Annealing-Induced Antibacterial Activity in TiO2 Under

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Annealing-Induced Antibacterial Activity in TiO Under Ambient Light Mu Yao Guo, Fangzhou Liu, Yu Hang Leung, Yanling He, Alan Man Ching Ng, Aleksandra B. Djurisic, Hangkong Li, Kaimin Shih, and Wai Kin Chan J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b07325 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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The Journal of Physical Chemistry

Annealing-induced Antibacterial Activity in TiO2 under Ambient Light Mu Yao Guo, †‡ Fangzhou Liu, ‡ Yu Hang Leung, ‡ Yanling He, † Alan Man Ching Ng, †,‡* Aleksandra B. Djurišić, ‡* Hangkong Li,# Kaimin Shih, # Wai Kin Chan• †

Department of Physics, Southern University of Science and Technology, Shenzhen, China ‡

#

Department of Physics, The University of Hong Kong, Pokfulam Road, Hong Kong

Dept. of Civil Engineering, the University of Hong Kong, Pokfulam Road, Hong Kong

•

Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong

ABSTRACT : We demonstrate that annealing at 850ºC in the presence of Cu universally results in robust antibacterial activity under ambient illumination for TiO2 nanoparticles, different from those annealed in quartz crucible without metal or in the presence of Ti. Resulting robust antibacterial activity occurred after annealing regardless of the initial properties and crystal structure of the starting samples (two anatase, one rutile, and P25). A clear difference in the powder color from white to gray and a pure rutile crystal structure is observed after annealing in all the samples. ESR measurements, however, reveal obvious differences in the defects present in the samples annealed under different conditions. Strong antibacterial activity is observed under ambient illumination for samples annealed in the presence of Cu, despite lower activity for photocatalytic degradation of common dyes such as methylene blue after annealing.

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Antibacterial activity could not be attributed neither to the presence of Cu (no activity in the dark), nor to the ROS production (none detected under ambient illumination). This indicates that other mechanisms, such as direct charge transfer involving defect levels induced by annealing in the presence of copper, may play a role in the observed antibacterial activity.


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Introduction There has been considerable interest in synthesizing visible-light active TiO2 for various photocatalytic applications, including antibacterial activity and water treatment.1-30 The strategies include doping with metal and non-metal impurities, preparation of non-stoichiometric TiO2, and various composites and heterostructures.1 Doping with different elements, for example carbon,25 nitrogen,29 copper,27 etc., can contribute to decreased bandgap and/or enhanced light absorption in the visible spectral range. In doped TiO2, changes in the properties can arise from the introduction of the dopant or from the changes in material composition, i.e. Ti:O ratio. In fact, visible-light absorption and/or visible-light induced photocatalytic activity can be achieved without introduction of dopants by changing the stoichiometry and inducing native defects. In non-stoichiometric TiO2, both oxygen deficiency (oxygen vacancies, Ti3+) and excess oxygen can contribute to the visible-light induced photocatalytic activity.1 Considerable efforts have been made to synthesize visible-light active titania photocatalysts, including so-called black titania with significantly enhanced absorption.3 Various methods have been reported for preparing black titania, such as hydrogenation,3,9 argon annealing,3 metal reduction,3,9 the use of organic reductants,3,9 plasma treatment,3 proton implantation,3 electrochemical reduction,3,9 incomplete oxidation of precursors,3

ultrasonic

treatment,3 and laser irradiation.3 The visible light absorption and/or photocatalytic activity of black titania is typically attributed to surface disordered structure, non-stoichiometry, and/or hydrogenation.3 Among different synthesis methods, metal reduction is of significant interest since it is a very simple, low cost and safe method.3 Similar to black titania produced by other methods except hydrogenation, the photocatalytic activity of metal-reduced TiO2 was attributed to non-stoichiometry, i.e. Ti3+ and/or oxygen vacancies4,5,10 and the presence of disordered


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surface.5 Metals reported to be capable of reducing TiO2 are aluminum,3,5,7,9,10 magnesium,3 and zinc.3,4,9 In terms of applications, the majority of applications of reduced or black titania have been focused on photocatalytic hydrogen generation,3,5,8 CO2 reduction,3 and/or pollutant degradation.3-7,10,11 While antibacterial action under visible illumination is of considerable interest for water treatment2 and the antibacterial activity of titania has been extensively studied for different titania nanostructured morphologies and crystal structures,2,14-30 studies on antibacterial activity of “black” non-stoichiometric titania under visible illumination have been scarce. The antibacterial activity of TiO2 under illumination is commonly attributed to the photocatalytic antibacterial action, mainly due to the generation of different reactive oxygen species (ROS) such as hydroxyl groups, superoxide ions, singlet oxygen, protonated superoxide radical, and hydrogen peroxide.2 In addition to ROS generation, shape-related deformational stress was also mentioned as possible contributor for elongated nanostructures.12 In many cases, antibacterial activity tests involved UV illumination,12-14

although there have also been reports on

antibacterial activity under fluorescent, ambient, or simulated solar illumination.15-25 Typically, no toxicity in the dark is observed.16,22 The antibacterial activity under illumination has been attributed to photocatalytic decomposition of cell wall by ROS produced,15,16,18-22,24-29 as well as the physical damage to the cell wall due to close contact between the bacteria and small nanoparticles.15 It was proposed that the antibacterial activity of titania under illumination depends not only on the ability of ROS production, but also on morphology and aggregation, as well as bacteria-TiO2 contact, due to the fact that only ROS produced near bacteria surface could cause phototoxicity.16 Furthermore, bacteria inactivation was found to be associated with redox conversion Ti4+/Ti3+,24 and thus it would likely depend on the sample properties in terms of


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native defects, which can also affect the ROS generation. Consequently, studying the antibacterial properties of non-stoichiometric titania is of significant interest. Here we demonstrate preparation of visible light active titania with robust antibacterial activity by annealing the samples in the presence of Cu metal as a reduction agent in argon atmosphere at 850ºC. The high annealing temperature results in pure rutile crystal structure, which is commonly considered less photocatalytically active compared to pure anatase or anatase/rutile mixed structures.18,19,30 Nevertheless, high antibacterial activity was reported previously for rutile titania, for example for TiO2 microspheres with exposed reactive {111} facets under UV illumination.14 In addition, it was proposed that the ROS production under visible illumination was dependent on the crystal structure, and that only for rutile TiO2 the production of singlet oxygen was observed.20 In our work, all the samples annealed in the presence of Cu exhibited visible light absorption and a robust antibacterial activity under ambient illumination despite large particle size and rutile crystal structure and the lack of detectable ROS produced under ambient illumination. Other rutile samples (annealed without Cu) did not exhibit significant antibacterial activity under ambient illumination. The samples were comprehensively characterized and the reasons for observed antibacterial activity are discussed. Experimental Section: Sample preparation: TiO2 anatase nanoparticles (99%, APS 15 nm, 1A-TiO2), TiO2 rutile (99.8%, APS 20-50 nm, R-TiO2), SnO2 (99.5%, APS 55 nm) and Al2O3 (99%, APS 30–40 nm) nanoparticles were obtained from Nanostructured & Amorphous Materials Inc. TiO2 anatase nanoparticles (95%, APS 7 nm, 2A-TiO2) were obtained from MK Impex Corp., Division MK


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Nano. TiO2 P25 was purchased from Evonik Industries. Metal oxide nanoparticles were annealed in a quartz crucible in a tube furnace in vacuum at 850 °C for 1 hour at Ar atmosphere without wrapping or wrapped with Ti or Cu foil. Sample characterization: The X-ray diffraction (XRD) data were collected using a D8 Advanced Diffractometer (Bruker AXS). Energy dispersive X-ray (EDX) mapping was done using a FEI Tecnai G2 F30 transmission electron microscope (TEM). X-ray photoelectron spectroscopy (XPS) spectra were obtained using Physical Electronics 5600 multitechnique system and Thermo Fisher Escalab 250Xi system. For FTIR measurements, the nanoparticles were mixed with KBr powder (infrared grade, Sigma-Aldrich) and pellets of the mixture were made. The measurement was performed on the pellets using PerkinElmer Spectrum Two IR spectrometer. Zeta potential and aggregation size of each sample were measured using Brookhaven BI-200SM research goniometer and laser light scattering system with the concentration of 0.1 mg/ml in de-ionized water. For absorption measurements, the nanoparticles were drop cast on quartz substrates at the concentration of 10 mg/ml. The absorption spectra were obtained using PerkinElmer Lambda 750s spectrometer with 60 mm integrating sphere. ESR measurements of the nanoparticle samples were carried out using Bruker EMXPlus electron spin resonance (ESR) spectroscopy at room temperature with solar illumination and at 5K without illumination. In addition to sample properties characterization, their ability to generate ROS was examined using ESR. The ROS generated by the nanoparticles were detected using Bruker EMXPlus ESR at room temperature with the addition of a spin trap molecule. Spin trap 5,5Dimethyl-1-pyrroline N-oxide (DMPO) was purchased from Sigma–Aldrich Co., and the solution for the measurement was prepared by placing 150 mL of 0.02M DMPO onto the


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substrates and illuminated illuminating with solar light or ambient light for 2 min. Then, the solution was transferred into an EPR tube and the measurement was performed immediately. Antibacterial activity experiments: A gram-negative bacterium Escherichia coli XL1-Blue (Stratagene) was used for the experiment. Luria-Bertani (LB) broth (Affymetrix/USB) was used as the culture medium, and the incubation temperature was 37 °C. 0.9% w/v sodium chloride (NaCl) solution was used as the dispersion medium for the bacteria. Nanoparticle coated substrates were prepared by drop-casting 1 mg/ml nanoparticle suspension onto the 2 cm × 2 cm glass substrate. The control samples were bare glass substrates. The substrates were sterilized with UV-C illumination before the antibacterial activity test. 0.5 mL of bacteria suspension (106 CFU/mL) were placed onto the nanoparticle coated substrates. The substrates were then exposed to ambient laboratory fluorescent light (0.7 mW/cm2 or kept in the dark) for 4 hours. For the testing of antibacterial activity under UV illumination, the samples were exposed to UV light (365 nm, 69.3 mW/cm2, Spectroline BIB-150P lamp) for 30 min. Serial dilution was then performed and 25 µL of dilution was pipetted onto a culture agar in triplicate. The plates were kept at 37 °C for 16 hours and the formation of colonies was observed. The bacteria cells with and without exposure to titania nanoparticles were examined by SEM. For SEM characterization, the cells were collected and fixed using 2.5% glutaraldehyde in cacodylate buffer (0.1M sodium cacodylate-HCl buffer pH 7.4) at 4 °C overnight. Then the fixation buffer was changed to cacodylate buffer with 0.1 M sucrose to stop fixation. The samples were rinsed with cacodylate buffer and serially dehydrated with ethanol. The samples were dried by critical point drying method. SEM was performed using a LEO 1530 FEG SEM. For ATR-FTIR measurements, after exposure to the samples, the cells were collected by centrifuge at 10000 rpm and dried by critical point drying method. The ATR-FTIR measurements were performed using a Bruker Vertex 70


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FTIR spectrometer. For the investigation of Cu ion release, nanoparticle coated substrates were prepared by drop-casting 15ml 1 mg/ml nanoparticle suspension onto the glass substrate. Then 15 ml of deionized water was placed onto the substrate for 4 hours (same duration as antibacterial tests), collected and diluted to 150 ml to reach the minimum quantity for Cu ion release test, and filtered through a 0.45 µm filter to remove any nanoparticles The suspension were filtered using 0.45 µm filter. The Cu ion release measurements were performed by ALS Technichem (HK) Pty Ltd using ICP-MS. Photocatalytic activity for dye degradation: Methylene blue (Sigma Aldrich) was dissolved in deionized water at 5 mg/L. 50 mg of the nanoparticles was mixed with 50 ml of dye solution in a Petri dish and stirred for 30 min in the dark to reach equilibrium. The mixture was then exposed to simulated solar AM 1.5 illumination (41.6 mW/cm2, Oriel solar simulator) or UV illumination (365 nm, 69.3 mW/cm2, Spectroline BIB-150P lamp). 3 mL of the mixture was withdrawn and filtered for absorption measurement immediately before exposure to illumination and at specified time intervals under illumination. Results and discussion XRD spectra of different samples before and after annealing are shown in Figure 1. Regardless of the starting crystal structure of the samples before annealing, all the samples exhibited rutile crystal structure after annealing. The sample properties (Cu to metal atomic ratio, aggregation size, zeta potential) and antibacterial activity were also investigated, and they are summarized in Table 1. Annealing at 850ºC in the presence of Cu results in a significant enhancement of antibacterial activity for all the titania samples. No obvious correlations are observed between the zeta potential values, aggregation sizes, and Cu content and antibacterial


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activity. The lack of correlation between the zeta potentials, aggregation sizes and antibacterial activity is not surprising since the tests were performed on nanoparticle coated surfaces rather than in suspension to avoid possible effect of nanoparticle sedimentation, reduced light penetration due to variation in turbidity of samples with different aggregation sizes, and nonlinear dependence of the toxicity on nanoparticle concentration.31 While for an individual pair of samples (not annealed, and annealed in the presence of Cu), annealing results in the Cu content increase and antibacterial activity increase, there is no correlation between the Cu content and antibacterial activity of the samples annealed in the presence of Cu. Furthermore, Cu content of the sample 2A-TiO2 Cu850 is similar to that of R-TiO2 without annealing which shows very low antibacterial activity. Bacterial cells exposed to 2A-TiO2 Cu850 exhibit cell membrane damage (see Supporting Information, Fig. S1), commonly observed in bacteria exposed to nanomaterial samples.31,32 However, we did not observe significant changes in the ATR-FTIR spectra of the bacteria exposed to the samples (see Supporting Information, Fig. S2), different from our previous work on non-ROS mediated toxicity of different metal oxides.31,32 There have been small changes in the relative intensities of different peaks corresponding to amide (amide I 1645 cm-1, amide II 1550 cm-1)33 bands and sugar vibrations33 at 1070 cm-1 (see Supporting Information, Table S1), but the difference between the control samples and samples exposed to 2A-TiO2 Cu 850 is negligible. While annealing in the presence of Cu resulted in significant antibacterial activity for all the titania samples, it failed to induce significant antibacterial activity in SnO2 and Al2O3 samples, although a small reduction in the survival rate of bacteria colonies is seen for the annealed samples. For the sample with the highest Cu content, SnO2 Cu 850, bacteria survival


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rate of 93% is obtained. This indicates that simple Cu incorporation is not sufficient to induce antibacterial activity, but the mechanism of the activity is not fully clear.

Figure 1. XRD pattern of different TiO2 nanoparticles before and after annealing in at 850ºC in the presence of Cu: (a) 2A-TiO2, (b) P25, (c) R-TiO2.


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Cu ions could be released from the samples, and this possibility was examined and the results are shown in Supporting Information, Table S1. While Cu ion release occurs both in titania and alumina samples annealed in the presence of Cu, only titania samples exhibit robust antibacterial activity. While some effect of Cu ion release could not be entirely excluded, it is obviously not sufficient to explain the observed results. Thus, to study the reasons for the observed enhancement of the antibacterial activity in titania samples after annealing we selected to perform a comprehensive characterization of the sample 2A-TiO2, and compare the antibacterial activity of samples annealed without metal and those annealed in the presence of Cu and the presence of Ti. We have selected 2A-TiO2 since these samples, in addition to P25, exhibited the lowest survival rate of bacterial colonies, but unlike P25 samples starting particles contained no Cu impurities and exhibited negative zeta potential (P25 without annealing was the only sample for which positive zeta potential was measured). Obtained absorption spectra, photos, and FTIR spectra of the samples are shown in Fig. 2. It can be observed that annealing at 850ºC increases the absorption of all the samples, and the sample colour changes from white to gray. The changes in the absorption spectra likely arise from the change in the stoichiometry and the corresponding gap states. The gap states extend the optical response into the visible spectral range,25,26 and they can also alter the lifetime of charge carriers and recombination pathways and kinetics.26 In the case of annealing in the presence of Cu, impurity contribution could also result in increase absorption in the visible range, as demonstrated previously for samples annealed in stainless steel crucibles.3,6 However, since all the samples exhibit color change and clear visible absorption, main contributors to the absorption in the visible spectral range are likely defect levels induced by non-stoichiometry. Another common feature of the samples after annealing is the change in the shape of FTIR spectra, as


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shown in Fig. 2b. Various peaks corresponding to different C-H and/or C-O vibrations in the spectral range 1000-1500 cm-1 disappear after annealing, and the peaks corresponding to vibration modes of OH groups at ~3000-3500 cm-1 and water at ~1623 cm-1 are significantly reduced after annealing.25 This indicates the removal of surface adsorbates by high temperature annealing. In addition, XPS spectra of the samples were measured, and they are shown in Fig. 3. We can observe a shift in the Ti 2p peak after annealing, and the change of the shape of O1s peak. The shift in Ti 2p peaks to lower energies, without any clearly resolved Ti3+ peak, is commonly observed.10,18,34 Since the binding energy of Ti3+ is lower compared to Ti4+,18 the peak shift to lower energies is consistent with the increased concentration of Ti3+ and/or increased oxygen defficiency. This is consistent with the change of colour of the samples from white to gray. We can also observe a small shift and a change in the shape of the O1s peak. The peak shifts can indicate a change in the chemical environments of surface elements.34 O1s peak consist of two peaks, labeled O1 and Os, with the peak at lower binding energy corresponding to the oxygen in Ti-O-Ti, while the higher binding energy peak corresponds to oxygen in Ti-O-H.34 An observed increase in O1/Os ratio for samples annealed in the presence of Cu and Ti is consistent with the significant reduction in the presence of surface hydroxyl groups observed in the FTIR spectra. The sample annealed without any metal, however, does not show an increase in O1/Os ratio and it shows more pronounced shift of the Ti 2p peaks to lower energies. This indicates that these samples are likely more oxygen deficient. It should also be noted that the O1s peaks in all samples are quite broad, which reduces the reliability of the fitting and the corresponding analysis. Nevertheless, the observed changes still convey useful information on surface properties and/or oxygen deficiency of the samples.


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Figure 2. a) Absorption spectra of the 2A-TiO2 samples annealed under different conditions. Photo of the samples is shown in the inset. b) FTIR spectra of the 2A-TiO2 samples annealed under different conditions. XPS was also used to examine the presence of Cu in 2A-TiO2 Cu 850 samples, and the obtained results are shown in Figure 3c. To examine whether copper was uniformly incorporated, EDX mapping was performed and the results are shown in Supporting Information, Figure S3. It can be observed that Cu distribution is uniform, with no evidence of clusters or particles. This is in agreement with the fact that only rutile TiO2 is observed in the XRD. From the XPS spectra, we can observe the presence of Cu2+ based on the position of Cu (2p3/2) peak at ~934.2 eV and the shake-up satellite peak of the Cu (2p) core level at ~942.4 eV.35,37 Cu+


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chemical state was previously assigned to a peak at ~ 932.8 eV.36,37 Thus, we can observe that our samples contain both Cu+ as well as Cu2+.

Figure 3. XPS peaks of different samples corresponding to a) Ti 2p b) O 1s levels c) Cu 2p levels.


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Since all the samples exhibit a change in the stoichiometry and the increase in visible light absorption, but only the samples annealed in the presence of Cu exhibited significant antibacterial activity, it is necessary to consider whether antibacterial activity occurs due to Cu impurity incorporation. It was previously reported that the doping of anatase TiO2 with Cu2+ resulted in increased absorption in the visible spectral range and antibacterial activity under visible light illumination.27 The antibacterial activity in the dark was negligible, and antibacterial activity under visible illumination was attributed to the increased ROS formation due to reduced recombination of photogenerated charge carriers by Cu doping.27 This was different from the previous report on F,Cu doped titania, where antibacterial activity was observed both in the dark and under illumination, and thus it was partly attributed to Cu toxicity.23 In addition, while enhancement of antibacterial activity was previously reported for different Cu- and CuOcontaining samples was reported to increase with illumination,35-37 the samples also showed significant antibacterial activity in the dark. Thus, we examined whether antibacterial activity for our samples occurs without illumination and the obtained results are summarized in Table 2. Only a small effect on the E. coli survival can be observed, indicating that Cu toxicity, if any, is a negligible contributor to the observed antibacterial activity. This is consistent with the lack of significant toxicity of SnO2 and Al2O3 annealed in the presence of Cu.


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Figure 4. Electron spin resonance (ESR) spectra for a) 2A-TiO2 with different annealing conditions under solar illumination b) SnO2 samples under solar illumination c) all samples under ambient illumination. It was previously proposed that Cu-doped anatase titania exhibited enhanced antibacterial activity due to increased ROS formation resulting from lower recombination losses. In general, antibacterial action in titania has been attributed to ROS, 15,16,18-22,24-29 as well as photogenerated charge carriers.25,29 Therefore, we examined the effect of illumination on the native defects in the samples, as well as their ROS generating capability. The obtained ESR spectra in the presence of a DMPO spin trap under simulated solar and ambient illumination are shown in in Figure 4. It


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can be observed that under simulated solar illumination all the samples exhibit pronounced peaks corresponding to OH• radicals, except 2A-TiO2 which exhibits only weak peaks. Similar to simulated solar illumination, comparable peak intensities are obtained for annealed samples, while 2A-TiO2 exhibits weak peaks under UV illumination (see Supporting Information, Fig. S4). Under ambient illumination, however, no signals can be observed in any of the samples, indicating that ROS generation under ambient illumination is below the detection limit. Thus, no correlation can be observed between ROS generation and antibacterial activity. In addition, bacteria survival rate under UV illumination also does not correlate with ROS production, since 2A-TiO2 Ti 850 samples do not exhibit any significant antibacterial activity under any illumination condition despite being capable of producing ROS. In addition, it should be noted that 2A-TiO2 samples without annealing do not exhibit significant antibacterial activity under UV illumination, which is unusual for titania. However, other TiO2 samples exhibit robust antibacterial activity under UV illumination (survival rate 0% for 1-TiO2), but do not exhibit significant antibacterial activity under ambient illumination. Since no antibacterial activity is observed in the dark, illumination obviously plays a significant role, but the process likely does not involve ROS production since no correlation with ROS production can be observed. To further examine the relationship between the illumination and charge transfer associated processes, we have tested the samples for photocatalytic degradation of MB dye, and the obtained results are shown in Figure 5. Dye adsorption data are given in Supporting Information, Table S1. All samples exhibit relatively low dye adsorption (below 10%), and there is no correlation between the amount of dye adsorbed and the photocatalytic degradation. It can be observed that the dye degradation under UV and simulated solar illumination follows the same trends, but the 2A-TiO2 sample exhibiting the fastest MB


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degradation also exhibited the lowest ROS generation under simulated solar illumination and negligible antibacterial activity under all conditions. Different trends in dye degradation and toxicity to E. coli have been previously obtained (MB dye degradation but the lack of toxicity to E. coli),30 but the observed behavior was opposite from our findings. The lack of correspondence between antibacterial and dye degradation photocatalytic activity was previously attributed to the differences in surface interactions between titania and the dye and titania and bacterial cells.30

Figure 5. Degradation curves of methylene blue for different TiO2 samples under a) simulated solar illumination and b) UV illumination. Thus, possible explanation for the observed antibacterial activity under ambient illumination in the absence of ROS production, toxicity in the dark, and lack of significant changes in ATR-FTIR spectra of the bacterial cells exposed to nanoparticles is the charge


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transfer of photogenerated charges involving gap states. Charge transfer between photoexcited TiO2 and metallic or carbon components has been shown previously to play a role in the enhanced antibacterial activity.38-40 In this case, we have doped material rather than a composite (no evidence of particle formation or Cu-rich microdomains from EDX mapping data), with Cu present both as Cu+ and Cu2+ (see Fig. 3c). It was previously reported that the oxidation state of Cu is dependent n TiO2 surface features which are in turn dependent on the synthesis conditions.41 However, different from the previous report,41 no Cu clusters are found in our samples. Nevertheless, other reports also indicate the importance of well-dispersed Cu2+ for photocatalytic activity of Cu:TiO2.41,42 However, reduced photocatalytic activity was also reported for Cu doped TiO2 despite Cu being incorporated mainly as Cu2+.43 Other works in the literature also reported that high photocatalytic activity is associated with the presence of Cu+,44 or co-existence of Cu+ and Cu2+.45 In general, there is a strong interaction between Ti and Cu in Cu doped TiO2,46 and very likely optimal dopant concentration and chemical state depend on the properties of TiO2 (surface properties, native defect concentrations) due to a complex interplay between native defects and the dopant which is in agreement with our results. From the obtained data, it cannot be concluded what is the exact nature of Cu incorporation, other than the fact there are likely multiple states due to the presence of both Cu+ and Cu2+. This could possibly occur due to differences in surface and bulk Cu incorporation, but other possibilities could not be excluded. Whether Cu is incorporated as substitutional or interstitial impurity, changes in the band structure of the material are expected. From the change in the optical properties and increased absorption in the visible spectral range, the appearance of the gap states is expected. Gap state involvement could result in the toxicity to bacteria in absence of MB degradation due to differences in energy level alignment


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and charge transfer dynamics. To examine the differences between the native defects in the samples, ESR spectra47-51 under UV illumination at room temperature, as well as low temperature ESR spectra without illumination were measured and the obtained results are shown in Figure 6. Let us examine first the spectra obtained at room temperature. Generally, EPR peaks in titania are much weaker at room temperature compared to low temperature.50 Only weak signals corresponding to surface hole trapping sites were previously observed for anatase titania,50 and the spectra are similar to those we obtain for samples without annealing. In addition, the peaks disappear without illumination, similar to the previous report.50 While it was previously reported that no room temperature EPR signals were detected in rutile titania,50 we can observe a clear signal at g=2.002 in sample annealed in quartz crucible without any metal, which corresponds to conduction electrons,51 or an electron trapped at an oxygen vacancy.10,34,48,50 This is in agreement with the XPS results, which indicate more significant oxygen deficiency in these samples. However, in samples annealed in presence of Ti and Cu we do not detect significant peaks at room temperature. Since the absence of EPR peaks at room temperature was previously attributed to fast recombination, we can conclude that the native defect populations and nonradiative decay pathways are likely quite different in rutile samples prepared under different conditions. At low temperature, we can observe a weak peak at g~1.997 in 2A-TiO2 samples. This peak is commonly assigned to Ti3+ (most commonly for g=1.996).49 We have not been able to observe any signals corresponding to Ti3+ in the room temperature EPR spectra of our samples, and at low temperature this peak is present only in 2A-TiO2 and it has low intensity. This implies a small concentration of Ti3+ defects in the starting samples Although XPS data indicate higher oxygen deficiency and/or Ti3+ in annealead samples, oxygen vacancies and Ti3+ are not


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unambiguously detectable from XPS data.10 Based on the lack of Ti3+ signals in annealed samples, detected oxygen deficiency is likely due to oxygen vacancies. In 2A-TiO2 850 Ti sample, we observe the most prominent ESR signal, located at g~1.962. The 2A-TiO2 850 Cu sample exhibits both signals at g~1.963 and a broad peak at g~2.147, while for 2A-TiO2 850 sample only a broad peak at ~g=2.106 can be observed. Broad EPR peaks could indicate the surface traps49 or unresolved hyperfine structure. The presence of a peaks above g~2.07 was previously reported for different Cu2+ in TiO2 states,51 but this does not explain the presence of such a peak in 2A-TiO2 850 sample. It was previously reported that low temperature signals at g=2.019, g=2.014 and g=2.002 correspond to the hole trapping sites, while signals at g=1.969 and g=1.947 correspond to the electron trapping sites.50 However, these signals required UV illumination in the previous report,50 while we could observe them without illumination at low temperature. If the broad feature at g>2.0 corresponds to surface hole traps, this would be consistent with the 2A-TiO2 850 Cu and 2A-TiO2 850 exhibiting the antibacterial activity, with the effect under UV illumination being less pronounced in 2A-TiO2 850. The co-existence of hole and electron traps in 2A-TiO2 850 Cu may also be significant for antibacterial activity, since it is expected to affect the lifetime of photogenerated charge carriers.


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Figure 6. ESR spectra of different TiO2 samples a) room temperature, with under UV illumination b) at 5 K, without illumination. It should also be noted that it was previously reported that different rutile samples with similar properties (rutile crystal structure, similar particle sizes, shapes and surface areas) exhibited different antibacterial activity.34 It was found that the inactive samples exhibited evidence of the oxygen deficiency (presence of Ti3+) based on XPS and EPR measurements, indicating that other factors play a role in photocatalytic activity.34 The active samples exhibited oxygen rich surface, evidenced by the presence of FTIR band at 687 cm-1 corresponding to Ti-OO bond vibrations.34 In our samples, no clear FTIR band at 687 cm-1 can be resolved. While the shape of the broad absorption band between 400-900 cm-1 is different for different samples,


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samples annealed in the presence of Cu and Ti are similar, which indicates that the reason for different behavior cannot be identified from FTIR spectra. The most prominent difference that we can identify from the comprehensive characterization of the samples is the existence of multiple peaks in the low temperature EPR spectra in 2A-TiO2 850 Cu samples, indicating likely co-existence of electron and hole surface traps. The energy level alignment differences and the differences in native defects in the samples likely play a key role in the observed differences in antibacterial and photocatalytic activity. Conclusions Annealing at 850ºC in the presence of Cu induced antibacterial activity in titania samples regardless of their starting crystal structure. High annealing temperature results in rutile crystal structure, desorption of surface adsorbates, and absorption in the visible spectral range regardless of the presence of the metal (none, Ti, or Cu). However, only the titania samples annealed in the presence of Cu exhibit significant antibacterial activity under ambient illumination. Since no antibacterial activity is observed in the dark, as well as for other metal oxides annealed in the presence of Cu, antibacterial activity could not be attributed to the copper toxicity. Comprehensive characterization of the titania samples before annealing, and after annealing in the presence of different metals (none, Ti, or Cu) indicates that despite similar optical absorption spectra different defect levels are present in the samples, and these defect levels affect the antibacterial activity. Supporting Information An electronic file is available that contains Supporting Information including SEM images, ATR-FTIR spectra of E. coli exposed to different TiO2 samples and ESR spectra with DMPO


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Page 24 of 35

spin-trap under UV illumination. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author To whom correspondence should be addressed. Tel: +852 2859 7946 Fax: +852 2559 9152 Email: [email protected]. AMCN : Tel: +86-755-88018211, Fax: +86-755-88018204, E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENTS Financial support from the Strategic Research Theme, University Development Fund, and Seed Funding of the University of Hong Kong are acknowledged. AMCN would like to acknowledge the support from a grant from Shenzhen Science and Technology Commission (Project no. JCYJ20160530184523244) and National Science Fund of China (Project No. 21403103). REFERENCES 1. Etacheri, V.; Di Valentin, C.; Schneider, J.; Bahnemann, D.; Pillai, S. C. Visible-light activation of TiO2 photocatalysts: advances in theory and experiments. J. Photochem. Photobiol., C 2015, 25, 1-29.


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Table 1. Copper content (determined from XPS measurements), Zeta potential and aggregation size of different nanoparticles. Number of E. coli bacteria colonies formed on agar plates for bacteria exposed to different nanoparticles in ambient illumination for 4 hours. SD denotes standard deviation, AS denotes aggregation size. Sample

Cu:M

Colonies counts

(M=Ti,

Mean ±

Survival

Zeta

AS

SD

Rate (%)

potential

(nm）

Sn, Al) Control

(mV) 992, 907, 804

901±94

100.0

1A-TiO2

1.5

1350, 1343, 1164

1286±105

142.7

-34±1

824

1A-TiO2 Cu

4.3

104, 124, 110

113±10

12.5

-29±4

855

2A-TiO2

0.0

810, 860, 909

860±50

95.4

-13±2

592

2A-TiO2 850

0.0

918, 1083, 966

989±85

109.8

-39±1

546

2A-TiO2 Cu

2.6

4, 0, 1

2±2

0.2

-39±1

963

2A-TiO2 Ti 850

0.0

864, 880, 1072

939±116

104.2

-35±2

1970

R-TiO2

2.3

664, 862, 923

816±135

90.6

-38±2

1087

R-TiO2 Cu 850

3.9

432, 453, 399

428±27

47.5

-29±2

308

850

850


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P25

1.5

832, 668, 702

734±87

81.5

3±1

270

P25 Cu 850

3.4

2, 2, 0

1±1

0.1

-15±5

836

SnO2

1.7

1013, 839, 1139

997±151

110.7

-33±1

310

SnO2 Cu 850

7.9

669, 963, 883

838±152

93.0

-25±2

405

Al2O3

0.8

735, 1002, 939

892±140

99.0

-18±1

746

Al2O3 Cu 850

1.8

734, 625, 609

656±68

72.8

-31±2

693


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Table 2. Number of E. coli bacteria colonies formed on agar plates for bacteria exposed to different nanoparticles in the dark for 4 hours and under UV illumination for 30 mins. SD denotes standard deviation. Sample

Control

Colonies counts,

Mean ±

Survival

Colonies

Mean ±

Survival

dark

SD, dark

Rate, dark

counts,

SD, UV

Rate, UV

(%)

UV

100.0

972,804,

3200, 2888, 2416

2834±395

(%) 957±147

100.0

796±115

83.2

474±138

49.5

1096 2A-TiO2

2800, 2296, 2920

2672±331

94.3

692,776, 920

2A-TiO2 850

2784, 2408, 2024

2405±380

84.9

362,432, 628

2A-TiO2 Cu 850

2440, 2608, 2560

2536±87

89.5

0,0,1

0±1

0

2A-TiO2 Ti 850

2776,2512, 3072

2787±280

98.3

844,616,

887±294

92.7

1200


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TOC Graphic


35

Annealing-Induced Antibacterial Activity in TiO2 Under

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