fits are in good agreement (R² > 0.90) with the LH model. The degradation rate constant (k) gradually increases with an increased oxidation temperature. However, a decrease in the photocatalytic activity and rate constant (k) was observed on Ti/ZnO thin films that oxidized at 400 °C. The decrease could be attributed to the existence of the (1 1 1) (2 0 0) Ti_0.895O_.77 phase, which also decreases the optical transmittance, as shown on XRD analysis (Fig. 2a). In the thin-film crystal structure, defect states (such as oxygen vacancies) are well-known to cause decreased photocatalytic activity.

A slight decrease in the rate constant was observed when TiO₂ was applied to the ZnO surface. The reduction may be ascribed to the blueshift of the TiO₂-coated ZnO film's absorption edge related to that of ZnO film (Butalid et al., 2020). It is known that the crystal structure influences photocatalytic efficiency. TiO₂ photocatalytic activity may be improved on its (1 0 1) and (0 0 1) faces, while ZnO is optimized on its zinc-terminated polar (0001) (Zhou et al., 2019). In addition, the band gap's energy is crucial to the thin film photocatalytic capabilities.

Semiconducting photocatalysts are highly dependent on the dissociation of the photogenerated hole–electron pairs and the transport of electrons from the photocatalyst into organic pollutants trapped inside oxygen vacancy defects on the surface (Yu et al., 2020). Yi Zhou et al. (Zhou et al., 2019) reported that the higher band conduction band (cb) of TiO₂ allowed charge transfer from ZnO to TiO₂ through a direct Z-scheme photocatalytic mechanism. A possible photocatalytic reaction process for TiO₂/ZnO bilayer thin film has been proposed in Fig. 8. TiO₂/ZnO is a heterogeneous semiconductor-based photocatalysis approach with a bandgap of around 3.2–3.34 eV (Hernández et al., 2014; Tekin et al., 2020). In the presence of UVA light, electrons will migrate from vb ( $h_{\mathit{vb}}^{+}$ ) to cb ( $e_{\mathit{cb}}^{-}$ ) (Eqs. (5) and (6)) due to higher energy photons than in the thin film bandgap. Photogenerated electrons ( ${e_{\mathit{cb}}^{-}\mathrm{a}\mathrm{n}\mathrm{d}h}_{\mathit{vb}}^{+}$ ) rapidly react with the adsorbed oxygen and water molecule to produce highly reactive hydroxyl radicals (^•OH) (Eq. (8)) and superoxide anions radical ( $O_2^{·-}$ ) (Eq. (7)) (Vinu and Madras, 2010). Accordingly, it can be inferred that the heterojunction defect levels do not significantly alter photocatalytic activity. In addition, the electron-hole pairs are separated because the electrons remain in the cb of TiO₂, where the hole remains in the vb of ZnO, which reduces the recombination rate and enhances the photocatalytic activity of TiO₂/ZnO bilayer thin films. The following equations describe how photocatalytic reaction of TiO₂/ZnO thin films (Eqs. (5)–(12)):

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Fig. 8

Proposed photocatalyst schematic diagram of the TiO₂/ZnO bilayer thin film.

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This study revealed that the photocatalytic of TiO₂/ZnO thin films depends on the oxidation temperature that affects the crystalline phase. It could be noted that the addition of ZnO as bottom layer thin films could promote increasing photocatalytic activity of TiO₂ thin films. These studies indicate that the oxidation temperature is crucial in obtaining the high photocatalytic performance of TiO₂/ZnO bilayer thin films.

A number of studies have demonstrated the photocatalytic activity of ZnO-TiO₂ thin films prepared with various methods, as listed in Table 4. However, the photocatalytic activity of TiO₂/ZnO bilayer thin films produced by magnetron sputtering combined with thermal oxidation techniques has been described in only a few published studies. At the same irradiation time, the photocatalytic activity obtained in this research is higher than TiO₂/ZnO developed with the sol–gel method (Hussin et al., 2019), (Pérez-González et al., 2015) and reactive magnetron sputtering (MS) (Pérez-González et al., 2017). However, this study's photocatalytic results were lower than Butalid et al. [37]. Several factors influence the photocatalytic activity of thin films, such as thin-film profile (thickness, morphology, element composition, crystallinity, band gap energy, etc.), the pollutant type and concentration, and irradiation condition (type, light intensity, time). This research revealed that the development of TiO₂/ZnO thin films by thermal oxidation could enhance the high photocatalytic activity of thin films.

Catalyst reusability is an important aspect of thin film catalysis. Five consecutive cycle tests were applied to show further the photostability of thin-film samples on the removal of MB solution. Fig. 9 showcases thin films' degradation rate stability profile under 300 min UV irradiation time. It can be observed that different thin films sample showed different MB degradation profiles. Among them, ZnO thin films demonstrated the weakest photostability, which promotes decrease of 25.952 and 57.698% degradation efficiency after three and five cycles, respectively, as shown in Fig. 9a. However, there were less significant changes in the degradation efficiency of the TiO₂ (6.570%) (Fig. 9b) and TiO₂/ZnO (4.029%) (Fig. 9c) bilayer thin film samples in five consecutive cycle tests. A comparison of the XRD patterns of the initial and 5 times reused TiO₂ and ZnO thin films is shown in Supplementary material B, Fig. B1. Fig. B1a (Supplementary material B) indicates that there were no additional phases, and this result is almost similar to the initial TiO₂ thin film. However, in ZnO thin films show there are decreasing peak intensity after 5 times used (Supplementary material B, Fig. B1b). This finding demonstrates that TiO₂ films are photochemically stable even after being reused 5 times, since the crystalline structure of the films remains the same despite subsequent degradations, with the intensity of the peak only slightly increasing.

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Fig. 9

Photocatalytic stability performance of (a) ZnO and (b) TiO₂ and (c) TiO₂/ZnO bilayer toward photodegradation of Methylene Blue solution under 300 min of UV irradiation.

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This finding indicates that the addition of TiO₂ as the upper layer on ZnO thin films was seen to decrease the photo corrosion of ZnO during photodegradation. Removing photocatalytic material by photo corrosion would decrease the photocatalytic efficiency due to a reduction in generated charge carriers. Butalid et al. (Butalid et al., 2020) have reported that Zn²⁺ was obtained in the MB solution, which had a high concentration on ZnO thin films that TiO₂/ZnO thin films after the third cycle of photodegradation of ZnO thin film. Furthermore, Ikizler and Peker (İkizler and Peker, 2016) investigated the spin coated TiO₂ on hydrothermally grown ZnO nanorods. These results showed that the concentration of Zn²⁺ ions decreased from 1.1 to 0.6 ppm when ZnO surfaces were coated with TiO₂. However, lower Zn²⁺ ions were still found on the samples due to the incompleteness of the TiO₂ coating, resulting in areas on the ZnO surface with pitting corrosion; hence the observed. This is attributed to the effect of photo corrosion that caused the dissolution of the ZnO thin films. Photo corrosion issue as a ZnO drawback could reduce the degradation efficiency and shorten the catalyst lifetime. furthermore, Zn²⁺ ions may also harm water-borne microbes if the concentration is above a certain threshold (Franklin et al., 2007), (İkizler and Peker, 2016). This result confirms that protective layers such as TiO₂ minimize the effect of photo corrosion which promotes high recyclability and photocatalytic activity of TiO₂/ZnO thin films. Further analysis was established to investigate the correlation between the photocatalytic activity and antimicrobial activity of ZnO, TiO₂ single layer, and TiO₂/ZnO (thermal oxidized 550 °C) bilayer thin films.

3.4 Antimicrobial activity

The antimicrobial behavior of synthesized thin-film samples against gram-negative (E. coli), positive bacteria (S. aureus), and pathogenic yeast (C. albicans) was evaluated using an agar diffusion test. The diameter of clear zones, which indicates inhibition of microbe growth, was used to determine the antimicrobial activity. The antimicrobial behavior was evaluated in the absence of light (dark), visible and UVA light with a 30-minute exposure time, as displayed in Supplementary material C, Fig. C1. In the dark condition, no inhibition zone is found in all samples. However, introducing a visible light condition for TiO₂/ZnO and TiO₂ thin film samples could somewhat inhibit the growth of E. coli and S. aureus. In addition, the inhibition zones have started to increase under UVA light exposures with a diameter zone of around 33.7, 28.13, and 33.23 mm for ZnO, TiO₂, and TiO₂/ZnO thin films, respectively (Supplementary material C, Table C1). These inhibition diameters are nearly equal to or even more extensive than the diameter of the antibiotic used as a positive control. Previous studies investigated the zone inhibition analysis of TiO₂/ZnO nanocomposite using different bacteria such as E.coli (19.7 mm) (Ali et al., 2021), (25 mm) (Vignesh et al., 2017), and S. aureus (21.38 mm) (Harun et al., 2018), (17.67 mm) (Siwińska-Stefańska et al., 2019) (Supplementary material C, Table C2). which showed relevance to the present study. The light has a significant advantage when comparing the antimicrobial activity of dark, light, and UVA inhibition zones. The results proved that assimilation of the photocatalyst with UVA light conditions significantly influenced antimicrobial behavior compared to visible light or dark conditions. Further analysis was established to examine the effectiveness time of UVA light exposure on thin films against three types of microbes.

Fig. 10 presents exposures of ZnO, TiO₂, and TiO₂/ZnO thin films under 10 min UVA irradiation could exhibit a significant inhibition rate (%) (p < 0.05) against three types of microbes with an average inhibition rate of 58.222, 44.444 and 77.111 %, respectively. The negative controls showed a low inhibitory effect (<5%), and antibiotics as positive controls showed a 94% inhibition rate. It could be seen that the TiO₂/ZnO inhibition rate at 10- and 20-min irradiation is significantly higher than in ZnO and TiO₂ thin films. However, after a 30-min irradiation time, the inhibition rate was not significantly different for ZnO and TiO₂/ZnO films. Under different irradiation times, TiO₂ exhibits less antimicrobial activity than ZnO and TiO₂/ZnO thin films. Furthermore, increasing the UVA exposure time to 30 min results in a high inhibition rate of 97.733 ± 3.05, 98.07 ± 2.41, and 95.800 ± 3.11% against E. coli, S. aureus, and C. albicans, respectively (Fig. 10). These inhibition rates are close to those of antibiotics as a positive control. Increased irradiation exposure time resulted in a substantial enhancement in the inhibition rate against the three pathogenic microbes. Although integrating photocatalyst with UVA light to decrease microbial growth is not new, this present study confirmed that exposure time responses of antimicrobial activity to increased inhibition values demonstrated the time-sensitive properties of UVA irradiation as a potential and fast disinfection.

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Fig. 10

Effect of ZnO, TiO₂ and TiO₂/ZnO thin films on antimicrobial activity against E. coli, S. aureus and C. albicans compared to the substrate (negative control) and antibiotic (positive control) under different UV light irradiation treatments. Means denoted by a different letter indicate significant differences between treatments (p < 0.05; Tukey HSD test). The data was expressed as mean ± standard deviation (n = 3).

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The antimicrobial effect on ZnO, TiO₂, and TiO₂/ZnO thin films against three types of pathogenic microbes could be due to several mechanisms, as illustrated in Fig. 11. First, the electrostatic interaction between the positive charge of TiO₂/ZnO and the negative charge of the microbe cell might result in instability of the microbial cell wall. The instability of the microbial cell wall could cause membrane disruption, morphological changes, and intracellular substance liberation (Brayner et al., 2006). Moreover, the release of Zn²⁺ ions from ZnO thin films under UV light irradiation (Butalid et al., 2020; Sapkal et al., 2012; Vignesh et al., 2017) has the potential to damage the integrity of microbial cells that serve as a crucial cellular defense mechanism for regulating zinc excess and deficiency (Kasemets et al., 2009) (Joe et al., 2017). It is noted that increasing irradiation time could increase Zn²⁺ ion concentration, which promotes high antimicrobial activity. However, applying TiO₂ as the upper layer on ZnO (TiO₂/ZnO) reduced the photocorrosion effect (Butalid et al., 2020), (Sapkal et al., 2012) and resulted in a decrease in Zn²⁺ ion concentration that could only cause minor antimicrobial activity.

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Fig. 11

Proposed antimicrobial based photocatalytic mechanism of (a) ZnO, (b) TiO₂, and (c) TiO₂/ZnO thin films.

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During the photocatalytic process, the reactive hydroxyl radical (•OH), superoxide anions radical ( $O_2^{·-}$ ), and hydrogen peroxide (H₂O₂), known as Reactive Oxygen Species (ROS), cause serious damage to microorganisms (Abebe et al., 2020; Espitia et al., 2012; Gordon et al., 2011). ROS promotes the oxidation of cellular structures that promote partial loss of cell membrane integrity, permeabilization of cell membrane, exposed cellular component. As a results, leads to the complete microbial inactivation (Equation (13)) (Afreen et al., 2020; Yemmireddy and Hung, 2017).

Srinivinas et al. (Srinivas et al., 2019) have shown that ROS-induced stress may disrupt microbe structure, leading to a lack of selective function and energy (ATP) synthesis, which impacts DNA replication.

The significant antimicrobial activity enhancement of TiO₂/ZnO thin films can be ascribed to the UV irradiation and the thin film properties (crystallinity and crystalline size). As mentioned in Table 2, TiO₂/ZnO thin films have a smaller crystalline size and a larger unit cell volume than ZnO and TiO₂; thus, this condition may promote a high number of photocatalytic reactions sites. The increased photocatalytic reaction sites forge a more considerable amount of ROS that enhances antimicrobial performance. In addition, oxidation of Ti/ZnO at 550 °C could promote the high crystal structure of ZnO wurtzite, anatase, and rutile TiO₂. Instead of the high antimicrobial activity of ZnO, the mixed phase of TiO₂ rutile and anatase can also promote higher photocatalytic activity than a single TiO₂ phase. The electrons from the rutile to anatase TiO₂ during photoexcitation may avoid charge recombination in the rutile phase (Kalarivalappil et al., 2019; Zhu et al., 2018), producing efficient photogenerated electron-hole pair separation and high photocatalytic and high antimicrobial performance. Furthermore, adding a TiO₂ layer could reduce rapid recombination on ZnO thin films, enhancing the efficient photocatalytic and antimicrobial activity of TiO₂/ZnO thin films. Antimicrobial inhibition and photocatalytic activity were found to have a strong linear relationship in the thin film sample. The antimicrobial activity of the thin film sample is related to various characteristics, including surface chemistry and microbe concentration, as well as the intensity of the light (Adams et al., 2006).

The photographs of the viable microbe colonies (white spots) on the different irradiation times of TiO₂/ZnO thin films are shown in Fig. 12. The viable microbe colony shows that TiO₂/ZnO thin film could inhibit bacterial growth, whereas increasing irradiation exposure time resulted in a decreased viable microbe colony. In particular, S. aureus showed considerably greater sensitivity (98.07%) to TiO₂/ZnO thin films than E. coli (97.73%) and C. albicans (95.8%) under 30 min irradiation. Similar to the study reported by Widyastuti et al. (Widyastuti et al., 2021) and Russell (Russell, 2003), which mentioned that E. coli is more resistant to ROS than S. aureus. Structural changes in the bacterial cell membrane significantly contribute to increased resistance. E. coli has an exterior cell membrane made mostly of lipopolysaccharide (LPS) and a very thin peptidoglycan layer (Epand and Epand, 2009), which serves as a barrier to block the entry of reactive oxygen species (ROS) into the cell. Moreover, this structure is more complex than S. aureus (Russell, 2003). Furthermore, Fig. 12 indicates that the antibacterial activity (E. coli and S. aureus) of TiO₂/ZnO seems stronger than antifungal activity (C. albicans). Then, it is widely known that fungi have a greater tolerance to the adverse external environment than bacteria due to their thicker cell walls and spore generation capacity (Asare et al., 2022; Probst et al., 2022).

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Fig. 12

The photograph of viable E. coli, S. aureus and C. albicans during different UV-irradiation times treated with TiO₂/ZnO bilayer thin film (thermally oxidized at 550 °C).

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Numerous researchers have assessed the antibacterial activity of TiO₂/ZnO in various morphologies, as listed in Table 5. However, there has been limited research on the antimicrobial activity of TiO₂/ZnO thin films. The antimicrobial activity of TiO₂/ZnO thin films that were developed in this work showed higher inhibition activity with a fast disinfection time than those reported formerly. Meanwhile, using UVA light as an irradiation lamp during photocatalyst contact time is promising to encourage fast and high antimicrobial activity.

These findings suggest that the thermal oxidation temperature of Ti/ZnO bilayer thin films plays a crucial role in generating good photocatalytic and antimicrobial performance. Moreover, Ti/ZnO thin film through 550 °C thermal oxidation could provide ZnO wurtzite, anatase, and rutile TiO₂ crystal phases with good photocatalytic activity and higher antimicrobial activity. This study reveals that incorporating the magnetron sputtering with the thermal oxidation method may enhance the efficacy of TiO₂/ZnO bilayer thin films as photocatalysts and antimicrobial agents toward pathogenic bacteria and fungus. Therefore, the synthesized TiO₂/ZnO thin films embodied with UV irradiation lamps appear to be a promising antimicrobial coating with good opportunity for industrial scaling up and being a cost-effective and time-effective material.