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TiO2 mischcrystal composites and antibacterial activities for Escherichia coli under visible light
Author’s Accepted Manuscript Preparation of novel Cu/TiO2 mischcrystal composites and antibacterial activities for Escherichia coli under visible light Yingchun Miao, Xiaolin Xu, Kaiquan Liu, Ninghong Wang www.elsevier.com/locate/ceri
PII: DOI: Reference:
S0272-8842(17)30743-5 http://dx.doi.org/10.1016/j.ceramint.2017.04.137 CERI15117
To appear in: Ceramics International Received date: 29 March 2017 Revised date: 17 April 2017 Accepted date: 24 April 2017 Cite this article as: Yingchun Miao, Xiaolin Xu, Kaiquan Liu and Ninghong Wang, Preparation of novel Cu/TiO 2 mischcrystal composites and antibacterial activities for Escherichia coli under visible light, Ceramics International, http://dx.doi.org/10.1016/j.ceramint.2017.04.137 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation of novel Cu/TiO2 mischcrystal composites and antibacterial activities for Escherichia coli under visible light Yingchun Miao*, Xiaolin Xu, Kaiquan Liu, NinghongWang Faculty of Chemical and environment Sciences, Qujing Normal University, Qujing 655000, China Abstract: In this paper, novel copper/titanium dioxide (Cu/TiO2) composites were facilely prepared by simple sol-gel hydrothermal method. The crystalline phases, surface morphology and antibacterial activity of Cu/TiO2 were investigated systematically. Interestingly, the as-prepared Cu/TiO2 materials were made up with three different crystalline phases, including cuprite Cu2Cl(OH)3, Cu2+1O, and anatase TiO2. Next, the ‘zone of inhibition’ experiment was performed against E. coli using Cu/TiO2 catalysts with different Cu doping concentration as the antibacterial agents. Our results show that the Cu/TiO2-3.00 catalyst possesses optimal antibacterial ability against E. coli, which may be connected with the strong oxidizing reactive oxygen species (ROS) destroying the bacteria and photogenerated charge separation and recombination. Key words: Titanium dioxide; Copper; Composites; Photocatalysis; Antibacterial activities. 1. Introduction Since favorable mechanical performance, excellent chemical stability, corrosion-resistance and good biocompatibility, Titanium dioxide and its composites have attracted wide scientific interest for photo-degradation of pollutants and antibacterial applications, such as degradation of organic contaminants and disinfection of microorganism[1-5]. After Matsunaga et al. first reported photocatalytic antibacterial performance of platinum-doped TiO2 in 1985[6], TiO2-based nanocomposites have been extensively investigated for various microorganism disinfections,
*[email protected]（Corresponding author: Yingchun Miao）
including viruses, bacteria, fungi, algae and protozoa[2, 7-11]. For example, Ag-TiO2 nanoparticles incorporated SiO2 films can inhibited the gram negative bacteria E. coli. TiO2/Ag2O heterostructure could simultaneously remove the organic pollutant and kill the bacterium under visible light irradiation[7]. Additionally, Kim et al reported the chitosan/AgCl–TiO2 colloid produced excellent antibacterial activity for S. aureus and E. coli[12]. Generally, photocatalytic antibacterial performance of these TiO2-based composites depends on the interaction between microorganisms and reactive oxygen species (ROSs) generated from the electron-hole pairs under light irradiation[13-15]. These metal nanoparticles doping can remedy the shortcoming of TiO2, owing to its wide band gap and fast recombination of photo-induced electron-hole pairs. Recently, numerous studies reported metal nanoparticles could also be used as antibacterial agents to kill microorganism, mainly including Au, Ag, Zn, Pd and Cu[16-21]. For instance, ZnO nanoparticles were found to exert bactericidal effect against S. aureus, Campylobacter jejuniand E. coli[20]. Cu nanoparticles could be employed as an efflux pump inhibitor to tackle drug resistant bacteria[22]. Remarkably, among all the metal species, Cu as an essential element in living organisms has aroused wide investigation because of its excellent antibacterial properties, low toxicity and low-cost[13, 15, 18, 23]. Interestingly, Cu-containing TiO2 composites prepared by different technologies showed better antibacterial activities than pure Cu nanoparticles or TiO2. For example, Cu-Ti-O NTAs possess excellent long-term antibacterial ability against Staphylococcus aureus (S. aureus)[17]. Although Cu is an excellent candidate for replacing noble metal materials in the antibacterial applications, the preparation and practical applications of Cu decorated TiO2 heterostructures with high antibacterial effect still suffer from huge challenges, such as complicated producing technology and low photoactivity. Few reports are available for
antibacterial application of the hybrid CuxO/TiO2 nanocomposites[24-26]. Hence, more researches about these Cu decorated TiO2 heterostructures should be conducted. Generally, the technologies prepared the Cu doping TiO2 nanocomposites (Cu/TiO2) mainly include solvothermal reaction, coprecipitation, and impregnation method[27-29]. In this work, the Cu/TiO2 composites were synthesized by a novel sol-gel hydrothermal method at low temperature. Amazedly, cuprite Cu2Cl(OH)3, and Cu2+1O crystals can be successfully decorated on the anatase TiO2. Various characterization techniques were employed to analyze the unique Cu/TiO2 mischcrystal nanomaterials. Additionally, the antibacterial activities of Cu/TiO2 nanomaterials were systematically studied and discussed. 2. Experimental 2.1 Materials and characterization All reagents were purchased from commercial companies and used without further purification, including titanium tetrachloride (TiCl4, AR), copper foil (purity: 99.5%), agar (AR), tryptone (LP), yeast extract (AR), NaOH(AR) and NaCl (AR). Powder X-ray diffraction (XRD) measurements were carried out on a Rigaku D/max 2000 single-crystal diffractometer using Cu Kα radiation (λ=0.15405nm).Energy dispersive spectroscopy (EDS) analysis was performed with HITACHI S-4800 SEM equipped with Oxford X-max spectrometer. Fourier transform infrared (FT-IR) spectroscopy was performed on NEXUS 470.The morphologies of the samples were analyzed by a HITACHI S-4800 scanning electron microscopy (SEM) and a JEM-2010 transmission electron microscope (TEM). The surface compositions of samples were measured by X-ray photoelectron spectroscopy (XPS, Versa Probe PHI 5000). The absorption spectra of the samples were recorded using a Varian Cary 500 UV-visible spectrophotometer. The magnetic properties of the materials
were studied by using avibrating sample magnetometer (LDJ 9600-1, USA). 2.2 Synthesis of various Cu/TiO2nanocomposites Typically, 10 mL titanium tetrachloride (TiCl4) was placed into a 25 mL beaker, and liquid nitrogen was added under stirring to obtain TiCl4 colloid. The precursor gel was kept static for 5 h at room temperature. Next, the colloid and copper foil were sealed within a 100 mL Teflon-lined autoclave, and heated at 160 °C for 24 h. After completion of the reaction, the products were obtained by filtration, and washed three times with water and ethanol respectively. Finally, the solid materials were dried under vacuum at 60 °C for 12 h. Other materials with different copper doping concentration were synthesized by same method, and labeled as Cu/TiO2-x (x represents the copper doping concentration in these materials). 2.3 Culture and treatment of microorganisms: Liquid medium: 2.5 g tryptone, 1.25 g yeast extract and 2.5 g sodium chloride were added into 25 ml Erlenmeyer flask and stirred for 10 min. Next, the pH value of this system was adjusted to 7.0 by 1mol/L sodium hydroxide solution. Finally, the mixture was disinfected in the high-handed sterilization pan to obtain a sterile, liquid medium. Solid medium: 5.875 g agar powder was added into 250 ml Erlenmeyer flask, and dissolved in 250 mL water by heating to 98 °C. Next, the solution was sealed by rubber plug and cooled to room temperature. Finally, the system was disinfected the high-handed sterilization pan to obtain a sterile, solid medium. Culture of microorganisms: The gram-negative Escherichia coli(ATCC8099) were used in this work. The zone of inhibition test was employed to evaluate the antibacterial activity of Cu/TiO2 materials. First, the gram-negative E. coliwere cultivated in the Luria Bertani (LB) media. Then,
the nutrient agar plates were in oculated with 1 mL of bacterial suspension containing around104 colony forming units (CFU) by using spread plate method. Next, the Cu/TiO2 photocatalysts with different Cu doping concentration were gently placed on the inoculated plates, and then the seplates were exposed on the visible light at 30oC for 40min. After that, these dishes were cultivated in the artificial bioclimatic test chamberfor 24 h. Finally, these tests were performed in triplicate. Before adsorption, pure P25 were used as control. 3. Results and analysis 3.1 Characterization of samples First, the crystalline phases of as-prepared Cu/TiO2-x materials were characterized by XRD technology. As seen from Figure 1, when the Cu doping concentration in the materials was below 2.0%, diffraction peak of the anatase TiO2 can only be observed at 2θ=27.5, 36.2, 41.3, 54.5, and 56.6° (JCPDS file Card No. 21-1272). No peaks associated with Cu species were detected in the XRD patterns, which was likely due to the low crystallinity and the high dispersion of Cu on TiO2 surfaces. Interestingly, with the increase in Cu doping concentration, two kind of crystalline phases Cu species appeared on the XRD pattern, which matched with the Cu2+1O (2θ=16.4, 28.9, and 61.5°; JCPDS file Card No. 05-0667) and cuprite Cu2Cl(OH)3 (2θ=16.3, 19.2, 32.5, and 47.6°; JCPDS file Card No. 50-1559). Results indicated that the three crystal forms (TiO2, Cu2+1O and Cu2Cl(OH)3) in the materials were generated by the simple hydrothermal treatment, which may enhance the antibacterial activity of these materials. To further identify the elemental composition, the EDX analysis was performed. As shown in figure 2a, Cu/TiO2-3.00 catalysts contained Ti, O, Cu, and Cl. No other elements were found in this sample. Additionally, the FT-IR was employed to investigate the chemical bond of samples in
Figure 2b. It can be clearly seen that -OH bending vibration and stretching vibration absorption peaksappeared around 1600 cm-1 and 3500 cm-1 respectively[30], indicating the existence of adsorbed water in these materials. The peaks at about 3356 cm-1and 3317 cm-1 can be attributed to the fundamental stretching vibration of surface hydroxyl groups in Cu/TiO2 samples [31], which are common characteristics of oxide semiconductors and essential conditions for photocatalysis. These groups can enhance the photocatalytic performance through the generation of hydroxyl radicals as a result of their trapping by the photogenerated holes [31]. At the same time, we found that the absorption peaks at 3356 cm-1, 3317 cm-1, 2900 cm-1, and 2800 cm-1 became stronger with the increase of Cu doping amount. These strong absorption peaks would generate new doping energy levels in the energy gaps of TiO2 and improve the photocatalysis antibacterial performance. However, it is difficult to distinguish some absorption peaks at 400 cm-1 to 800 cm-1 wavenumber due to mischcrystal structure of samples. Next, the morphology of the Cu/TiO2-3.00 catalyst as typical sample was studied by SEM and TEM. It is clearly seen that most of the Cu nanoparticles were well immobilized on the TiO2 nanorods surface, but some fragmentary Cu nanoparticles can also be observed in Figure 3a and b. The crystalline structure of the Cu/TiO2-3.00 catalyst was detected by HRTEM in Figure 3c and d. Notably, the TiO2 (101) (110), Cu2Cl(OH)3 (013), and Cu2+1O (110) crystalline lattices can be clearly observed, corresponding to the XRD analysis in Figure 1. Interestingly, TiO2, Cu2Cl(OH)3 and Cu2+1O crystal planes were linked by the way of head or terminal conjunction, which may be conducive to the electron-transfer between Cu species and TiO2. To further study the micro component of Cu/TiO2-3.00 andobserve the valence of different elements, the XPS spectra of Cu/TiO2-3.00 catalyst was shown in Figure 4. The four major peaks
at about 198.4, 458.4, 531.6, and 934.6 eVshould belong to Cl 2p, Ti 2p, O 1s and Cu 2p in Figure 4a. In Ti 2p spectrum (Figure 4b), the bindingenergies of 458.4 and 464.1 eV were indexed to Ti 2p3/2 and Ti 2p1/2, respectively[7]. For Cu 2p spectrum (Figure 4c), to the best of our knowledge, Cu0 and Cu2+ have more than 2 eVseparations. Therefore, the peaks at 934.6 eV and 954.5 eV could be assigned to Cu2+[32]. The O 1s XPSspectrum of Cu/TiO2-3.00 was separated into two spectral components in Figure 4d, indicating Cu−O (531.6 eV) and Ti−O (529.5 eV)[33]. Additionally, the Cl 2p peak at 198.4 can also be clearly seen in Figure 4e. All the above analyses further verified the combination of Cu2Cl(OH)3 and Cu2+1O with TiO2. Finally, the optical properties of the Cu/TiO2-3.00 photocatalysts were studied by UV–vis spectrometry. Optical absorption analysis revealed that Cu/TiO2-3.00 exhibited absorption edges at 517 nm in Figure 4f, which indicated the good visible response of Cu species onthe surface of TiO2.We inferred that the enhanced response to visible light can be attributed to the embedded metallic Cu species, such as Cu2Cl(OH)3 and Cu2+1O, which promoted the formation of oxygen vacancies in TiO2 through the metal-oxide interaction [34], thus increasing the visible light absorption of Cu/TiO2. 3.2 Antibacterial assay 3.1The zone of inhibition test The ‘zone of inhibition’ experiment was performed against E. coli using Cu/TiO2 catalysts with different Cu doping concentration as the antibacterial agents. First, the dosage of Cu/TiO2-3.00 chose as a typical antibacterial agent was evaluated in Figure 5. Obviously, with the increase of dosage, stronger antibacterial effects on the E. colican be obtained by visible light irradiation for 40 min. Hence, we chose 0.15 mg dosage of catalysts to conduct the subsequent experiments.
Next, antibacterial activities of different Cu/TiO2 catalysts were investigated. As shown in Figure 6, the Cu/TiO2-3.00 photocatalyst exhibited inhibition zones of 25 mm diameter by visible light irradiation for 30 min, which was superior to others. To further compared with antibacterial activities of various Cu/TiO2 catalysts, the bactericidal effects on E. coli at different irradiation times, and changes in inhibition zones were shown in Figure 7. Firstly, the commercial P25 catalyst was chose as the control group. Obviously, all the Cu/TiO2 catalysts exhibited partly bactericidal effects on E. coli, while control plates with P25 showed almost noinhibition zone. Hence, we can conclude that antimicrobial activity of TiO2-based materials can be well improved by the immobilizing Cu species. Meanwhile, with the increase of Cu doping concentration, the antibacterial performance became stronger and the peak appeared at Cu doping concentration of about 3.0% for 10 min irradiation. These results indicated that the Cu/TiO2-3.00 catalyst possessed optimal antibacterial activity. Generally, the antibacterial mechanisms were put forward based on comprehensive consideration for both the photosensitivity of TiO2-based materials and the antibacterial activity of metal-ions[13, 35]. Firstly, the photocatalytic antibacterial mechanism may be involved with the strong oxidizing reactive oxygen species (ROS). For example, hydroxyl radical (·OH), superoxide anion radical (·O-2 ) and hydrogen peroxide (H2O2) are generated in the photocatalytic reaction of TiO2-based catalysts. The chemical formulas can be expressed as follows: + Cu⁄TiO2 + ℎ𝜐 → e− CB (TiO2 ) + hVB (Cu)
(1)
− e− CB + O2 →∙ O2
(2)
− h+ VB + OH →∙ OH
(3)
+ h+ VB + H2 O →∙ OH + H
(4)
∙ + ∙ O− 2 + H → HO2
(5)
∙ − ∙ O− 2 + H2 O → HO2 + OH
(6)
HO∙2 + H2 O → H2 O2 +∙ OH
(7)
H2 O2 → 2 ∙ OH
(8)
According to the above analysis, the TiO2 samples doped with Cu species can improve the response to the visible light, and elevate the photosensitivity of TiO2. Therefore, Cu ions in the Cu/TiO2 catalysts may stabilize the photo-produced electron–hole pairs, which can remarkably prolong the time of ROS destroying the bacteria and improve the antibacterial activities of the Cu/TiO2 nanocomposites. Moreover, some Cu ionsreleased from the Cu/TiO2 nanomaterials can be adsorbed to the surface of microorganism, and plenty of negative charges will be carried by electrostatic force,which can destroy the charge balance of microorganism, and lead to cells deformed seriously until bacteria die by bacteriolysis. Meanwhile, Cu ions can get into the bacteria via cell membrane and interact with some functional groups on the protease, such as sulfhydryl group (ASH), amino group (ANH2), hydroxylgroup (AOH), it will result in the change of the structure and function of protein. In order to identify the above hypothesis, the transient photocurrent response of Cu/TiO2-3.00 photocatalysts was conducted. As shown in Figure 8, among all the as-prepared materials, higher and more stable photocurrent was clearly observed on the Cu/TiO2-3.00 catalyst. This result indicated that photogenerated charge separation could be effectively promoted. At the same time, 3.0% Cu loading was more favourable for charge recombination than others. According to above the antibacterial mechanisms, we concluded that the Cu/TiO2-3.00 catalyst
might maximally prolong the time of ROS destroying the bacteria and the abundance of Cu ions were fully released from the nanocomposite. Hence, the Cu/TiO2-3.00 catalyst showed the excellent antibacterial performance. 4. Conclusion In summary, novel copper/titanium dioxide (Cu/TiO2) composites were facilely prepared by simple sol-gel hydrothermal method. The crystalline phases and surface morphology of Cu/TiO2 were characterized systematically by XRD, FT-IR, XPS, SEM and TEM. Interestingly, the as-prepared Cu/TiO2 materials were made up with three different crystalline phases, containing cuprite Cu2Cl(OH)3, Cu2+1O, and anatase TiO2. Next, the antibacterial experiment was performed against E. coli using Cu/TiO2 catalysts with different Cu doping concentration. Our results showed that the antibacterial activity of Cu/TiO2 catalysts became stronger with the increase of the Cu doping concentration. The Cu/TiO2-3.00 catalyst possessed optimal antibacterial ability against E. coli, which may be connected with the strong oxidizing reactive oxygen species (ROS) destroying the bacteria and photogenerated charge separation and recombination. More investigation of metal/TiO2 nanocomposites will be carried out in the future. Acknowledgments Yunnan Applied Basic Research Project of Province(C0120150543), Key Projects of Yunnan Provincial Department of Education(2015Z183，2016ZZX207), Innovation and Entrepreneurship Project of College Students in Yunnan Province and Open Project of Environmental Chemistry Key Laboratory of Ministry of Education and Shanghai Normal University. Reference [1] A. Mukhopadhyay, S. Basak, J.K. Das, S.K. Medda, K. Chattopadhyay, G. De, Ag−TiO 2
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(2014) 13502-13509. [33] R.A. Rather, S. Singh, B. Pal, A Cu+1/Cu0-TiO2 mesoporous nanocomposite exhibits improved H2 production from H2O under direct solar irradiation, Journal of Catalysis 346 (2017) 1-9. [34] J. Zhao, Y. Li, Y. Zhu, Y. Wang, C. Wang, Enhanced CO 2 photoreduction activity of black TiO2−coated Cu nanoparticles under visible light irradiation: Role of metallic Cu, Applied Catalysis A: General 510 (2016) 34-41. [35] W. Fan, T. Liu, X. Li, R. Peng, Y. Zhang, Nano-TiO2 affects Cu speciation, extracellular enzyme activity, and bacterial communities in sediments, Environmental pollution 218 (2016) 77-85. Figure 1. XRD patterns of the Cu/TiO2 material with different Cu doping amount. Figure 2.EDX analysis (a) of Cu/TiO2-3.00 and FT-IR (b) of all the samples. Figure 3.SEM (a) and TEM (b, c, and d) images of the Cu/TiO2-3.00 catalyst. Figure 4.XPS analysis (a-e) and UV-Vis DRS spectra (f) of the Cu/TiO2-3.00 catalyst. Figure 5.The antibacterial activity of Cu/TiO2-3.00 with different dosage. Figure 6.Zone of inhibition observed around various Cu/TiO2 in nutrient agarplates inoculated with bacterial suspension of E. coli.
Figure 7. The comparison of the antibacterialzones of various Cu/TiO2 catalysts Figure 8.Transient photocurrent response of photocatalysts for two20 s light-on-off cycles in 1M Na2SO4 aqueous solution under solar light irradiation with a bias potential of 1.0 V.
PII: DOI: Reference:
S0272-8842(17)30743-5 http://dx.doi.org/10.1016/j.ceramint.2017.04.137 CERI15117
To appear in: Ceramics International Received date: 29 March 2017 Revised date: 17 April 2017 Accepted date: 24 April 2017 Cite this article as: Yingchun Miao, Xiaolin Xu, Kaiquan Liu and Ninghong Wang, Preparation of novel Cu/TiO 2 mischcrystal composites and antibacterial activities for Escherichia coli under visible light, Ceramics International, http://dx.doi.org/10.1016/j.ceramint.2017.04.137 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation of novel Cu/TiO2 mischcrystal composites and antibacterial activities for Escherichia coli under visible light Yingchun Miao*, Xiaolin Xu, Kaiquan Liu, NinghongWang Faculty of Chemical and environment Sciences, Qujing Normal University, Qujing 655000, China Abstract: In this paper, novel copper/titanium dioxide (Cu/TiO2) composites were facilely prepared by simple sol-gel hydrothermal method. The crystalline phases, surface morphology and antibacterial activity of Cu/TiO2 were investigated systematically. Interestingly, the as-prepared Cu/TiO2 materials were made up with three different crystalline phases, including cuprite Cu2Cl(OH)3, Cu2+1O, and anatase TiO2. Next, the ‘zone of inhibition’ experiment was performed against E. coli using Cu/TiO2 catalysts with different Cu doping concentration as the antibacterial agents. Our results show that the Cu/TiO2-3.00 catalyst possesses optimal antibacterial ability against E. coli, which may be connected with the strong oxidizing reactive oxygen species (ROS) destroying the bacteria and photogenerated charge separation and recombination. Key words: Titanium dioxide; Copper; Composites; Photocatalysis; Antibacterial activities. 1. Introduction Since favorable mechanical performance, excellent chemical stability, corrosion-resistance and good biocompatibility, Titanium dioxide and its composites have attracted wide scientific interest for photo-degradation of pollutants and antibacterial applications, such as degradation of organic contaminants and disinfection of microorganism[1-5]. After Matsunaga et al. first reported photocatalytic antibacterial performance of platinum-doped TiO2 in 1985[6], TiO2-based nanocomposites have been extensively investigated for various microorganism disinfections,
*[email protected]（Corresponding author: Yingchun Miao）
including viruses, bacteria, fungi, algae and protozoa[2, 7-11]. For example, Ag-TiO2 nanoparticles incorporated SiO2 films can inhibited the gram negative bacteria E. coli. TiO2/Ag2O heterostructure could simultaneously remove the organic pollutant and kill the bacterium under visible light irradiation[7]. Additionally, Kim et al reported the chitosan/AgCl–TiO2 colloid produced excellent antibacterial activity for S. aureus and E. coli[12]. Generally, photocatalytic antibacterial performance of these TiO2-based composites depends on the interaction between microorganisms and reactive oxygen species (ROSs) generated from the electron-hole pairs under light irradiation[13-15]. These metal nanoparticles doping can remedy the shortcoming of TiO2, owing to its wide band gap and fast recombination of photo-induced electron-hole pairs. Recently, numerous studies reported metal nanoparticles could also be used as antibacterial agents to kill microorganism, mainly including Au, Ag, Zn, Pd and Cu[16-21]. For instance, ZnO nanoparticles were found to exert bactericidal effect against S. aureus, Campylobacter jejuniand E. coli[20]. Cu nanoparticles could be employed as an efflux pump inhibitor to tackle drug resistant bacteria[22]. Remarkably, among all the metal species, Cu as an essential element in living organisms has aroused wide investigation because of its excellent antibacterial properties, low toxicity and low-cost[13, 15, 18, 23]. Interestingly, Cu-containing TiO2 composites prepared by different technologies showed better antibacterial activities than pure Cu nanoparticles or TiO2. For example, Cu-Ti-O NTAs possess excellent long-term antibacterial ability against Staphylococcus aureus (S. aureus)[17]. Although Cu is an excellent candidate for replacing noble metal materials in the antibacterial applications, the preparation and practical applications of Cu decorated TiO2 heterostructures with high antibacterial effect still suffer from huge challenges, such as complicated producing technology and low photoactivity. Few reports are available for
antibacterial application of the hybrid CuxO/TiO2 nanocomposites[24-26]. Hence, more researches about these Cu decorated TiO2 heterostructures should be conducted. Generally, the technologies prepared the Cu doping TiO2 nanocomposites (Cu/TiO2) mainly include solvothermal reaction, coprecipitation, and impregnation method[27-29]. In this work, the Cu/TiO2 composites were synthesized by a novel sol-gel hydrothermal method at low temperature. Amazedly, cuprite Cu2Cl(OH)3, and Cu2+1O crystals can be successfully decorated on the anatase TiO2. Various characterization techniques were employed to analyze the unique Cu/TiO2 mischcrystal nanomaterials. Additionally, the antibacterial activities of Cu/TiO2 nanomaterials were systematically studied and discussed. 2. Experimental 2.1 Materials and characterization All reagents were purchased from commercial companies and used without further purification, including titanium tetrachloride (TiCl4, AR), copper foil (purity: 99.5%), agar (AR), tryptone (LP), yeast extract (AR), NaOH(AR) and NaCl (AR). Powder X-ray diffraction (XRD) measurements were carried out on a Rigaku D/max 2000 single-crystal diffractometer using Cu Kα radiation (λ=0.15405nm).Energy dispersive spectroscopy (EDS) analysis was performed with HITACHI S-4800 SEM equipped with Oxford X-max spectrometer. Fourier transform infrared (FT-IR) spectroscopy was performed on NEXUS 470.The morphologies of the samples were analyzed by a HITACHI S-4800 scanning electron microscopy (SEM) and a JEM-2010 transmission electron microscope (TEM). The surface compositions of samples were measured by X-ray photoelectron spectroscopy (XPS, Versa Probe PHI 5000). The absorption spectra of the samples were recorded using a Varian Cary 500 UV-visible spectrophotometer. The magnetic properties of the materials
were studied by using avibrating sample magnetometer (LDJ 9600-1, USA). 2.2 Synthesis of various Cu/TiO2nanocomposites Typically, 10 mL titanium tetrachloride (TiCl4) was placed into a 25 mL beaker, and liquid nitrogen was added under stirring to obtain TiCl4 colloid. The precursor gel was kept static for 5 h at room temperature. Next, the colloid and copper foil were sealed within a 100 mL Teflon-lined autoclave, and heated at 160 °C for 24 h. After completion of the reaction, the products were obtained by filtration, and washed three times with water and ethanol respectively. Finally, the solid materials were dried under vacuum at 60 °C for 12 h. Other materials with different copper doping concentration were synthesized by same method, and labeled as Cu/TiO2-x (x represents the copper doping concentration in these materials). 2.3 Culture and treatment of microorganisms: Liquid medium: 2.5 g tryptone, 1.25 g yeast extract and 2.5 g sodium chloride were added into 25 ml Erlenmeyer flask and stirred for 10 min. Next, the pH value of this system was adjusted to 7.0 by 1mol/L sodium hydroxide solution. Finally, the mixture was disinfected in the high-handed sterilization pan to obtain a sterile, liquid medium. Solid medium: 5.875 g agar powder was added into 250 ml Erlenmeyer flask, and dissolved in 250 mL water by heating to 98 °C. Next, the solution was sealed by rubber plug and cooled to room temperature. Finally, the system was disinfected the high-handed sterilization pan to obtain a sterile, solid medium. Culture of microorganisms: The gram-negative Escherichia coli(ATCC8099) were used in this work. The zone of inhibition test was employed to evaluate the antibacterial activity of Cu/TiO2 materials. First, the gram-negative E. coliwere cultivated in the Luria Bertani (LB) media. Then,
the nutrient agar plates were in oculated with 1 mL of bacterial suspension containing around104 colony forming units (CFU) by using spread plate method. Next, the Cu/TiO2 photocatalysts with different Cu doping concentration were gently placed on the inoculated plates, and then the seplates were exposed on the visible light at 30oC for 40min. After that, these dishes were cultivated in the artificial bioclimatic test chamberfor 24 h. Finally, these tests were performed in triplicate. Before adsorption, pure P25 were used as control. 3. Results and analysis 3.1 Characterization of samples First, the crystalline phases of as-prepared Cu/TiO2-x materials were characterized by XRD technology. As seen from Figure 1, when the Cu doping concentration in the materials was below 2.0%, diffraction peak of the anatase TiO2 can only be observed at 2θ=27.5, 36.2, 41.3, 54.5, and 56.6° (JCPDS file Card No. 21-1272). No peaks associated with Cu species were detected in the XRD patterns, which was likely due to the low crystallinity and the high dispersion of Cu on TiO2 surfaces. Interestingly, with the increase in Cu doping concentration, two kind of crystalline phases Cu species appeared on the XRD pattern, which matched with the Cu2+1O (2θ=16.4, 28.9, and 61.5°; JCPDS file Card No. 05-0667) and cuprite Cu2Cl(OH)3 (2θ=16.3, 19.2, 32.5, and 47.6°; JCPDS file Card No. 50-1559). Results indicated that the three crystal forms (TiO2, Cu2+1O and Cu2Cl(OH)3) in the materials were generated by the simple hydrothermal treatment, which may enhance the antibacterial activity of these materials. To further identify the elemental composition, the EDX analysis was performed. As shown in figure 2a, Cu/TiO2-3.00 catalysts contained Ti, O, Cu, and Cl. No other elements were found in this sample. Additionally, the FT-IR was employed to investigate the chemical bond of samples in
Figure 2b. It can be clearly seen that -OH bending vibration and stretching vibration absorption peaksappeared around 1600 cm-1 and 3500 cm-1 respectively[30], indicating the existence of adsorbed water in these materials. The peaks at about 3356 cm-1and 3317 cm-1 can be attributed to the fundamental stretching vibration of surface hydroxyl groups in Cu/TiO2 samples [31], which are common characteristics of oxide semiconductors and essential conditions for photocatalysis. These groups can enhance the photocatalytic performance through the generation of hydroxyl radicals as a result of their trapping by the photogenerated holes [31]. At the same time, we found that the absorption peaks at 3356 cm-1, 3317 cm-1, 2900 cm-1, and 2800 cm-1 became stronger with the increase of Cu doping amount. These strong absorption peaks would generate new doping energy levels in the energy gaps of TiO2 and improve the photocatalysis antibacterial performance. However, it is difficult to distinguish some absorption peaks at 400 cm-1 to 800 cm-1 wavenumber due to mischcrystal structure of samples. Next, the morphology of the Cu/TiO2-3.00 catalyst as typical sample was studied by SEM and TEM. It is clearly seen that most of the Cu nanoparticles were well immobilized on the TiO2 nanorods surface, but some fragmentary Cu nanoparticles can also be observed in Figure 3a and b. The crystalline structure of the Cu/TiO2-3.00 catalyst was detected by HRTEM in Figure 3c and d. Notably, the TiO2 (101) (110), Cu2Cl(OH)3 (013), and Cu2+1O (110) crystalline lattices can be clearly observed, corresponding to the XRD analysis in Figure 1. Interestingly, TiO2, Cu2Cl(OH)3 and Cu2+1O crystal planes were linked by the way of head or terminal conjunction, which may be conducive to the electron-transfer between Cu species and TiO2. To further study the micro component of Cu/TiO2-3.00 andobserve the valence of different elements, the XPS spectra of Cu/TiO2-3.00 catalyst was shown in Figure 4. The four major peaks
at about 198.4, 458.4, 531.6, and 934.6 eVshould belong to Cl 2p, Ti 2p, O 1s and Cu 2p in Figure 4a. In Ti 2p spectrum (Figure 4b), the bindingenergies of 458.4 and 464.1 eV were indexed to Ti 2p3/2 and Ti 2p1/2, respectively[7]. For Cu 2p spectrum (Figure 4c), to the best of our knowledge, Cu0 and Cu2+ have more than 2 eVseparations. Therefore, the peaks at 934.6 eV and 954.5 eV could be assigned to Cu2+[32]. The O 1s XPSspectrum of Cu/TiO2-3.00 was separated into two spectral components in Figure 4d, indicating Cu−O (531.6 eV) and Ti−O (529.5 eV)[33]. Additionally, the Cl 2p peak at 198.4 can also be clearly seen in Figure 4e. All the above analyses further verified the combination of Cu2Cl(OH)3 and Cu2+1O with TiO2. Finally, the optical properties of the Cu/TiO2-3.00 photocatalysts were studied by UV–vis spectrometry. Optical absorption analysis revealed that Cu/TiO2-3.00 exhibited absorption edges at 517 nm in Figure 4f, which indicated the good visible response of Cu species onthe surface of TiO2.We inferred that the enhanced response to visible light can be attributed to the embedded metallic Cu species, such as Cu2Cl(OH)3 and Cu2+1O, which promoted the formation of oxygen vacancies in TiO2 through the metal-oxide interaction [34], thus increasing the visible light absorption of Cu/TiO2. 3.2 Antibacterial assay 3.1The zone of inhibition test The ‘zone of inhibition’ experiment was performed against E. coli using Cu/TiO2 catalysts with different Cu doping concentration as the antibacterial agents. First, the dosage of Cu/TiO2-3.00 chose as a typical antibacterial agent was evaluated in Figure 5. Obviously, with the increase of dosage, stronger antibacterial effects on the E. colican be obtained by visible light irradiation for 40 min. Hence, we chose 0.15 mg dosage of catalysts to conduct the subsequent experiments.
Next, antibacterial activities of different Cu/TiO2 catalysts were investigated. As shown in Figure 6, the Cu/TiO2-3.00 photocatalyst exhibited inhibition zones of 25 mm diameter by visible light irradiation for 30 min, which was superior to others. To further compared with antibacterial activities of various Cu/TiO2 catalysts, the bactericidal effects on E. coli at different irradiation times, and changes in inhibition zones were shown in Figure 7. Firstly, the commercial P25 catalyst was chose as the control group. Obviously, all the Cu/TiO2 catalysts exhibited partly bactericidal effects on E. coli, while control plates with P25 showed almost noinhibition zone. Hence, we can conclude that antimicrobial activity of TiO2-based materials can be well improved by the immobilizing Cu species. Meanwhile, with the increase of Cu doping concentration, the antibacterial performance became stronger and the peak appeared at Cu doping concentration of about 3.0% for 10 min irradiation. These results indicated that the Cu/TiO2-3.00 catalyst possessed optimal antibacterial activity. Generally, the antibacterial mechanisms were put forward based on comprehensive consideration for both the photosensitivity of TiO2-based materials and the antibacterial activity of metal-ions[13, 35]. Firstly, the photocatalytic antibacterial mechanism may be involved with the strong oxidizing reactive oxygen species (ROS). For example, hydroxyl radical (·OH), superoxide anion radical (·O-2 ) and hydrogen peroxide (H2O2) are generated in the photocatalytic reaction of TiO2-based catalysts. The chemical formulas can be expressed as follows: + Cu⁄TiO2 + ℎ𝜐 → e− CB (TiO2 ) + hVB (Cu)
(1)
− e− CB + O2 →∙ O2
(2)
− h+ VB + OH →∙ OH
(3)
+ h+ VB + H2 O →∙ OH + H
(4)
∙ + ∙ O− 2 + H → HO2
(5)
∙ − ∙ O− 2 + H2 O → HO2 + OH
(6)
HO∙2 + H2 O → H2 O2 +∙ OH
(7)
H2 O2 → 2 ∙ OH
(8)
According to the above analysis, the TiO2 samples doped with Cu species can improve the response to the visible light, and elevate the photosensitivity of TiO2. Therefore, Cu ions in the Cu/TiO2 catalysts may stabilize the photo-produced electron–hole pairs, which can remarkably prolong the time of ROS destroying the bacteria and improve the antibacterial activities of the Cu/TiO2 nanocomposites. Moreover, some Cu ionsreleased from the Cu/TiO2 nanomaterials can be adsorbed to the surface of microorganism, and plenty of negative charges will be carried by electrostatic force,which can destroy the charge balance of microorganism, and lead to cells deformed seriously until bacteria die by bacteriolysis. Meanwhile, Cu ions can get into the bacteria via cell membrane and interact with some functional groups on the protease, such as sulfhydryl group (ASH), amino group (ANH2), hydroxylgroup (AOH), it will result in the change of the structure and function of protein. In order to identify the above hypothesis, the transient photocurrent response of Cu/TiO2-3.00 photocatalysts was conducted. As shown in Figure 8, among all the as-prepared materials, higher and more stable photocurrent was clearly observed on the Cu/TiO2-3.00 catalyst. This result indicated that photogenerated charge separation could be effectively promoted. At the same time, 3.0% Cu loading was more favourable for charge recombination than others. According to above the antibacterial mechanisms, we concluded that the Cu/TiO2-3.00 catalyst
might maximally prolong the time of ROS destroying the bacteria and the abundance of Cu ions were fully released from the nanocomposite. Hence, the Cu/TiO2-3.00 catalyst showed the excellent antibacterial performance. 4. Conclusion In summary, novel copper/titanium dioxide (Cu/TiO2) composites were facilely prepared by simple sol-gel hydrothermal method. The crystalline phases and surface morphology of Cu/TiO2 were characterized systematically by XRD, FT-IR, XPS, SEM and TEM. Interestingly, the as-prepared Cu/TiO2 materials were made up with three different crystalline phases, containing cuprite Cu2Cl(OH)3, Cu2+1O, and anatase TiO2. Next, the antibacterial experiment was performed against E. coli using Cu/TiO2 catalysts with different Cu doping concentration. Our results showed that the antibacterial activity of Cu/TiO2 catalysts became stronger with the increase of the Cu doping concentration. The Cu/TiO2-3.00 catalyst possessed optimal antibacterial ability against E. coli, which may be connected with the strong oxidizing reactive oxygen species (ROS) destroying the bacteria and photogenerated charge separation and recombination. More investigation of metal/TiO2 nanocomposites will be carried out in the future. Acknowledgments Yunnan Applied Basic Research Project of Province(C0120150543), Key Projects of Yunnan Provincial Department of Education(2015Z183，2016ZZX207), Innovation and Entrepreneurship Project of College Students in Yunnan Province and Open Project of Environmental Chemistry Key Laboratory of Ministry of Education and Shanghai Normal University. Reference [1] A. Mukhopadhyay, S. Basak, J.K. Das, S.K. Medda, K. Chattopadhyay, G. De, Ag−TiO 2
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(2014) 13502-13509. [33] R.A. Rather, S. Singh, B. Pal, A Cu+1/Cu0-TiO2 mesoporous nanocomposite exhibits improved H2 production from H2O under direct solar irradiation, Journal of Catalysis 346 (2017) 1-9. [34] J. Zhao, Y. Li, Y. Zhu, Y. Wang, C. Wang, Enhanced CO 2 photoreduction activity of black TiO2−coated Cu nanoparticles under visible light irradiation: Role of metallic Cu, Applied Catalysis A: General 510 (2016) 34-41. [35] W. Fan, T. Liu, X. Li, R. Peng, Y. Zhang, Nano-TiO2 affects Cu speciation, extracellular enzyme activity, and bacterial communities in sediments, Environmental pollution 218 (2016) 77-85. Figure 1. XRD patterns of the Cu/TiO2 material with different Cu doping amount. Figure 2.EDX analysis (a) of Cu/TiO2-3.00 and FT-IR (b) of all the samples. Figure 3.SEM (a) and TEM (b, c, and d) images of the Cu/TiO2-3.00 catalyst. Figure 4.XPS analysis (a-e) and UV-Vis DRS spectra (f) of the Cu/TiO2-3.00 catalyst. Figure 5.The antibacterial activity of Cu/TiO2-3.00 with different dosage. Figure 6.Zone of inhibition observed around various Cu/TiO2 in nutrient agarplates inoculated with bacterial suspension of E. coli.
Figure 7. The comparison of the antibacterialzones of various Cu/TiO2 catalysts Figure 8.Transient photocurrent response of photocatalysts for two20 s light-on-off cycles in 1M Na2SO4 aqueous solution under solar light irradiation with a bias potential of 1.0 V.