Abstract
We have successfully synthesised one-dimensional single crystals of monoclinic phase titanium dioxide nanostructures (TiO2-B), prepared by a hydrothermal process. Morphological characterization was carried out by atomic force and scanning and transmission electron microscopy techniques. In order to study the crystalline structure, samples were calcined at 500°C in an air-filled chamber. X-ray diffraction results indicated that as-prepared samples presented diffraction patterns of hydrate hydrogen titanate and those calcined at 500°C exhibited the TiO2-B and anatase phases, confirmed by Raman spectroscopy. Scanning electron microscopy results showed that the one-dimensional nanostructures had high contrast and uniform widths for those synthesised and calcined, indicating the formation of a phase of monocrystals. Besides, a proof of the antibacterial effect was carried out of the monoclinic phase of TiO2-B on Escherichia coli pure cultures, where the viability of the bacterium decreases in presence of TiO2-B nanostructures plus UV illumination. Monocrystals did not change after photocatalytic tests, suggesting a possible application as long-term antibacterial protection.
1. Background
Titanium dioxide (TiO2) is a polymorph ceramic material with well-known stable phases called anatase and rutile. Other metastable phases are brookite and monoclinic. The monoclinic phase of titanium dioxide is usually called TiO2-B, exhibiting more open-structure than rutile, anatase, and brookite [1, 2]. This TiO2-B has been synthesised by three different methods. Marchand et al. in 1980 [3] used hydrolysis of K2Ti4O9 followed by heating. A second method introduced modifications to the Marchand method with K2TiF6 solution instead of K2Ti4O9 and mixing with ammonium hydroxide solution in order to obtain metastable compounds [4]. Recently, hydrothermal reaction of titania in a concentrated NaOH solution with moderate heating has been reported [5].
Research of new antibacterial materials is a general interest based on the rise of multiresistant pathogens. In this sense, antibacterial effects of TiO2 by photocatalysis have been reviewed [6], driven by the production of reactive species against microorganisms [7]. This reaction occurs when particles are illuminated by light with energy higher than its band gap [8]. Electrons in TiO2 jump from the valence band to the conduction band, and negative charges (e−) with the positive electric-hole (h+) pairs are formed on the surface of the photocatalyst. Absorbed water molecules are oxidized by the positive electric-holes (h+), forming hydroxyl free radicals, with a high oxidation power [9]. Moreover, negative charges react with the oxygen in water, generating superoxide radicals, producing ion homeostasis deregulation, coenzyme-independent respiration, and cell wall structure disruption [10–12].
By particle-size reduction at nanoscale level of TiO2 crystals, improvements in the antibacterial properties of spherical particles (320 nm) in Escherichia coli cultures have been demonstrated [13]. To contribute with the above, this work reports the synthesis of one-dimensional TiO2-B (1D-TiO2-B) nanostructures including wires and platelets shapes [14] by one-step hydrothermal reaction and their antibacterial property, which is due to photocatalysis. Samples were calcined and characterized by a battery of techniques, such as X-ray diffraction (XRD), atomic force microscopy (AFM), Raman spectroscopy, field electron gun scanning electron microscopy (FEG-SEM), and transmission electron microscopy (TEM). In this work, 1D-TiO2-B antibacterial activity was compared to spherical anatase nanoparticles, where the viability of Escherichia coli cultures exposed to 1D-TiO2-B plus ultraviolet radiation was reduced. Furthermore, Raman analysis and light absorption test were run to confirm that structural characteristics do not change after antibacterial experiences.
2. Methods
Synthesis of hydrogen titanate hydrated was performed hydrothermally in a Teflon® beaker containing 25 mL NaOH (Aldrich, 99.99%) at a concentration of 10 M, placed in a sealed reactor together with 0.21 g of TiO2 in the anatase phase (Aldrich, 99.8%). The resulting solution was stirred for 1 h at room temperature. The mixture was then heated at 130°C and treated for 6 or 8 days. The treated powders were washed with 0.1 M HCl (Sigma) aqueous solution and distilled water and were subsequently separated from the solution by centrifugation. This procedure was repeated until the washing water showed pH about 7. Powders were calcined at 500°C for an evolution of crystal structure study.
For morphology analyses, samples were ultrasonically dispersed in isopropanol and deposited onto a grid and examined with a Field Emission Gun Scanning Electron Microscope (FEG-SEM) model JSM-6330F (Jeol, Japan) and with an Atomic Force Microscope (AFM), alpha 300A (WiTec, Germany). By D5000 powder diffractometer with nonmonochromatized CuKα radiation (40 kV, 30 mA) (Siemens, Germany), 1D nanostructures were characterized. Also, using 514.5 nm Ar and 632.8 nm He-Ne lasers by a CRM 200 Confocal Raman Microscopy (WiTec, Germany), vibrational Raman spectra were collected. Energy dispersive X-ray (EDX) spectroscopy spectra were acquired with a Low Vacuum Scanning Electron Microscope (LV-SEM) model JSM-5900LV equipped with X-ray Microanalysis (Jeol, Japan). High-resolution images were obtained using a TEM model 3010 operating at 300 kV with a point resolution of 0.17 nm, which is equipped with EDX and selected area electron diffraction (SAED) (Jeol, Japan). Diffuse reflectance was obtained with a lambda 20 UV-Vis spectrometer (Perkin Elmer, USA).
For antibacterial tests, Escherichia coli ATCC 25922® cultures were grown aerobically in 10 mL glass test tube containing 9 mL of liquid broth and 1 mL of inoculum containing cells at 35°C for 20 h. Loss of viability was studied using 10 mg of TiO2-B and anatase per sample. The final cell concentration was determined by a viable cell count procedure using colony forming units (CFU) counting technique. A tube containing E. coli without nanostructures was irradiated as control. Also dark test was carried out. In order to perform photocatalytic experiment, plates containing 9 mL of NaCl 0.9% and 1 mL of cell solution were prepared. Following that, test plates were UV-illuminated using a HL 200 HybriLinker oven with wavelength λ = 254 nm and 120000 μJ (UVP, USA). 10 μL of sample was taken in periodic intervals (0, 10, 20, 30, and 40 min) in triplicate and then inoculated into plates containing tryptone soy agar and incubated for 16 h at 35°C. In order to analyse the loss of viability, CFU count was performed. Finally, Raman analysis tests were run to demonstrate that main properties of TiO2-B nanostructures did not change after antibacterial test. One-dimensional nanostructures were washed with 0.5 M KOH and deionized water and then dried for 6 h at room temperature before the spectroscopic analysis.
3. Results and Discussion
3.1. Morphology and Compositional Analysis
Based on similar synthesis of TiO2-B nanofibers, nanowires, and nanotubes conducted by hydrothermal processes before [15, 16], morphology of the samples using FEG-SEM and AFM analyses was performed. The shapes of the starting anatase material were particles between 50 nm and 300 nm in diameter (Figure 1(a)). After hydrothermal treatment at 130°C for 8 days, anatase particles were transformed into platelet-like mineral, as shown in Figures 1(b) and 1(c), with a length of 0.2 to 1.2 micrometres and an average thickness of 100 nm (Figure 1(d)). Other samples were obtained after a shorter hydrothermal treatment at 130°C for 6 days. The anatase nanoparticles were completely transformed into wire-like shapes, the hydrothermal process time being a variable that can mould the final morphology of the 1D structures (Figures 1(e) and 1(f)).
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Compositional analysis was carried out using energy dispersive X-ray spectroscopy, attached to a high-resolution transmission electron microscope (TEM). Spectra of as-prepared and calcined samples presented Ti, O, Cu, and Al, as shown in Figure 2(a). Copper came from the sample holder and aluminium from the calcination process. The O Kα and Ti Kα peaks are observed at 0.520 and 4.51 keV. The chemical composition of the TiO2 platelets was approximately 64.9 at% O and 35.1 at% Ti. These values are near the ideal stoichiometry of the TiO2 compound, which is around 66 at% O and 33 at% Ti. Element weight and atomic percentage of TiO2 bulk sample are shown in Figure 2(b). Considering experimental data, theoretical atomic weight, and the low copper presence due to the sample holder, the calculated composition of the sample was Ti(1) O(1.73) Al(0.07) Cu(0.40).
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3.2. Structural Analysis by Transmission Electron Microscopy
To study the architectural properties of 1D-TiO2-B materials, structural analysis was performed by high-resolution microscopy. Figure 3(a) shows a low magnification TEM bright field image without annealing at 500°C in an air-filled chamber and the nanostructures synthesised by the hydrothermal process at 130°C for 8 days. Several platelets from 100 nm up to a maximum of 2 microns of thickness are shown, whose lengths vary from 500 nm up to several microns. The dark field image is oriented in the 110 plane of hydrogen titanate (Figure 3(b)). Figure 3(c) corresponds to an HR-TEM image of a 1D-TiO2-B 58 nm in diameter displaying a layered structure indicating that the rod grew along the (010) direction of TiO2-B (Figure 3(d)) with an interplanar spacing of about 0.616 nm, a value consistent with previous reports [17]. The crystalline structure of the nanoplatelets and nanowires belongs to the family of titanium dioxides with monoclinic phase with the parameters 16 nm, nm, nm, and ° which allows classifying this compound into the space group C2/m [3]. The rod in Figure 3(d) exhibits a set of intershell spaces, with distances of 0.376 and 0.325 nm, corresponding to interplanar distances along the (111) and (133) directions of 1D-TiO2-B, respectively. The spots of the selected area electron diffraction (SAED) can be interpreted as caused by the three different crystalline orientations (Figure 3(e)). The uniform diameter and contrast of the wires are properties indicative of single crystal structures [18]. A simulation of the atomic structure of 1D-TiO2-B in Figure 3(f) is shown.
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3.3. X-Ray Diffraction and Raman Spectroscopy Analyses
Diffractograms and Raman spectroscopy analyses of the as-prepared and calcined at 500°C samples are shown in Figure 4. X-ray diffraction analysis pattern shows that crystallization of the samples varied due to the heat treatment. TiO2-B phase was formed after 500°C calcination for hydrothermal process for 6th day (Figure 4(a)). After a revision of XRD database patterns and available literature, unidentified peaks were labelled as titanate phase (T). For these peaks it was not possible to associate any known structure. As-prepared sample showed a high presence of water molecules in the entire diffraction pattern. Anatase (A) and titanate (T) phases showed a low presence of peaks. Samples annealed at 500°C clearly show how the crystalline structures change dramatically after the heat treatment. This change mainly occurred due to the water evaporation and the heating exposure time that helped to generate the structural changes that create the B phase of TiO2 and also eliminate the presence of impurities. No organic contaminants were found. Comparing both XRD patterns, certain anatase and titanate peaks (see grey bar in Figure 4(a)) have a predominant behaviour in the two obtained patterns. Observed anatase phase present in both patterns helped to generate B phase peaks after sample calcination. On the other hand, some of the titanate peaks observed close to the 10 degrees in the as-synthetized spectrum changed its structure after the heat treatment generating B phase of TiO2. To characterize the observed pattern, comparison of results with previous reports was performed. Up to the 90% of similarity of the patterns was found and the remaining 10% did not match with lower intensity peaks shown in the literature guide [4, 19–21].
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The confirmation of the synthesis of TiO2-B crystal structure by Raman spectroscopy spectra is shown in Figure 4(b). This result confirms the characterization obtained by XRD and shows typical peaks well defined of the sample after calcination. Moreover, titanates bands do not correspond to rutile, anatase, or brookite phase. Calcined samples at 500°C show similar main peaks patterns localized at 195, 248, 408, 633, and 858 cm−1 as is described by Kolen’ko et al. in their experiment with TiO2-B nanorods [21].
3.4. Antibacterial Test
Diverse approaches of TiO2-based antibacterial nanomaterials are broadening nowadays [22], including polymers [23] and particle mixtures [24]. In specific, 1D-TiO2-B structures have been tested in Klebsiella pneumoniae [25]. However, the rise of Escherichia coli multiresistant strains is a growing worldwide concern [26], and confirmed antibacterial behaviour of new materials is needed.
To address an application of 1D-TiO2-B material, a bacterial-growth control performance was tested (Figure 5). UV-illuminated Petri dishes clearly showed decreased colonies after treatment. Quickly, differences are noticed in UV + TiO2-B at 10 minutes. Controls in dark did not result in a significant loss of viability (Figure 5(a)). Quantification of the colony forming units (CFU) of E. coli showed reduction of viable cells when they are in contact with TiO2-B and anatase and/or UV-illuminated. However, UV + TiO2-B produced a faster CFU decay than UV + anatase or UV only. (Figure 5(b)). Statistics of normalized data demonstrated that 10-minute incubation showed the most significant differences among conditions (Figure 5(c)). Less substantial differences, in all late UV-illuminated samples, were noticed. Clearly, normalized CFU data showed the best reduction slope under UV + TiO2-B treatment (Figure 5(c)). One-way analysis of variance confirms slope differences between UV + TiO2-B and UV only with a value < 0.0001 at 10 minutes. Results for sets on dark TiO2-B showed small initial reduction viability at 10 minutes but remain constant later indicating no toxicity of nanostructures, confirming that antibacterial process only occurs driven by photocatalysis (Figures 5(a), 5(b), and 5(c)). Results for loss of viability test for anatase nanoparticles are similar to previous reports [13]. The specific role of free radical in the model presented here remains unclear, which could be addressed in further research.
Finally, the properties of TiO2-B nanostructures after testing were evaluated by Raman spectroscopy (Figure 5(d)). Nanowires were analysed before and after antibacterial experiences, where those Raman spectra did not change substantially. Furthermore, photocatalytic properties appear at the same wavelength (303 nm) (data not shown). Results obtained confirm that the structural and optical properties of the TiO2-B nanowires synthesised did not change after being tested for antibacterial activity.
4. Conclusions
This work reports the successful synthesis of titanium dioxide-B nanowires and platelets starting from a hydrothermal process with pure TiO2 and NaOH at concentrations of 10 M. The as-prepared samples exhibited the titanate phase. These samples were calcined at 500°C and presented the monoclinic phase of titanium dioxide. Main properties of the TiO2-B platelets and nanowires were characterized utilizing a battery of analytical techniques such as XRD, Raman spectroscopy, AFM, SEM, HRTEM, and EDX analysis, and light absorption tests. These studies indicated that annealed samples presented diffraction patterns and vibration modes corresponding to the titanium dioxide indexed as the monoclinic phase. The XRD study indicated in addition the presence of anatase and titanate phases in low concentration. Features of these nanostructures go from 25 nm to 218 nm in diameter, with a mean of 102.8 nm, and from 200 nm up to 2.6 micrometers in length, with a mean of 0.85 μm.
In this paper, we propose 1D-TiO2-B as an antimicrobial material against Escherichia coli. In our case, the inherited action of free radicals produced by UV-TiO2-B on the bacterial surface remains unclear. This synergic effect of reactive species needs further research at molecular level, comparing with other pathogens, under different light doses and also other TiO2 polymorphs. This report also demonstrates the optical and structural stability of 1D-TiO2-B under high energy light, offering potential long-term bactericide applications especially in surface sterilization.
Competing Interests
The authors have declared that there are no competing interests.
Authors’ Contributions
Sergio León-Ríos, Rodrigo Espinoza González, Sandra Fuentes, Emigdio Chávez Ángel, and Alex Echeverría performed all experimental procedures. Sergio León-Ríos, Rodrigo Espinoza González, Alex Echeverría, and Antonio E. Serrano analysed the data. Sergio León-Ríos, Alex Echeverría, Antonio E. Serrano, Cecilia S. Demergasso, and R. Antonio Zárate wrote the paper and participated in all discussions.
Acknowledgments
This work has been partially financed by Fundación Andes grant under Contract no. C-13876, FONDECYT grant under Contract no. 1080401, Anillo ACT1204, and FIC-R 2015: “Transferencia Desarrollo de Nanotransistores Basados en Cables Bacterianos del Desierto,” Cod. BIP 30412822-0. This work was supported by the DGIP of the Universidad Católica del Norte. Special thanks are due to Laboratorio de Microscopia Electrónica (LME) at Laboratorio Nacional de Luz del Sincrotron (LNLS), Campinas, Brazil, for providing access to the FEG-SEM and HR-TEM. The authors thank the FCFM, Universidad de Chile, for the use of their XRD facility. They also thank Mr. Jaime Llanos, of the Laboratorio Inorgánica, Departamento de Ciencias Químicas y Farmacéuticas, Facultad de Ciencias, UCN.