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Mesoporous TiO2 synthesis using a semi-hard biological template
Accepted Manuscript Mesoporous TiO2 synthesis using a semi-hard biological template Armin Hernández-Gordillo, Antonio Campero, L.Irais Vera-Robles PII:
S1387-1811(18)30258-0
DOI:
10.1016/j.micromeso.2018.05.014
Reference:
MICMAT 8916
To appear in:
Microporous and Mesoporous Materials
Received Date: 28 March 2018 Revised Date:
8 May 2018
Accepted Date: 10 May 2018
Please cite this article as: A. Hernández-Gordillo, A. Campero, L.I. Vera-Robles, Mesoporous TiO2 synthesis using a semi-hard biological template, Microporous and Mesoporous Materials (2018), doi: 10.1016/j.micromeso.2018.05.014. 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 proof before it is published in its final 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.
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Mesoporous TiO2 synthesis using a semi-hard biological template Armin Hernández-Gordillo*a,b, Antonio Camperoa, L. Irais Vera-Robles*b a
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Departamento de Química, Área de Química Inorgánica, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco No.186, Col. Vicentina, 09340 CDMX, Mexico b Departamento de Química, Área de Biofisicoquímica, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco No.186, Col. Vicentina, 09340 CDMX, Mexico. *
Corresponding authors: [email protected] (L.I. V-R.); [email protected]
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Abstract
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The bacteriophage M13, a rod-shaped virus, was used as a semi-rigid template to synthesize crystalline mesoporous TiO2. This strategy allowed us to modulate solgel reactions on the virus surface without the need of chemical or genetic manipulation. Important parameters as pH influence and concentration were investigated. The studies by which the pH was varied were the principal route to obtain materials with pores of around 6.1 and 8.2 nm. When an M13 phage concentration of 0.1 mg/mL was employed, anatase mesopores presented specific surface areas from 80 to 130 m2/g and pore volumes from 0.18 to 0.21 cm3/g. It should be mentioned that surface area is due exclusively to mesopores, and from N2 adsorption/desorption experiments, no evidence of micropores was detected. This absence of micropores results from the stable protein arrangement on M13 phage, which contrasts with the disruption of micellar assemblies of soft templates. These results show that anatase mesophases are obtained in an easy and rapid route at room temperature and could be expanded to synthesize other metal oxide mesostructures tuning M13 phage properties.
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Keywords Mesoporous, TiO2, Template, Virus M13 1. Introduction
Titanium dioxide is a material with multiple uses in environmental and energy fields.[1] Applications of TiO2 are closely related to its physicochemical properties such as size, porosity, crystallinity, etc. Thus, its adequate control is an important field of research. Of special interest are TiO2 mesostructures not only because the homogeneous pore size distribution and larger surface area but also the homogeneous distribution of active sites through the mesoporous network. In comparison, with bulk or nanometric particles, mesoporous materials facilitate their recovery and recycling, moreover, they are friendly with the environment due to low cytotoxicity in living organisms.[2] Thus, synthesis of mesoporous TiO2 is a subject
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of study in development. Many efforts have been focused on finding suitable templates for a strict control on the shape and size of the pores, and at the same time producing organized structures. These kinds of materials are obtained mainly by two approaches, namely the soft-templating and the hard-templating.
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The soft-templating route was originally proposed for the synthesis of silica mesopores. Surfactants are used to form micellar arrangements on which condensation reactions of silicon alkoxides are carried out to form SiO2 mesopores of various sizes and shapes.[3] However, the adaptation of this method to the synthesis of TiO2 has some issues due to the high chemical reactivity of the precursors and the disruption of the micellar arrangement, producing high microporosity.[4] Another drawback is the collapse of the porous network during the elimination of the template by thermal treatment, however, this step is necessary to obtain crystalline structures.[5] To overcome these issues, variations in the methodology have been proposed, for example, improving the micelle formation by using evaporation-induced self-assembly (EISA) where the solvent is evaporated slowly at mild temperatures,[6, 7] also by modifying the atmosphere during the thermal treatment (crystallization process)[8] or exploring nonconventional surfactants like ionic liquids crystals among others[9].
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In contrast, the use of hard templates (preformed mesoporous solids) has been successful to produce crystalline mesopores of TiO2 with novel structures.[10] Unlike the soft-templating method, this one allows the use of highly hydrolyzable precursors in extreme synthesis conditions. In addition, it prevents the collapse of mesopores even to high temperatures, both porous and crystalline frameworks are preserved.[11, 12] However, the main problems with this method are the excessive time employed in order to obtain the rigid template and its further elimination.
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In the last years, new molecules have been proposed as templates to create mesostructures.[13] Specifically, viruses which are genetic and protein materials hierarchically assembled have been used with this purpose, producing mesoporous silica with cylindrical[14, 15] or icosahedral[16] pores depending on the shape of the virus. In particular, filamentous viruses Ff (Ff phages) have been used as template because of their easy replication and rapid purification.[17] These rod-shaped viruses (6.6 nm in diameter) are semi-flexible.[18] Inside of rod is located the DNA molecule, outside is formed mainly for hundreds of subunits of protein p8 (α-helix, 50 residues) that cover the whole longitudinal body of the phage (each protein covers ~2.3 nucleotides)[19], as well as other four types of proteins cover the ends.[20] The Ff phages are stable between pH values 3−11 and tolerate up to 80 °C to neutral pH.[19, 21, 22] Their resistance to organic solvents depend on their polarity, due to the hydrophobic residues in the protein p8. Ff phages are stable in aqueous mixtures that contain methanol, ethanol, or isopropanol up to 60, 50 and 30% (v/v) respectively.[23] Ff phages exhibit liquid crystal behavior in function of their concentration,[24, 25] adopting a nematic phase even at concentrations as low as 0.1 mg/mL.[26] Phage organization also depends on its length, pH and ionic strength of the medium.[27] This Ff phages family includes M13, fd, and f1, which share similar physicochemical properties.[19, 20] In
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particular, M13 phage particles have an isoelectric point of 4.3[28]. Then, they are able to aggregate at pH < 4.4, organize at 4.7 ≤ pH ≤ 5.5 or repel each other at pH > 5.5 with the concomitant change on the surface charge of M13 phage.[29-31] In comparison with micellar arrangements, Ff phages have a very stable assembly of p8 proteins and their tubular morphology is under genetic control; neither solvents nor precise concentrations or temperatures can alter their shape, which allows us to classify them as a semi-hard template. Moreover, their capacity to tolerate mixtures that contain a high amount of alcohols facilitates their use in sol-gel reactions.
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Silica ordered mesopores are the only structures reported by using M13 phage as semi-rigid template.[14, 32] Then, the use of M13 phage to obtain TiO2 porous structures is being explored. For example, layer by layer technique was performed to manufacture TiO2 film using as platform M13 phage which was previously engineered with three glutamates to encourage the electrostatic interaction between phage and (NH4)2TiF6 precursor. These reactions are carried out to 50 ºC by several hours, followed by calcination.[33] Other approach to synthesize TiO2 nanowires was obtained employing the same genetically engineered M13 phage as scaffold and Ti(OBu)4 as precursor. Low temperature (-40 ºC) was necessary to control the high reactivity of the precursor. These nanowires were mixed with a sacrificial polymer and deposited on a substrate.[34] In both cases, the only function of the phage was molding the nanowires, which created the porous network as a result of space between them resulting in a wide pore size distribution. Thus, pore size was not related to the diameter of the phage. In spite of the fact some success has been recently achieved, still, it is a challenge to use M13 phage to obtain fast and easily TiO2 mesoporous structures.
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Herein, we optimized a methodology to synthesize TiO2 mesoporous structures with anatase pore walls. The novelty of this approach is to use a surfactant-like molecule that naturally is assembled as a cylindrical micelle, looking more like a semi-hard template, namely the bacteriophage M13. Our main goal was to adapt the synthesis conditions to the chemical nature of the native phage, without genetic or chemical manipulation and at room temperature. Due to a large number of variables to control, we adopted a method inspired by the genetic algorithm that allowed us to explore more general reaction conditions. Based on this approach we obtain mesoporous TiO2 and we observed that not only M13 concentration but pH is a vital parameter to control pore size. As a result, the average pores sizes of these materials were around of 6.1 and 8.2 nm with specific surfaces areas of ∼130 and 80 m2/g, respectively. 2. Experimental 2.1 Phage replication and purification The M13 phage used in this work was derived from vector M13mp18 (New England BioLabs). These phages have a length of ~995 nm and ~3150 p8 proteins, corresponding to the number of nucleotides of the vector (7249 bases). For phage amplification, 5 mL of an overnight culture of E. coli (XL1-Blue strain) was diluted 100 times in a flask (2 L) with 2xYT medium broth (Invitrogen)
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previously sterilized. The diluted culture was infected adding a stock solution of phage (~1014 pfu) and incubated for 16 h at 32 °C and 200 rpm to maximize the yield. Tetracycline was added to all media before incubation (10 µg/mL). After incubation, the culture was centrifuged for 10 min, 6000 rpm at 4 °C (Avanti J-30I centrifuge, Beckman Coulter), the supernatant was recovered and mixed with polyethylene glycol (PEG, ~8000 g/mol) and NaCl to a final concentration 20% w/v and 2.0 M respectively, stored for 1 h at 4 °C and centrifuged for 15 min, at 8000 rpm and 4 °C. The pellet was resuspended in distilled water and the precipitation with PEG-NaCl was repeated once again. Finally, to ensure the recuperation of fully assembled phage and discard PEG and salt residues, the phage was precipitate by isoelectric point adjusting the pH to 4.3, stored and centrifuged, the pellet was resuspended in water and neutralized with dilute NaOH. Quantification of phage was performed by spectrophotometry (NanoDrop 2000, Thermo Scientific), considering the specific absorptivity coefficient at 269 nm as 3.84 cm2*mg-1 for Ff bacteriophages,[35] the path length was automatically adjusted to avoid dispersion.
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2.2 Synthesis of TiO2 In general, reactions were as follows: titanium isopropoxide (97 %, Sigma-Aldrich) was added in absolute ethanol previously acidified with concentrated hydrochloric acid. The molar ratio between the alkoxide and ethanol (Ti:OH) was calculated from the maximum amount of ethanol supported by the phage (50% v/v). This alcoholic solution was added to a phage solution keeping vigorous agitation and aging for 10 minutes. Then, the mixture was then centrifuged for 15 min (12000 rpm), the gelatinous pellet was washed with ethanol technical grade (2 x 10 mL) and dried at 50 °C. All samples were calcined at 500 °C for 2 h with a heating rate of 1 °C/min. To find the optimal conditions for the synthesis of mesoporous TiO2 (molar ratios, phage concentration, and pH), we used a methodology inspired by the genetic algorithm which is presented in Appendix A (Figure S1). The nomenclature for samples is as follow: TiX_Yz were Ti refers the TiO2, X means the pH of the alkoxide solution, Y means the phage concentration in mg/mL and finally, z means the Ti:OH molar ratio being z: a=100, b=250, c=500.
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2.3 Material characterization Removal template was monitored by thermal gravimetric analysis (Pyris Diamond, Perkin Elmer) heating in air (using nitrogen as the carrier gas, 50 mL/min) a dry sample from 25 to 500 °C at a heating rate of 1 °C/min, held at this temperature for 2 h and finally heated to 1000 °C. The synthesized TiO2 was observed by transmission electron microscopy (JEOL 2000), samples were suspended in water, and an aliquot of 2 µL was placed on a copper grid covered with carbon type-B (300 mesh). X-ray diffraction patterns were recorded using a Bruker D8 Advance diffractometer (λ=1.54 Å), measurement intervals were 10-70° and 0.6-7° in 2θ for normal and low angle measurements respectively. The crystallite size was estimated by applying the Scherrer equation using the principal diffraction peak corresponding to the plane (101) of anatase and the Scherrer constant K=0.9.[36] Raman spectroscopy was used to study the vibrational features of the materials; the Raman spectra were acquired using a Horiba-Jobin Yvon LabRam
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800 system equipped with an Olympus BX40 confocal microscope. Nitrogen adsorption/desorption measurements at 77 K were performed on an ASAP 2020 (Micromeritics) volumetric adsorption analyzer; before the measurements, the samples were outgassed for 12 h at 373 K. The specific surface area of solid samples was calculated by multiple-point Brunauer–Emmett–Teller (BET) method in the relative pressure range of P/P0 = 0.05–0.25. Pore volume was calculated at P/P0=0.99. Pore size distribution curves were computed using the non-local density functional theory (NLDFT) method, (using the nitrogen adsorption branch, assuming cylindrical pores on silica). T-plot (P/P0 = 0.2–0.5) was used to calculate the microporosity in samples with the high specific surface area.
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3. Results and discussion
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Synthesis of mesoporous structures depends on the kind of template, in this work the template is a semi-rigid structure (M13 phage), whose cylindrical shape is inherent, but liquid crystal arrangements are a function of the concentration, pH, and temperature, among others. On the other hand, the width of pore wall can be related to the precursor amount. Molar ratios Ti alkoxide:protein p8 (Ti:p8) larger than 500:1 produced big aggregates instantaneously. On the contrary, small amounts of precursor were able to diffuse across the liquid and reach surface phages, but it was not enough to completely cover them, as we observed from the UV-vis spectrum (not shown). Then, we set the molar ratio Ti:p8 100:1, to have enough Ti species and to control its condensation and build the pore walls.
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Thermogravimetric (TG) and differential scanning calorimetry (DSC) analysis were performed to determine the content of phage template and the stability of TiO2 phases. TiO2 material showed a loss in weight between 200−500º C due to the decomposition of protein and nucleic acids coming from phage, this represents around 30.0%, however, this value is below the expected (∼40%) suggesting that not all phage was trapped in the final network. It must be noted that TGA analysis was made in a similar way to that of the calcination step, in other words when sample reached 500 ºC the temperature was kept constant for two hours and we observed still a loss in weight (1.0 %) due to the thermal stability of DNA. Then, the temperature was raised up to 1000 ºC and no change in mass was observed. This demonstrated that keeping the temperature to 500 ºC for two hours suffices to eliminate completely the semi-hard template. From the Figure 1, two exothermic peaks to 430 and 960 ºC are registered, which should correspond to phase transition for anatase and rutile, respectively, as we will demonstrate below. 3.1 Importance of pH in the alkoxide solution While solubility of titanium species is favored to low pH (< 3), reducing the rate condensation of Ti alkoxides[37], M13 phages are spontaneously assembled in two-dimensional structures in a narrow interval of pH (4.7−5.5). Outside this range of pH, competitive electrostatic interactions between viral particles result in their aggregation or repulsion. Taking into account this behavior, experiments were
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conducted to different pH values and setting phage concentration as low as 0.10 mg/mL. When the pH of the alkoxide solution was > 3.0, the sol-gel reaction was very rapid, developing big aggregates that did not interact with the surface phage. Conversely, when the pH was < 1.0, titanium clusters formed very stables sols and no interaction with M13 particles was observed, instead, M13 was agglomerated due to pH (pH
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Therefore, TiO2 materials were obtained and characterized, starting from alkoxide solution adjusted to 1.0 and 1.5 pH values. Synthesis to pH 1.0 (sample Ti1.0_0.1c), produced mesoporous structures with a specific surface area of 132 m2/g and an H2b hysteresis cycle type (Figure 2a), where the desorption process is affected by blocking effects along the pore, caused probably by a partial collapse during calcination step. This sample also showed a narrow pore size distribution (Figure 2b) with an average pore diameter of 6.1 nm in agreement with the phage diameter. When pH was slightly increased to 1.5 (sample Ti1.5_0.1b), the specific surface area decreased to 85 m2/g and the hysteresis cycle change to type H1 (Figure 2c), indicating that this network is made by regular cylindrical pores with open ends whose average pore size is 8.2 nm (Figure 2d), suggesting a higher thermal stability of the walls in the structure.
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In spite that the M13 phage has a well-established structure because of the ionic interactions between proteins and DNA, environmental conditions can affect its size. When M13 phage is dried its diameter size decreases to 5.5 nm, permitting a diameter expansion up to 9.0 nm in solution.[38, 39] Besides, the solvent polarity is a factor involved in the p8 protein packaging.[31] As observed in Figure 3, Nterminal residues are exposed to the solvent (cyan color) and have random coil structure, which means that they can move freely, on the contrary of the residues that comprise the α-helix structure (purple color). Thus, this freedom in motion should be the responsible for the increase in the phage diameter and therefore would explain the pore diameter of 8.2 nm. In fact, α-helix structure stability was verified by infrared spectroscopy before calcination. Amide I and amide II appear at 1648, and 1540 cm−1, respectively (Figure S2).[40, 41] Nonetheless, the possible encapsulation of bundles phages cannot be discarded. Interestingly, when the sol-gel reaction was performed without M13 phage, a very stable sol was obtained, whereas in presence of the phage an immediate formation of fibrous particles was observed (TiO2 covered phages).[42] This indicates that the alkoxide in acidic conditions only polymerizes in the presence of phage, confirming its role as template. The catalytic effect of the phage on titanium oxide condensation may be caused by the lateral groups of the residues on the major coat protein. Figure 3c shows these eight superficial residues for the p8 protein (AEGDDDPAK), where the carboxylate (from glutamate and aspartate) and amine groups (from the N-terminal and lysine) can interact with titanium species promoting their reactivity.[43, 44]. In this way, small titanium clusters with positive charge surrounded the negatively charged surface of phages (Scheme 1b). In all
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experiments under acidic conditions without phage (negative control) it was never observed the formation of a precipitate, even after aging the sample for one week (Scheme 1a).
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3.2 Effect of phage concentration
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As can be seen in both cases, mesoporous structures were obtained according to adsorption isotherms type IVa. Interestingly, no sorption to low relative pressure was observed because of the absence of micropores, in contrast with surfactants (soft template), which can form disrupted micellar arrangements producing microporous cavities when trapped in the oxide framework; phage particles do not disassemble because of the interaction protein-DNA, and their use as template leads exclusively to mesoporous materials.
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Previously, it has been demonstrated that silica mesostructures were obtained when phage concentration is ≥ 4 mg/mL[14, 32], but this is not valid for metal alkoxides because their high hydrolysis rate and moreover their condensation is raised by the presence of the phage, as we discussed above. Thus, the effect of phage concentration was tested to study their liquid-like crystal behavior and its templating effect in the synthesis of ordered mesopores. When phage concentrations ˃ 0.1 mg/mL were used, sol-gel reactions were catalyzed by p8 proteins, avoiding a proper homogenization even to pH as low as 1.0. This was evidenced by the decrease of the specific surface area when increased the concentration of phage, producing specific surface areas of 130, 67, 55 and 22 m2/g for concentrations of 0.10, 1.00, 3.00 and 6.00 mg/mL respectively.
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Figure 4a and c display the nitrogen adsorption/desorption isotherms for samples Ti1.0_3.0b and Ti1.0_1.0c obtained at 3.00 and 1.00 mg/mL respectively, which are similar in shape. In both cases, adsorption continues up to relative pressures of 1.0, showing a hysteresis cycle of type H3 suggesting the existence of macropores. According to our hypothesis, TiO2 formation is undertaken on the surface of bundles of phages, inhibiting the complete diffusion of the titanium precursor. This aggregates of TiO2 can be analyzed as plates more than as mesoporous structures, with no well-defined pore size distribution (Figure 4b and d). When the phage concentration was higher (sample Ti1.0_6.0a), it was not possible to control the reaction and no porosity can be assigned to the material. In contrast, sample Ti1.2_0.1b synthesized to lower phage concentration (0.10 mg/mL) exhibited a marked hysteresis cycle type H2a (Figure 4e), typical of mesoporous structures and free of micropores according to the t-plot of samples (Figure S3) with an average pore diameter of 6.1 nm (Figure 4f) and narrower pore size distribution. To gain insight information about the framework, samples were studied by transmission electron microscopy (TEM). In the Figure 5a, the sample Ti1.2_0.1b shows pores 6.5 nm in size according to the pore size distribution (Figure 4f), whereas, in sample Ti1.0_1.0c porous channel layouts of 8.0 nm in size were distinguished (Figure 5b) in agreement with the maximum phage diameter. This
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confirms liquid-like crystal behavior for the native phage and more importantly its role as a semi-hard template to obtain mesopores of metal oxides. In spite that the phage acted as template, it was not able to create perfect ordered structures, as was demonstrated by low-angle X-ray diffraction (XRD), where only a low-intensity peak was visible in sample Ti3.0_1.0b (Fig. S4) whereas in sample Ti1.2_0.1b this signal disappeared. Thus, we can obtain a local order of pores and macropores when phage concentrations ≥ 0.10 mg/mL are used (Scheme 1d), and disordered pores with a narrow pore size distribution if low concentration is used (Scheme 1c), in line with the self-organization of M13 phage.
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Low phage concentrations were able to produce materials with two pores sizes, as mentioned in section 3.1, a possible explanation could be that two or more phages are trapped between TiO2 walls. To investigate this hypothesis, we used the overlap concentration (C*). This concentration C* (0.05 mg/mL for Ff phage family [46]) ensures that particles do not interact with each other and only individual virions must be trapped in the TiO2 network. The material obtained at this C* showed an average pore size of 8.2 nm. Noticeably, bigger concentrations (0.10) gave the same results, implying that even to this phage concentration particles are so scarce that they do not overlap. Thus, phage agglomeration is discarded and the possible explanation should be the shrinking-swelling of p8 proteins provoked by small pH variations and cosolvent amount.
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Nitrogen adsorption/desorption characterization of these mesoporous structures (Ti1.5_0.1a and Ti1.5_0.05b) was performed. Figures 6a and c show practically the same nitrogen adsorption/desorption isotherms and hysteresis cycles of type H1, typical of regular cylindrical pores with the ends accessible to the gas molecules, describing perfectly the phage geometry, which is reflected in good pore homogeneity. The collapse of the pores is avoided by the continuous and smooth template removal by calcination (Figure 1). So, samples Ti1.5_0.1a and Ti1.5_0.05b present similar specific surface areas of 78 and 83 m2/g, respectively.
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Although three-dimensional arrays have been observed in phage solutions at concentrations ≥ 0.10 mg/mL,[26] with the synthesis method used here, concentrated phage solutions were not suitable to obtain ordered mesopores, due to the catalytic effect of p8 protein on titanium precursor. Therefore, it can be concluded that the maximum phage concentration for the synthesis of mesopores with a narrow pore size, using alkoxides, is 0.10 mg/mL. Furthermore, all experiments at pH ∼1.5 produces invariably mesopores with an average diameter of 8.2 nm. 3.3 Pore wall thickness and thermal stability The phage has a tunable diameter because of the flexibility of p8 protein, this reveals important information about the two pore size distributions obtained. Another parameter to explain this result is related to the crystal size of the walls of the network. Thus, crystalline structure and size of different samples were analyzed using the X-ray diffraction. After calcination at 500 ºC for two hours, TiO2
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samples obtained using smaller phage concentration show diffraction peaks corresponding to anatase phase, however, if phage concentration is increased (6 mg/mL, sample Ti1.0_6.0a), a new peak to 27.5° in 2 theta is assigned to the rutile phase (Figure 7). Crystal size was calculated according to Scherrer equation and results are shown in Table 1, it can be noted that mesoporous structures increase the crystal size as surface area decreases.
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From Table 1, it can be observed that mesoporous materials of 6.1 nm in diameter have higher specific surface area than those of 8.2 nm. It would seem that either the number or the length of pores are different in the samples. However, as pore volume is almost constant (0.18−0.21 cm3/g) and the same number of phages were used in all samples (molar ratio Ti:p8), it is more viable attributing this behavior to the width of the wall because the same number of pores can produce lighter or heavier materials. Even, when phage concentration is 6.00 mg/mL the sample is no longer porous and two TiO2 phases co-habit. This phase transition is associated with the crystal size so that the increment in crystal size yields a conversion to rutile phase at a lower temperature.[47]
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Once the mesoporous structures were obtained at 500 ºC, we study the influence of temperature to gain more information about the crystallization process. Samples Ti1.0_0.1c and Ti1.5_0.1b were heated to 650 and 800 ºC and evaluated by DRX, N2 adsorption/desorption, and Raman spectroscopy. Raman spectra of sample Ti1.0_0.1c heated to 500, 650 and 800 ºC show bands to 141, 397, 515 and 637 cm-1, characteristic of anatase phase, no bands corresponding to proteins or ssDNA groups are evident, confirming a whole elimination of the template. Sample Ti1.5_0.1b presents same behavior to 500 and 650º C, however, an increase in calcination temperature to 800º C revealed two new bands to 445 and 615 cm-1 distinctive of rutile structure (Figure S5). This rutile transition also is consistent with the correlation between the crystal size and the transition temperature as it was seen in sample Ti1.0_6.0a.
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Thermal stability of mesostructures was tested for Ti1.0_0.1c and Ti1.5_0.1b, representative samples for pore size of 6.1 and 8.2 nm respectively. After calcination at 650 °C for 2 h, the specific surface area for both samples decreases to ∼30 m2/g and remains a hysteresis cycle (type H1) (Figure 8 a and c). On the contrary to silica mesostructures, where walls shrink, TiO2 walls sinter partially increasing the pore size up to 26 and 20 nm for Ti1.0_0.1c and Ti1.5_0.1b respectively (Figure 8b and d). When samples were calcined at 800 °C, surface area of Ti1.0_0.1c sample decreased to 11 m2/g without phase transition, whereas Ti1.5_0.1b sample practically loses all mesoporosity (4 m2/g), due to extended sintering induced by anatase to rutile phase transition giving as results bigger crystal as was observed in TEM (Fig. S6).
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4. Conclusions
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In addition, our strategy to use M13 phage as semi-hard template results in homogeneous mesoporous materials having specific surface areas of about 80 and 130 m2/g, and average pore diameters of 8.2 and 6.1 nm, respectively. These values agree with those found in the literature for TiO2 mesoporous structures, however, authors take advantage of the slow evaporation of THF solvent[48] or they use three-dimensional ordered macroporous carbon as scaffold[49]. In addition, several authors obtain porosity from micro to mesoscale, which contributes to reach values of surface area up to 1200 m2/g[50], but they do not distinguish between them and detailed observation of adsorption isotherms shows nitrogen volume adsorbed at low relative pressure, indicating that micropores contribute to the total surface area, in some cases around 50 % [51, 52]. In this work, TiO2 materials have surface areas due to mesopores exclusively. In summary, the phage was successfully used as template for the synthesis of TiO2 mesopores with narrow pore size distribution, although the synthesis of TiO2 with ordered pores was limited by the rate of reaction of the precursor. To solve this limitation, water stable precursors such as titanium(IV) bis(ammonium lactate) dihydroxide (TiBALDH) could be used. It is also possible to modify the phage capsid to have functional groups to control its isoelectric point and therefore its three-dimensional arrangement. Moreover, the phage diameter and length could be controlled by DNA engineering, in order to synthesize tailored mesostructures. This genetic or chemical versatility of the capsid could allow the synthesis of crystalline products to room temperature, opening new possibilities for the use of these filamentous viruses as templates for the synthesis of diverse mesoporous structures and organized materials.
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We demonstrate that mesoporous TiO2 structures with crystalline walls can be obtained using a biological semi-hard template. M13 phage has advantages under soft templates due to its shape like a rod is highly stable and avoids disturbance, which is reflected in the lack of micropores. This finding could be helpful in terms of applications, where micropores are responsible for the poisoning of the material, diminishing its efficiency. This biological template controls the condensation alkoxide in the sol-gel process making possible the synthesis of mesopores with adequate control of the pore diameter adjusting only the pH. Most remarkably, the products exhibit exceptional thermostability of the anatase phase. This is the predominant phase even after heated at 800 ºC accompanied by an increase in the pore and crystal size. The versatility physicochemical properties of the phage and its high resistance to various chemical environments provides a template with remarkable adaptability to different requirements for the synthesis of porous materials. Acknowledgments A. H-G. gratefully acknowledge the support from Consejo Nacional de Ciencia y Tecnología (CONACyT) for the scholarship granted in pursuit of his doctoral studies (265459). We thank Laboratorio Central de Microscopía Electrónica UAM-I for TEM images, Laboratorio de Difracción de Rayos X (T-128) UAM-I for XRD
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measurements and Laboratorios de Docencia (T-044) for the thermogravimetric analysis. Dr. Marcos Esparza-Schulz is acknowledged for the nitrogen isotherm adsorption/desorption experiments.
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Appendix A. Supplementary data Supplementary data related to this article can be found at References
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Table 1. Summary of some synthesized TiO2 using the M13 phage and calcined at 500 °C. Sample [Ti]/ Final DCrystal/ SBET/ Dp(NLDFT) / VT/ -1 mol*L pH nm m2*g-1 nm cm3*g-1 Ti1.2_0.1b 0.067 4.9 130 6.1 0.19 8.1 Ti1.0_0.1c 0.163 5.0 132 6.1 0.21 9.3 Ti1.5_0.1a 0.034 6.0 78 8.2 0.18 12.7 Ti1.5_0.1b 0.067 6.0 85 8.2 0.19 16.0 Ti1.5_0.05b 0.067 6.5 83 8.2 0.21 16.3 Ti1.0_6.0a 0.163 22 31.3 [Ti] alkoxide concentration in alcoholic solution, DCrystal anatase crystal diameter by Scherrer equation, SBET BET specific surface area, Dp(NLFDT) average pore diameter by NLDFT, VT total pore volume.
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Figure 1. Thermal gravimetric (continue line) and differential scan calorimetry (discontinue line) analysis of the Ti1.0_0.1c sample. Figure 2. Nitrogen adsorption/desorption isotherm and pore size distribution of the samples a)-b) Ti1.0_0.1c and c)-d) Ti1.5_0.1b.
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Figure 3. a) and b) Representation of assembly of protein p8 of phage capsid, DNA is omitted (PDB 2MJZ), in purple α-helix residues, in cyan solvent-exposed residues. c) Detail of the first eight residues of protein p8; horizontal arrows indicate the carboxylic groups, vertical arrows indicate amine groups. Hydrogen atoms were omitted for clarity. Visualizations made using VMD.[45]
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Scheme 1. Proposed Mechanism for the formation of mesoporous TiO2 using the semi-hard template M13.
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Figure 4. Nitrogen adsorption/desorption isotherms and pore size distribution of samples a)-b) Ti1.0_3.0b; c)-d) Ti1.0_1.0c; e)-f), Ti1.2_0.10b. Figure 5. TEM image of samples after calcination a) Ti1.2_0.10b, b) Ti1.0_1.0c. Figure 6. Nitrogen adsorption/desorption isotherm and pore size distribution of samples prepared with different phage concentration: a)-b) Ti1.5_0.1a (0.10 mg/mL); c)-d) Ti1.5_0.05b (0.05 mg/mL).
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Figure 7. X-ray powder diffraction patterns of TiO2 mesostructures using M13 phage as template. Synthesis details are shown in Table 1. Triangle and circle designate anatase and rutile phases respectively.
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Figure 8. Nitrogen adsorption/desorption isotherms and pore size distribution of samples a), b) Ti1.0_0.1c and c), d) Ti1.5_0.1b calcined at 650 °C (circles) and 800°C (triangles).
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The bacteriophage M13 was used as a semi-hard template for the synthesis of mesoporous TiO2 pH and capsid proteins can control the sol-gel reaction to give distinct pore size distribution Microporosity was avoided because of the very stable assembly of the virus
S1387-1811(18)30258-0
DOI:
10.1016/j.micromeso.2018.05.014
Reference:
MICMAT 8916
To appear in:
Microporous and Mesoporous Materials
Received Date: 28 March 2018 Revised Date:
8 May 2018
Accepted Date: 10 May 2018
Please cite this article as: A. Hernández-Gordillo, A. Campero, L.I. Vera-Robles, Mesoporous TiO2 synthesis using a semi-hard biological template, Microporous and Mesoporous Materials (2018), doi: 10.1016/j.micromeso.2018.05.014. 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 proof before it is published in its final 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.
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Mesoporous TiO2 synthesis using a semi-hard biological template Armin Hernández-Gordillo*a,b, Antonio Camperoa, L. Irais Vera-Robles*b a
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Departamento de Química, Área de Química Inorgánica, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco No.186, Col. Vicentina, 09340 CDMX, Mexico b Departamento de Química, Área de Biofisicoquímica, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco No.186, Col. Vicentina, 09340 CDMX, Mexico. *
Corresponding authors: [email protected] (L.I. V-R.); [email protected]
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(A. H-G.)
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The bacteriophage M13, a rod-shaped virus, was used as a semi-rigid template to synthesize crystalline mesoporous TiO2. This strategy allowed us to modulate solgel reactions on the virus surface without the need of chemical or genetic manipulation. Important parameters as pH influence and concentration were investigated. The studies by which the pH was varied were the principal route to obtain materials with pores of around 6.1 and 8.2 nm. When an M13 phage concentration of 0.1 mg/mL was employed, anatase mesopores presented specific surface areas from 80 to 130 m2/g and pore volumes from 0.18 to 0.21 cm3/g. It should be mentioned that surface area is due exclusively to mesopores, and from N2 adsorption/desorption experiments, no evidence of micropores was detected. This absence of micropores results from the stable protein arrangement on M13 phage, which contrasts with the disruption of micellar assemblies of soft templates. These results show that anatase mesophases are obtained in an easy and rapid route at room temperature and could be expanded to synthesize other metal oxide mesostructures tuning M13 phage properties.
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Keywords Mesoporous, TiO2, Template, Virus M13 1. Introduction
Titanium dioxide is a material with multiple uses in environmental and energy fields.[1] Applications of TiO2 are closely related to its physicochemical properties such as size, porosity, crystallinity, etc. Thus, its adequate control is an important field of research. Of special interest are TiO2 mesostructures not only because the homogeneous pore size distribution and larger surface area but also the homogeneous distribution of active sites through the mesoporous network. In comparison, with bulk or nanometric particles, mesoporous materials facilitate their recovery and recycling, moreover, they are friendly with the environment due to low cytotoxicity in living organisms.[2] Thus, synthesis of mesoporous TiO2 is a subject
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of study in development. Many efforts have been focused on finding suitable templates for a strict control on the shape and size of the pores, and at the same time producing organized structures. These kinds of materials are obtained mainly by two approaches, namely the soft-templating and the hard-templating.
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The soft-templating route was originally proposed for the synthesis of silica mesopores. Surfactants are used to form micellar arrangements on which condensation reactions of silicon alkoxides are carried out to form SiO2 mesopores of various sizes and shapes.[3] However, the adaptation of this method to the synthesis of TiO2 has some issues due to the high chemical reactivity of the precursors and the disruption of the micellar arrangement, producing high microporosity.[4] Another drawback is the collapse of the porous network during the elimination of the template by thermal treatment, however, this step is necessary to obtain crystalline structures.[5] To overcome these issues, variations in the methodology have been proposed, for example, improving the micelle formation by using evaporation-induced self-assembly (EISA) where the solvent is evaporated slowly at mild temperatures,[6, 7] also by modifying the atmosphere during the thermal treatment (crystallization process)[8] or exploring nonconventional surfactants like ionic liquids crystals among others[9].
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In contrast, the use of hard templates (preformed mesoporous solids) has been successful to produce crystalline mesopores of TiO2 with novel structures.[10] Unlike the soft-templating method, this one allows the use of highly hydrolyzable precursors in extreme synthesis conditions. In addition, it prevents the collapse of mesopores even to high temperatures, both porous and crystalline frameworks are preserved.[11, 12] However, the main problems with this method are the excessive time employed in order to obtain the rigid template and its further elimination.
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In the last years, new molecules have been proposed as templates to create mesostructures.[13] Specifically, viruses which are genetic and protein materials hierarchically assembled have been used with this purpose, producing mesoporous silica with cylindrical[14, 15] or icosahedral[16] pores depending on the shape of the virus. In particular, filamentous viruses Ff (Ff phages) have been used as template because of their easy replication and rapid purification.[17] These rod-shaped viruses (6.6 nm in diameter) are semi-flexible.[18] Inside of rod is located the DNA molecule, outside is formed mainly for hundreds of subunits of protein p8 (α-helix, 50 residues) that cover the whole longitudinal body of the phage (each protein covers ~2.3 nucleotides)[19], as well as other four types of proteins cover the ends.[20] The Ff phages are stable between pH values 3−11 and tolerate up to 80 °C to neutral pH.[19, 21, 22] Their resistance to organic solvents depend on their polarity, due to the hydrophobic residues in the protein p8. Ff phages are stable in aqueous mixtures that contain methanol, ethanol, or isopropanol up to 60, 50 and 30% (v/v) respectively.[23] Ff phages exhibit liquid crystal behavior in function of their concentration,[24, 25] adopting a nematic phase even at concentrations as low as 0.1 mg/mL.[26] Phage organization also depends on its length, pH and ionic strength of the medium.[27] This Ff phages family includes M13, fd, and f1, which share similar physicochemical properties.[19, 20] In
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particular, M13 phage particles have an isoelectric point of 4.3[28]. Then, they are able to aggregate at pH < 4.4, organize at 4.7 ≤ pH ≤ 5.5 or repel each other at pH > 5.5 with the concomitant change on the surface charge of M13 phage.[29-31] In comparison with micellar arrangements, Ff phages have a very stable assembly of p8 proteins and their tubular morphology is under genetic control; neither solvents nor precise concentrations or temperatures can alter their shape, which allows us to classify them as a semi-hard template. Moreover, their capacity to tolerate mixtures that contain a high amount of alcohols facilitates their use in sol-gel reactions.
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Silica ordered mesopores are the only structures reported by using M13 phage as semi-rigid template.[14, 32] Then, the use of M13 phage to obtain TiO2 porous structures is being explored. For example, layer by layer technique was performed to manufacture TiO2 film using as platform M13 phage which was previously engineered with three glutamates to encourage the electrostatic interaction between phage and (NH4)2TiF6 precursor. These reactions are carried out to 50 ºC by several hours, followed by calcination.[33] Other approach to synthesize TiO2 nanowires was obtained employing the same genetically engineered M13 phage as scaffold and Ti(OBu)4 as precursor. Low temperature (-40 ºC) was necessary to control the high reactivity of the precursor. These nanowires were mixed with a sacrificial polymer and deposited on a substrate.[34] In both cases, the only function of the phage was molding the nanowires, which created the porous network as a result of space between them resulting in a wide pore size distribution. Thus, pore size was not related to the diameter of the phage. In spite of the fact some success has been recently achieved, still, it is a challenge to use M13 phage to obtain fast and easily TiO2 mesoporous structures.
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Herein, we optimized a methodology to synthesize TiO2 mesoporous structures with anatase pore walls. The novelty of this approach is to use a surfactant-like molecule that naturally is assembled as a cylindrical micelle, looking more like a semi-hard template, namely the bacteriophage M13. Our main goal was to adapt the synthesis conditions to the chemical nature of the native phage, without genetic or chemical manipulation and at room temperature. Due to a large number of variables to control, we adopted a method inspired by the genetic algorithm that allowed us to explore more general reaction conditions. Based on this approach we obtain mesoporous TiO2 and we observed that not only M13 concentration but pH is a vital parameter to control pore size. As a result, the average pores sizes of these materials were around of 6.1 and 8.2 nm with specific surfaces areas of ∼130 and 80 m2/g, respectively. 2. Experimental 2.1 Phage replication and purification The M13 phage used in this work was derived from vector M13mp18 (New England BioLabs). These phages have a length of ~995 nm and ~3150 p8 proteins, corresponding to the number of nucleotides of the vector (7249 bases). For phage amplification, 5 mL of an overnight culture of E. coli (XL1-Blue strain) was diluted 100 times in a flask (2 L) with 2xYT medium broth (Invitrogen)
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previously sterilized. The diluted culture was infected adding a stock solution of phage (~1014 pfu) and incubated for 16 h at 32 °C and 200 rpm to maximize the yield. Tetracycline was added to all media before incubation (10 µg/mL). After incubation, the culture was centrifuged for 10 min, 6000 rpm at 4 °C (Avanti J-30I centrifuge, Beckman Coulter), the supernatant was recovered and mixed with polyethylene glycol (PEG, ~8000 g/mol) and NaCl to a final concentration 20% w/v and 2.0 M respectively, stored for 1 h at 4 °C and centrifuged for 15 min, at 8000 rpm and 4 °C. The pellet was resuspended in distilled water and the precipitation with PEG-NaCl was repeated once again. Finally, to ensure the recuperation of fully assembled phage and discard PEG and salt residues, the phage was precipitate by isoelectric point adjusting the pH to 4.3, stored and centrifuged, the pellet was resuspended in water and neutralized with dilute NaOH. Quantification of phage was performed by spectrophotometry (NanoDrop 2000, Thermo Scientific), considering the specific absorptivity coefficient at 269 nm as 3.84 cm2*mg-1 for Ff bacteriophages,[35] the path length was automatically adjusted to avoid dispersion.
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2.2 Synthesis of TiO2 In general, reactions were as follows: titanium isopropoxide (97 %, Sigma-Aldrich) was added in absolute ethanol previously acidified with concentrated hydrochloric acid. The molar ratio between the alkoxide and ethanol (Ti:OH) was calculated from the maximum amount of ethanol supported by the phage (50% v/v). This alcoholic solution was added to a phage solution keeping vigorous agitation and aging for 10 minutes. Then, the mixture was then centrifuged for 15 min (12000 rpm), the gelatinous pellet was washed with ethanol technical grade (2 x 10 mL) and dried at 50 °C. All samples were calcined at 500 °C for 2 h with a heating rate of 1 °C/min. To find the optimal conditions for the synthesis of mesoporous TiO2 (molar ratios, phage concentration, and pH), we used a methodology inspired by the genetic algorithm which is presented in Appendix A (Figure S1). The nomenclature for samples is as follow: TiX_Yz were Ti refers the TiO2, X means the pH of the alkoxide solution, Y means the phage concentration in mg/mL and finally, z means the Ti:OH molar ratio being z: a=100, b=250, c=500.
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2.3 Material characterization Removal template was monitored by thermal gravimetric analysis (Pyris Diamond, Perkin Elmer) heating in air (using nitrogen as the carrier gas, 50 mL/min) a dry sample from 25 to 500 °C at a heating rate of 1 °C/min, held at this temperature for 2 h and finally heated to 1000 °C. The synthesized TiO2 was observed by transmission electron microscopy (JEOL 2000), samples were suspended in water, and an aliquot of 2 µL was placed on a copper grid covered with carbon type-B (300 mesh). X-ray diffraction patterns were recorded using a Bruker D8 Advance diffractometer (λ=1.54 Å), measurement intervals were 10-70° and 0.6-7° in 2θ for normal and low angle measurements respectively. The crystallite size was estimated by applying the Scherrer equation using the principal diffraction peak corresponding to the plane (101) of anatase and the Scherrer constant K=0.9.[36] Raman spectroscopy was used to study the vibrational features of the materials; the Raman spectra were acquired using a Horiba-Jobin Yvon LabRam
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800 system equipped with an Olympus BX40 confocal microscope. Nitrogen adsorption/desorption measurements at 77 K were performed on an ASAP 2020 (Micromeritics) volumetric adsorption analyzer; before the measurements, the samples were outgassed for 12 h at 373 K. The specific surface area of solid samples was calculated by multiple-point Brunauer–Emmett–Teller (BET) method in the relative pressure range of P/P0 = 0.05–0.25. Pore volume was calculated at P/P0=0.99. Pore size distribution curves were computed using the non-local density functional theory (NLDFT) method, (using the nitrogen adsorption branch, assuming cylindrical pores on silica). T-plot (P/P0 = 0.2–0.5) was used to calculate the microporosity in samples with the high specific surface area.
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3. Results and discussion
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Synthesis of mesoporous structures depends on the kind of template, in this work the template is a semi-rigid structure (M13 phage), whose cylindrical shape is inherent, but liquid crystal arrangements are a function of the concentration, pH, and temperature, among others. On the other hand, the width of pore wall can be related to the precursor amount. Molar ratios Ti alkoxide:protein p8 (Ti:p8) larger than 500:1 produced big aggregates instantaneously. On the contrary, small amounts of precursor were able to diffuse across the liquid and reach surface phages, but it was not enough to completely cover them, as we observed from the UV-vis spectrum (not shown). Then, we set the molar ratio Ti:p8 100:1, to have enough Ti species and to control its condensation and build the pore walls.
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Thermogravimetric (TG) and differential scanning calorimetry (DSC) analysis were performed to determine the content of phage template and the stability of TiO2 phases. TiO2 material showed a loss in weight between 200−500º C due to the decomposition of protein and nucleic acids coming from phage, this represents around 30.0%, however, this value is below the expected (∼40%) suggesting that not all phage was trapped in the final network. It must be noted that TGA analysis was made in a similar way to that of the calcination step, in other words when sample reached 500 ºC the temperature was kept constant for two hours and we observed still a loss in weight (1.0 %) due to the thermal stability of DNA. Then, the temperature was raised up to 1000 ºC and no change in mass was observed. This demonstrated that keeping the temperature to 500 ºC for two hours suffices to eliminate completely the semi-hard template. From the Figure 1, two exothermic peaks to 430 and 960 ºC are registered, which should correspond to phase transition for anatase and rutile, respectively, as we will demonstrate below. 3.1 Importance of pH in the alkoxide solution While solubility of titanium species is favored to low pH (< 3), reducing the rate condensation of Ti alkoxides[37], M13 phages are spontaneously assembled in two-dimensional structures in a narrow interval of pH (4.7−5.5). Outside this range of pH, competitive electrostatic interactions between viral particles result in their aggregation or repulsion. Taking into account this behavior, experiments were
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conducted to different pH values and setting phage concentration as low as 0.10 mg/mL. When the pH of the alkoxide solution was > 3.0, the sol-gel reaction was very rapid, developing big aggregates that did not interact with the surface phage. Conversely, when the pH was < 1.0, titanium clusters formed very stables sols and no interaction with M13 particles was observed, instead, M13 was agglomerated due to pH (pH
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Therefore, TiO2 materials were obtained and characterized, starting from alkoxide solution adjusted to 1.0 and 1.5 pH values. Synthesis to pH 1.0 (sample Ti1.0_0.1c), produced mesoporous structures with a specific surface area of 132 m2/g and an H2b hysteresis cycle type (Figure 2a), where the desorption process is affected by blocking effects along the pore, caused probably by a partial collapse during calcination step. This sample also showed a narrow pore size distribution (Figure 2b) with an average pore diameter of 6.1 nm in agreement with the phage diameter. When pH was slightly increased to 1.5 (sample Ti1.5_0.1b), the specific surface area decreased to 85 m2/g and the hysteresis cycle change to type H1 (Figure 2c), indicating that this network is made by regular cylindrical pores with open ends whose average pore size is 8.2 nm (Figure 2d), suggesting a higher thermal stability of the walls in the structure.
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In spite that the M13 phage has a well-established structure because of the ionic interactions between proteins and DNA, environmental conditions can affect its size. When M13 phage is dried its diameter size decreases to 5.5 nm, permitting a diameter expansion up to 9.0 nm in solution.[38, 39] Besides, the solvent polarity is a factor involved in the p8 protein packaging.[31] As observed in Figure 3, Nterminal residues are exposed to the solvent (cyan color) and have random coil structure, which means that they can move freely, on the contrary of the residues that comprise the α-helix structure (purple color). Thus, this freedom in motion should be the responsible for the increase in the phage diameter and therefore would explain the pore diameter of 8.2 nm. In fact, α-helix structure stability was verified by infrared spectroscopy before calcination. Amide I and amide II appear at 1648, and 1540 cm−1, respectively (Figure S2).[40, 41] Nonetheless, the possible encapsulation of bundles phages cannot be discarded. Interestingly, when the sol-gel reaction was performed without M13 phage, a very stable sol was obtained, whereas in presence of the phage an immediate formation of fibrous particles was observed (TiO2 covered phages).[42] This indicates that the alkoxide in acidic conditions only polymerizes in the presence of phage, confirming its role as template. The catalytic effect of the phage on titanium oxide condensation may be caused by the lateral groups of the residues on the major coat protein. Figure 3c shows these eight superficial residues for the p8 protein (AEGDDDPAK), where the carboxylate (from glutamate and aspartate) and amine groups (from the N-terminal and lysine) can interact with titanium species promoting their reactivity.[43, 44]. In this way, small titanium clusters with positive charge surrounded the negatively charged surface of phages (Scheme 1b). In all
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experiments under acidic conditions without phage (negative control) it was never observed the formation of a precipitate, even after aging the sample for one week (Scheme 1a).
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3.2 Effect of phage concentration
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As can be seen in both cases, mesoporous structures were obtained according to adsorption isotherms type IVa. Interestingly, no sorption to low relative pressure was observed because of the absence of micropores, in contrast with surfactants (soft template), which can form disrupted micellar arrangements producing microporous cavities when trapped in the oxide framework; phage particles do not disassemble because of the interaction protein-DNA, and their use as template leads exclusively to mesoporous materials.
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Previously, it has been demonstrated that silica mesostructures were obtained when phage concentration is ≥ 4 mg/mL[14, 32], but this is not valid for metal alkoxides because their high hydrolysis rate and moreover their condensation is raised by the presence of the phage, as we discussed above. Thus, the effect of phage concentration was tested to study their liquid-like crystal behavior and its templating effect in the synthesis of ordered mesopores. When phage concentrations ˃ 0.1 mg/mL were used, sol-gel reactions were catalyzed by p8 proteins, avoiding a proper homogenization even to pH as low as 1.0. This was evidenced by the decrease of the specific surface area when increased the concentration of phage, producing specific surface areas of 130, 67, 55 and 22 m2/g for concentrations of 0.10, 1.00, 3.00 and 6.00 mg/mL respectively.
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Figure 4a and c display the nitrogen adsorption/desorption isotherms for samples Ti1.0_3.0b and Ti1.0_1.0c obtained at 3.00 and 1.00 mg/mL respectively, which are similar in shape. In both cases, adsorption continues up to relative pressures of 1.0, showing a hysteresis cycle of type H3 suggesting the existence of macropores. According to our hypothesis, TiO2 formation is undertaken on the surface of bundles of phages, inhibiting the complete diffusion of the titanium precursor. This aggregates of TiO2 can be analyzed as plates more than as mesoporous structures, with no well-defined pore size distribution (Figure 4b and d). When the phage concentration was higher (sample Ti1.0_6.0a), it was not possible to control the reaction and no porosity can be assigned to the material. In contrast, sample Ti1.2_0.1b synthesized to lower phage concentration (0.10 mg/mL) exhibited a marked hysteresis cycle type H2a (Figure 4e), typical of mesoporous structures and free of micropores according to the t-plot of samples (Figure S3) with an average pore diameter of 6.1 nm (Figure 4f) and narrower pore size distribution. To gain insight information about the framework, samples were studied by transmission electron microscopy (TEM). In the Figure 5a, the sample Ti1.2_0.1b shows pores 6.5 nm in size according to the pore size distribution (Figure 4f), whereas, in sample Ti1.0_1.0c porous channel layouts of 8.0 nm in size were distinguished (Figure 5b) in agreement with the maximum phage diameter. This
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confirms liquid-like crystal behavior for the native phage and more importantly its role as a semi-hard template to obtain mesopores of metal oxides. In spite that the phage acted as template, it was not able to create perfect ordered structures, as was demonstrated by low-angle X-ray diffraction (XRD), where only a low-intensity peak was visible in sample Ti3.0_1.0b (Fig. S4) whereas in sample Ti1.2_0.1b this signal disappeared. Thus, we can obtain a local order of pores and macropores when phage concentrations ≥ 0.10 mg/mL are used (Scheme 1d), and disordered pores with a narrow pore size distribution if low concentration is used (Scheme 1c), in line with the self-organization of M13 phage.
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Low phage concentrations were able to produce materials with two pores sizes, as mentioned in section 3.1, a possible explanation could be that two or more phages are trapped between TiO2 walls. To investigate this hypothesis, we used the overlap concentration (C*). This concentration C* (0.05 mg/mL for Ff phage family [46]) ensures that particles do not interact with each other and only individual virions must be trapped in the TiO2 network. The material obtained at this C* showed an average pore size of 8.2 nm. Noticeably, bigger concentrations (0.10) gave the same results, implying that even to this phage concentration particles are so scarce that they do not overlap. Thus, phage agglomeration is discarded and the possible explanation should be the shrinking-swelling of p8 proteins provoked by small pH variations and cosolvent amount.
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Nitrogen adsorption/desorption characterization of these mesoporous structures (Ti1.5_0.1a and Ti1.5_0.05b) was performed. Figures 6a and c show practically the same nitrogen adsorption/desorption isotherms and hysteresis cycles of type H1, typical of regular cylindrical pores with the ends accessible to the gas molecules, describing perfectly the phage geometry, which is reflected in good pore homogeneity. The collapse of the pores is avoided by the continuous and smooth template removal by calcination (Figure 1). So, samples Ti1.5_0.1a and Ti1.5_0.05b present similar specific surface areas of 78 and 83 m2/g, respectively.
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Although three-dimensional arrays have been observed in phage solutions at concentrations ≥ 0.10 mg/mL,[26] with the synthesis method used here, concentrated phage solutions were not suitable to obtain ordered mesopores, due to the catalytic effect of p8 protein on titanium precursor. Therefore, it can be concluded that the maximum phage concentration for the synthesis of mesopores with a narrow pore size, using alkoxides, is 0.10 mg/mL. Furthermore, all experiments at pH ∼1.5 produces invariably mesopores with an average diameter of 8.2 nm. 3.3 Pore wall thickness and thermal stability The phage has a tunable diameter because of the flexibility of p8 protein, this reveals important information about the two pore size distributions obtained. Another parameter to explain this result is related to the crystal size of the walls of the network. Thus, crystalline structure and size of different samples were analyzed using the X-ray diffraction. After calcination at 500 ºC for two hours, TiO2
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samples obtained using smaller phage concentration show diffraction peaks corresponding to anatase phase, however, if phage concentration is increased (6 mg/mL, sample Ti1.0_6.0a), a new peak to 27.5° in 2 theta is assigned to the rutile phase (Figure 7). Crystal size was calculated according to Scherrer equation and results are shown in Table 1, it can be noted that mesoporous structures increase the crystal size as surface area decreases.
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From Table 1, it can be observed that mesoporous materials of 6.1 nm in diameter have higher specific surface area than those of 8.2 nm. It would seem that either the number or the length of pores are different in the samples. However, as pore volume is almost constant (0.18−0.21 cm3/g) and the same number of phages were used in all samples (molar ratio Ti:p8), it is more viable attributing this behavior to the width of the wall because the same number of pores can produce lighter or heavier materials. Even, when phage concentration is 6.00 mg/mL the sample is no longer porous and two TiO2 phases co-habit. This phase transition is associated with the crystal size so that the increment in crystal size yields a conversion to rutile phase at a lower temperature.[47]
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Once the mesoporous structures were obtained at 500 ºC, we study the influence of temperature to gain more information about the crystallization process. Samples Ti1.0_0.1c and Ti1.5_0.1b were heated to 650 and 800 ºC and evaluated by DRX, N2 adsorption/desorption, and Raman spectroscopy. Raman spectra of sample Ti1.0_0.1c heated to 500, 650 and 800 ºC show bands to 141, 397, 515 and 637 cm-1, characteristic of anatase phase, no bands corresponding to proteins or ssDNA groups are evident, confirming a whole elimination of the template. Sample Ti1.5_0.1b presents same behavior to 500 and 650º C, however, an increase in calcination temperature to 800º C revealed two new bands to 445 and 615 cm-1 distinctive of rutile structure (Figure S5). This rutile transition also is consistent with the correlation between the crystal size and the transition temperature as it was seen in sample Ti1.0_6.0a.
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Thermal stability of mesostructures was tested for Ti1.0_0.1c and Ti1.5_0.1b, representative samples for pore size of 6.1 and 8.2 nm respectively. After calcination at 650 °C for 2 h, the specific surface area for both samples decreases to ∼30 m2/g and remains a hysteresis cycle (type H1) (Figure 8 a and c). On the contrary to silica mesostructures, where walls shrink, TiO2 walls sinter partially increasing the pore size up to 26 and 20 nm for Ti1.0_0.1c and Ti1.5_0.1b respectively (Figure 8b and d). When samples were calcined at 800 °C, surface area of Ti1.0_0.1c sample decreased to 11 m2/g without phase transition, whereas Ti1.5_0.1b sample practically loses all mesoporosity (4 m2/g), due to extended sintering induced by anatase to rutile phase transition giving as results bigger crystal as was observed in TEM (Fig. S6).
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4. Conclusions
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In addition, our strategy to use M13 phage as semi-hard template results in homogeneous mesoporous materials having specific surface areas of about 80 and 130 m2/g, and average pore diameters of 8.2 and 6.1 nm, respectively. These values agree with those found in the literature for TiO2 mesoporous structures, however, authors take advantage of the slow evaporation of THF solvent[48] or they use three-dimensional ordered macroporous carbon as scaffold[49]. In addition, several authors obtain porosity from micro to mesoscale, which contributes to reach values of surface area up to 1200 m2/g[50], but they do not distinguish between them and detailed observation of adsorption isotherms shows nitrogen volume adsorbed at low relative pressure, indicating that micropores contribute to the total surface area, in some cases around 50 % [51, 52]. In this work, TiO2 materials have surface areas due to mesopores exclusively. In summary, the phage was successfully used as template for the synthesis of TiO2 mesopores with narrow pore size distribution, although the synthesis of TiO2 with ordered pores was limited by the rate of reaction of the precursor. To solve this limitation, water stable precursors such as titanium(IV) bis(ammonium lactate) dihydroxide (TiBALDH) could be used. It is also possible to modify the phage capsid to have functional groups to control its isoelectric point and therefore its three-dimensional arrangement. Moreover, the phage diameter and length could be controlled by DNA engineering, in order to synthesize tailored mesostructures. This genetic or chemical versatility of the capsid could allow the synthesis of crystalline products to room temperature, opening new possibilities for the use of these filamentous viruses as templates for the synthesis of diverse mesoporous structures and organized materials.
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We demonstrate that mesoporous TiO2 structures with crystalline walls can be obtained using a biological semi-hard template. M13 phage has advantages under soft templates due to its shape like a rod is highly stable and avoids disturbance, which is reflected in the lack of micropores. This finding could be helpful in terms of applications, where micropores are responsible for the poisoning of the material, diminishing its efficiency. This biological template controls the condensation alkoxide in the sol-gel process making possible the synthesis of mesopores with adequate control of the pore diameter adjusting only the pH. Most remarkably, the products exhibit exceptional thermostability of the anatase phase. This is the predominant phase even after heated at 800 ºC accompanied by an increase in the pore and crystal size. The versatility physicochemical properties of the phage and its high resistance to various chemical environments provides a template with remarkable adaptability to different requirements for the synthesis of porous materials. Acknowledgments A. H-G. gratefully acknowledge the support from Consejo Nacional de Ciencia y Tecnología (CONACyT) for the scholarship granted in pursuit of his doctoral studies (265459). We thank Laboratorio Central de Microscopía Electrónica UAM-I for TEM images, Laboratorio de Difracción de Rayos X (T-128) UAM-I for XRD
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measurements and Laboratorios de Docencia (T-044) for the thermogravimetric analysis. Dr. Marcos Esparza-Schulz is acknowledged for the nitrogen isotherm adsorption/desorption experiments.
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Appendix A. Supplementary data Supplementary data related to this article can be found at References
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Table 1. Summary of some synthesized TiO2 using the M13 phage and calcined at 500 °C. Sample [Ti]/ Final DCrystal/ SBET/ Dp(NLDFT) / VT/ -1 mol*L pH nm m2*g-1 nm cm3*g-1 Ti1.2_0.1b 0.067 4.9 130 6.1 0.19 8.1 Ti1.0_0.1c 0.163 5.0 132 6.1 0.21 9.3 Ti1.5_0.1a 0.034 6.0 78 8.2 0.18 12.7 Ti1.5_0.1b 0.067 6.0 85 8.2 0.19 16.0 Ti1.5_0.05b 0.067 6.5 83 8.2 0.21 16.3 Ti1.0_6.0a 0.163 22 31.3 [Ti] alkoxide concentration in alcoholic solution, DCrystal anatase crystal diameter by Scherrer equation, SBET BET specific surface area, Dp(NLFDT) average pore diameter by NLDFT, VT total pore volume.
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Figure 1. Thermal gravimetric (continue line) and differential scan calorimetry (discontinue line) analysis of the Ti1.0_0.1c sample. Figure 2. Nitrogen adsorption/desorption isotherm and pore size distribution of the samples a)-b) Ti1.0_0.1c and c)-d) Ti1.5_0.1b.
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Figure 3. a) and b) Representation of assembly of protein p8 of phage capsid, DNA is omitted (PDB 2MJZ), in purple α-helix residues, in cyan solvent-exposed residues. c) Detail of the first eight residues of protein p8; horizontal arrows indicate the carboxylic groups, vertical arrows indicate amine groups. Hydrogen atoms were omitted for clarity. Visualizations made using VMD.[45]
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Scheme 1. Proposed Mechanism for the formation of mesoporous TiO2 using the semi-hard template M13.
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Figure 4. Nitrogen adsorption/desorption isotherms and pore size distribution of samples a)-b) Ti1.0_3.0b; c)-d) Ti1.0_1.0c; e)-f), Ti1.2_0.10b. Figure 5. TEM image of samples after calcination a) Ti1.2_0.10b, b) Ti1.0_1.0c. Figure 6. Nitrogen adsorption/desorption isotherm and pore size distribution of samples prepared with different phage concentration: a)-b) Ti1.5_0.1a (0.10 mg/mL); c)-d) Ti1.5_0.05b (0.05 mg/mL).
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Figure 7. X-ray powder diffraction patterns of TiO2 mesostructures using M13 phage as template. Synthesis details are shown in Table 1. Triangle and circle designate anatase and rutile phases respectively.
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Figure 8. Nitrogen adsorption/desorption isotherms and pore size distribution of samples a), b) Ti1.0_0.1c and c), d) Ti1.5_0.1b calcined at 650 °C (circles) and 800°C (triangles).
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The bacteriophage M13 was used as a semi-hard template for the synthesis of mesoporous TiO2 pH and capsid proteins can control the sol-gel reaction to give distinct pore size distribution Microporosity was avoided because of the very stable assembly of the virus