Biomimetic Sol–Gel Synthesis of TiO2 and SiO2 Nanostructures

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Biomimetic Sol−Gel Synthesis of TiO2 and SiO2 Nanostructures Armin Hernández-Gordillo,†,‡ Andrés Hernández-Arana,† Antonio Campero,‡ and L. Irais Vera-Robles*,† Departamento de Química, Á rea de Biofisicoquímica, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco No. 186, Col. Vicentina, 09340 México D.F., Mexico ‡ Departamento de Química, Á rea de Química Inorgánica, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco No.186, Col. Vicentina, 09340 México D.F., Mexico †

S Supporting Information *

ABSTRACT: We report the heptapeptide-mediated biomineralization of titanium dioxide nanoparticles from titanium alkoxides. We evaluated the influence of pH on the biomineralized products and found that nanostructured TiO2 was formed in the absence of external ions (water only) at pH ∼ 6.5. Several variants (mutants) of the peptides with different properties (i.e., different charges, isoelectric points (pIs), and sequences) were designed and tested in biomineralization experiments. Acid-catalyzed experiments were run using the H1 (HKKPSKS) peptide at room temperature, which produced anatase nanoparticles (∼5 nm in size) for the first time via a heptapeptide and sol−gel approach. In addition, the peptide H1 was used to synthesize SiO2 nanoparticles. The influence of the pH and the added ions were monitored: at higher pH levels (8−9), SiO2 nanoparticles (20−30 nm in size) were obtained. In addition, whereas borate and Tris ions allowed the formation of colloidal systems, phosphate ions were unable to produce sols. The results presented here demonstrate that biomineralization depends on the sequence and charge of the peptide, and ions in solution can optimize the formation of nanostructures.

1. INTRODUCTION During the past decade, biomolecules have been a focus of basic research and technological applications, especially in fields related to biomineralization and biomolecule-based nanomaterials. Proteins and peptides are the most important building blocks (BBs) used to create novel nanomaterials because they have outstanding properties, including the ability for molecular recognition, hierarchic organization, and self-assembly, among others. Furthermore, all these characteristics are under precise genetic control, which provides the advantage of enabling their modification through genetic-engineering techniques. In nature, these BBs can recognize, bind and induce biomineralization of specific inorganic materials. Moreover, proteins and peptides use ions available in the aqueous environment at room temperature, atmospheric pressure, and approximately neutral pH. Consequently, their use as biotemplates offers an environmentally friendly alternative route to the synthesis of nanomaterials. One of the most studied structures made by organisms are silica structures, which are formed from silicatein1−3 (from Euplectella aspergillum) and silafin4−6 (from Cylindrothecafusiformis) proteins. These proteins are able to catalyze and polymerize intricate shapes of SiO2, and they have been widely used to induce mineralization in vitro since their discovery. In addition, silafin and its related R5-peptide have been shown to be able to induce the formation of amorphous TiO2 structures.6 Moreover, Kröger et al.7 have reported using recombinant © 2014 American Chemical Society

native silafin (rich in K and R residues) as biotemplates and titanium(IV) bis-(ammonium lactato)-dihydroxide (TiBALDH) as a source of titanium to promote the formation of rutile-structured TiO2 at room temperature. They also constructed several mutants (shorter in length or richer in D and E residues than wild-type silafin), which were unable to produce crystalline structures. They concluded that the distribution of positive charges, the composition and the three-dimensional conformation were vital factors that contributed to rutile mineralization. Even when natural proteins can be used to induce biomineralization, small peptides are becoming an attractive scaffold for this task because they can maintain the molecular recognition ability while being smaller and simpler molecules. In this regard, commercially available peptide phage and cell display libraries are useful tools for identifying new peptides that are able to bind to a particular material. Because of its chemical stability and nontoxicity,8 titanium dioxide has numerous technological applications, particularly as a (photo)catalyst for water splitting9 and water decontamination,10 and as a pigment. Performance of titanium dioxide in such applications depends upon its size, shape, crystallinity, and purity; therefore, the development of approaches that allow Received: January 15, 2014 Revised: March 14, 2014 Published: April 2, 2014 4084

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templated by self-assembly of ligands (MTSALs).17 The peptides used in our biomineralization approach appear to act as heteroligands, allowing for control of the size, morphology, and crystallinity of the final material. Moreover, the H1 peptide also exhibits affinity toward SiO2, and we therefore studied some of the factors that affect the biomineralization of nanostructured silica.

these parameters to be controlled is a challenge. Because of the importance and numerous potential applications of titanium oxide, its synthesis using biomimetic approaches has been explored. In particular, the use of phage display libraries (present on the surface peptides of 12 and 7 residues) has become an instrument for identifying new peptides that can bind to the surface of TiO2. Dickerson and co-workers11 found several dodecapeptides capable of binding to and recognizing distinct crystalline orientations of rutile crystals. Furthermore, they observed that these peptides, in the presence of the titanium precursor TiBALDH, were able to induce TiO2 biomineralization, which was related to the peptides’ K and R residue content. On the basis of these results, they designed a peptide 4-fold repeat of RKK that exhibited high biomineralization activity; this dodecapeptide was even able to produce fine anatase crystals trapped in amorphous materials. Incomplete crystallization was explained in terms of the peptides’s lack of spatial organization and cooperative effects between residues. In another study, Sano et al.12 found a 12-mer peptide that exhibited high affinity for the surface of TiO2 particles. The authors constructed several peptide variants to investigate the role of each amino acid in the binding phenomenon. They observed that K, P, and D residues in positions 1, 4, and 6, respectively, were essential for binding. More importantly, they observed that only the first 6 residues of the N-terminal end were sufficient for binding activity. Chen et al.13 have found a remarkable looplike peptide (CHKKPSKSC) capable of binding to either SiO2 or TiO2 surfaces. To understand the influence of each amino acid on the binding activity, they designed several mutants by changing each residue by A.14 When three K residues were conserved, the binding force remained intact; however, the absence of one of the K residues decreased the peptide avidity to the oxide surface. Furthermore, the same group found that linear peptides were unable to biomineralize TiO2; however, dissolution of the peptides in phosphate buffer (PB) promoted the formation of amorphous TiO2.15 Because of the lack of consensus on the extent to which peptide sequences can bind to TiO2 as well as the lack of consensus on the influence of the residues in the mechanism of biomineralization, we here used a peptide reported in the literature to be capable of serving as a biotemplate for TiO2, specifically, the peptide HKKPSKS13 (referred to as H1 in this work). In addition, our sequence was linear instead of being constrained to allow the free movement of the backbone and thus take advantage of the possible interaction between the Ti precursor and the peptide.16 We used a heptapeptide because Sano and Shiba have reported that 6 residues are sufficient to bind to TiO2;12 as a consequence, we reduced the complexity of the system. We designed several mutants to investigate the influence of the basic residues on the mechanism of mineralization. In addition, we used several buffer systems to investigate the effects of the ions present in solution and the solution’s pH. As far as we know, all previously reported experiments concerning the biomimetic production of TiO2 have been conducted using TiBALDH as the Ti source. Here, we introduce Ti alkoxides as effective molecular precursors to control the final characteristics of the titanium oxide. Recent progress in understanding the sol−gel process has clearly shown that carboxylate or amine-containing compounds are able to chelate and stabilize sols of oxometallates; these ligands promote hydrolysis−condensation reactions that lead to formation of structures known as micelles

2. EXPERIMENTAL SECTION 2.1. Biomineralization of TiO2. All peptides were obtained from GeneScript (purity >96%). The biomineralization of TiO2 was performed with the peptides shown in Table 1, and Table 1. Properties of the Peptides Designed for Biomineralization Experimentsa

a

key

sequence

MW (g mol−1)

pI22

H1 R1 L1 H2 H3 H5

HKKPSKS RKKPSKS LKKPSKS HHKPSKS HKHPSKS HKKPHKS

810.94 829.94 786.97 819.91 819.91 861.01

10.30 11.26 10.30 10.00 10.00 10.30

Mutations are marked in bold type.

titanium isopropoxide or titanium n-propoxide (Sigma-Aldrich, 97%) was used as the precursor. The influence of the pH on the biomineralization process was studied using several buffers: 2(N-morpholino)ethanesulfonic acid (MES, pH = 3.6), sodium citrate (pH = 4.7), sodium acetate (pH = 3.6), barium acetate (pH = 4.7), potassium phosphate (PB, pH = 6.8), tris(hydroxymethyl)aminomethane (TB, pH = 8.0), and sodium borate (pH = 9.2). Peptide solutions were prepared in different 50 mM buffer systems. To control the sol−gel reactions, different [alkoxide:alcohol:water] molar ratios as well as various concentrations of the peptide solution were tested. In the first step, a mixture of isopropyl alcohol and a titanium source was prepared. A suitable quantity of peptide solution was then added dropwise at room temperature. Finally, the formed TiO2 was separated by centrifugation (13000 rpm) and washed twice with ethanol and water. All materials and water used in the experiments were previously sterilized to prevent peptide degradation. For comparison, TiO2 was also synthesized according to the same procedure without the use of a template (blank). For the synthesis of crystalline TiO2 particles, reactions were acid promoted by the addition of 0.5 μL of concentrated HCl, which lowered the pH value to approximately 2.0 2.2. Biomineralization of SiO2. For the synthesis of biomineralized SiO2, we used tetraethyl orthosilicate (TEOS) (Sigma-Aldrich, 99%) as a precursor; the addition of alcohol was therefore not necessary. For each peptide, 50 mM solutions were prepared for each of the different buffers tested. Typically, 6.5 μL of the silicon alkoxide was added to 50 μL of the peptide solution; the mixture was homogenized using a vortex mixer and left standing at room temperature until a colloidal suspension or a precipitate formed. After the SiO2 was obtained, it was washed three times with water, dried, and characterized. 2.3. Materials Characterization. The particle size and morphology of TiO2 and SiO2 were observed using transmission electron microscopy (TEM) performed on a JEOL 2010 transmission electron microscope operated at 200 kV. Two microliters of the sample was deposited onto a copper grid 4085

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covered with Formvar (300 mesh); in the case of SiO2, the sample was first diluted with water. Selected samples were annealed at 700 °C for 1 h using a Lindberg Blue M furnace. Xray diffraction (XRD) analyses were performed with Cu Kα (1.5406 Å) radiation using a Siemens D5000 or Bruker D-8 Advance diffractometers. Infrared spectra (IR) of the samples were recorded on a Perkin-Elmer Spectrum GX FT-IR spectrometer. UV−vis spectra were recorded on an Agilent 8453 Hewlett-Packard spectrometer. The peptide absorption band at 215 nm was used to determine peptide concentration. The size distribution of particles formed in alcoholic and alcoholic-aqueous sols, either before or after acid addition, was measured by dynamic light scattering (ZetaSizer ZS, Malvern). Figure 1. Charge−pH diagram for the distinct peptides used in biomineralization experiments.22

3. RESULTS AND DISCUSSION 3.1. Influence of the Titanium Precursor in the Biomineralization of TiO2. The formation of TiO2 via sol− gel methods using the alkoxide route has been recently studied;18 however, the use of biomolecules with these titanium sources has been almost unexplored. The use of TiBALDH as a Ti source for TiO2 formation is relatively recent.19 This precursor is completely stable in water and has been used as a common source of titanium in biomineralization experiments by several groups.1,15,20 In contrast, titanium alkoxides react quickly in aqueous media. In our approach, we used alkoxides rather than TiBALDH as a titanium source to study the ability of peptides to control the sol−gel process. We hypothesize that, in our biomimetic approach, the peptide itself controls the sol− gel process through its functional groups. Notably, the normal behavior of these precursors is modified in the presence of peptides. In the case of TiBALDH, peptides promote the formation of a solid, as reported by several groups,1,21 whereas, in the case of Ti alkoxides, the rate of formation of precipitate was strongly reduced due to the presence of the peptide. We presume that the peptide has two functions: first, its amine and carboxyl groups can coordinate to titanium, thereby stabilizing the colloid and slowing the formation of a precipitate; second, it acts as a structure-directing template, controlling the final morphology of the TiO2. In contrast, the addition of Ti alkoxide to water, in the absence of a peptide, facilitates the rapid and fully uncontrollable formation of a white precipitate, as we will show later. The 7-mer sequence (HKKPSKS), referred as H1 in this work, was used as a starting template; point changes were introduced to investigate the influence of the basic residues in the biomineralization process. The physical properties of the peptides are shown in Table 1. All of them have isoelectric points (pIs) between 10.00 and 11.26 and are positively charged in the pH range of 2−10, as evident in Figure 1. To test the ability of H1 to biomineralize, we first studied the influence of the buffer used in the reaction. Several buffers that ranged from slightly acidic to basic (i.e., acetate, phosphate, and borate) were tested. In a typical reaction, a mixture of titanium propoxide [Ti(n-PrO)4] and propanol (n-PrOH) with a molar ratio [1:50] was added to a peptide solution (2 mg/mL, in 50 mM buffer); a precipitate was quickly formed in all of the investigated buffers. In the case of the blanks (i.e., samples in which no peptide was present), the precipitation was even faster and could be observed with the naked eye. Further reactions with titanium alkoxides were performed in water unless otherwise indicated. When the reaction conditions were adjusted to a peptide concentration of 4 mg/mL and a [Ti:nPrOH:H2O] molar ratio of [1:50:40], a solid was obtained,

which proved to be formed by amorphous nanoparticles (NPs) with diameters of approximately 50 nm, as visualized by TEM (results not shown). In accordance with these results, we obtained nanostructured TiO2 when we used Ti(n-PrO)4 as a titanium source and the H1 peptide as a template. The reactivity of titanium alkoxides is well-known to also depend on their R alkyl group. We consequently used titanium isopropoxide [Ti(i-PrO)4] to study the influence of the precursor structure in the biomineralization process. This compound reacted in a manner similar to that of Ti(n-PrO)4 in the presence of different buffers (i.e., a precipitate was instantaneously formed). Consequently, the influence of the pH on this alkoxide could not be determined, and all further reactions were conducted in water. Because of the different rate of hydrolysis of this precursor, we adjusted the [Ti:iPrOH:H2O] molar ratio to [1:66:5] and maintained the concentration of the peptide solutions at 4 mg/mL in all experiments. This solution was added to the H1 peptide and then vortexed; after several minutes, turbidity was observed, which evolved into the formation of a solid. This solid was washed, centrifuged, and further analyzed. TEM images of the obtained product show an interconnected network of TiO2 nanoparticles approximately 50 nm in size (Figure 2a). Highresolution TEM images revealed that the material consists of small crystalline nanoparticles trapped in the amorphous phase (inset of Figure 2a), and the lattice fringe spacing of 2.33 Å is consistent with the (103) plane of the anatase phase. Other authors have also reported TEM images showing small crystalline structures inside the amorphous oxide; however, they performed the reaction in the presence of phosphates and used TiBALDH as a precursor.11,23 Nevertheless, according to XRD results, such crystalline structures were not detected in our samples, possibly because their small size and low abundance rendered them undetectable due to the sensitivity limitations of our diffractometer. Thus, in this section, we showed that H1 was able to both bind to Ti precursors and induce the formation of nanostructured TiO2. 3.2. Study of the Influence of the Mutants in the Morphology of the Final Material. The binding phenomenon between biomolecules and surface solids is a complex interaction that involves several factors, such as electrostatic interactions and hydrogen bonds, among others.24 Furthermore, the size and topographic aspects can be important in the biomineralization process. Although some small peptides with certain specific sequences could, in principle, be capable of joining at the surface of TiO2, this factor is not sufficient to guarantee biomineralization because the mechanism is also 4086

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Figure 2. TEM images of TiO2 samples biomineralized by various peptides: (a) H1 (inset HR-TEM); (b) H2; (c) H3; (d) R1; (e) L1; (f) H5; and the (g) blank.

induce the formation of TiO2 particles must be satisfied. Thus, mutants H2 and H3 (see Table 1) were evaluated for biotemplating. In these mutants, one K residue was replaced by one H residue to reduce the positive charge of the peptide. Both mutants generated white precipitates. However, when the precipitates were observed by TEM (Figure 2, panels b and c), different results were obtained. The first peptide resulted in aggregates without a defined morphology, whereas the second peptide produced a particle network with a morphology similar to that of the products obtained using the H1 peptide, although

governed by various additional factors, such as charge, sequence order, size, and pH, among others. Several groups have studied the influence of the binding ability of sequences identified by biopanning and have further introduced point changes (primarily positive charges found in basic residues). Thus, the existence of the three K residues in H1 has been postulated14 as being necessary to allow H1 to bind to the TiO2 surface. Our goal was to produce several mutants of H1 to study their effect on the biomineralization mechanism; however, in our case, not only must the binding ability be covered but also the capacity to 4087

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particles were slightly larger (approximately 100 nm). Unfortunately, in both cases, the electron diffraction (ED) patterns indicated that the materials were amorphous. Thus, two K residues appear to be sufficient to induce biomineralization, and both mutants exhibit the same properties (charge and pI) (i.e., they have the same residues), although the sequence in the second and third positions is inverted. Therefore, the peptide sequence critically affects the size and morphology of the final product. The importance of the H residues to the binding affinity has already been documented;11,14 we, therefore, replaced H in the first position by R and L amino acids. Products obtained by the mutants R1 and L1 were visualized by electron microscopy. When H was substituted with a basic residue (R), the nanoparticles were very similar to those obtained with the H1 peptide. However, HR-TEM images did not show any evidence of crystalline areas [i.e., the particles were amorphous (Figure 2d)]. Substitution of H with an aliphatic residue (L) resulted in amorphous aggregates (Figure 2e). All of the peptides have a net positive charge at pH 6.5 (i.e., the pH of water without any buffer system). The positive charge of the R1 mutant is greater than that of H1, and that of H1 is greater than that of L1 (4+, 3.5+ and 3+, respectively). Consequently, we suggest that the interaction between the precursor and R1 and H1 is sufficient to induce binding and that the peptide− alkoxide adduct serves as a nucleation site that controls the size and morphology of TiO2. On the basis of these results, we propose that the presence of basic residues in specific positions must be critical for the nanoscale arrangement of TiO2. Finally, we prepared the H5 mutant by substituting an S for an H residue into the fifth position. Reaction of the Ti precursor Ti(i-PrO)4 and H5 generated a white powder. The TEM images (Figure 2f) of the white powder revealed TiO2 nanoparticles approximately 100 nm in size interconnected in a manner similar to that shown in Figure 2a; however, this sample consisted exclusively of amorphous particles. H5 has a larger positive charge (4+) than H1. The results obtained in this section indicate that charge is a key parameter in obtaining nanostructured TiO2. Because H1 was the only peptide able to induce the formation of small crystalline regions of TiO2, it was characterized further and used in additional experiments. In most cases, small peptides do not show any structure in solution; in the case of H1, we verified its lack of structure by circular dichroism measurements. As we expected, H1 produced a spectrum typical of a random coil conformation (not shown). The same results were obtained by molecular dynamics simulations, which also predicted no secondary structure of the peptides (see Supporting Information), thus demonstrating that all of the peptides have a random coil conformation. Given that no particular regular conformation of the peptide can explain the formation of small nanocrystals, we postulate that the sequence is the responsible factor in the development of ordered structures. The aforementioned H1 peptide was the only one among those investigated with the ability to induce the formation of some anatase nanocrystals at room temperature (see Figure 2a). Thus, to establish the importance of the peptide sequence in the crystallization process of biotemplated TiO2, the products obtained from H1, L1, and the blank were annealed to 700 °C. Figure 3 shows the diffraction patterns collected. For the blank and L1 products, the peaks observed correspond exclusively to those of the rutile phase, in agreement with

Figure 3. Diffraction patterns of TiO2 biotemplated with H1, L1 peptide, and the blank. ▲ and ● indicate peak positions corresponding to rutile and anatase phases, respectively.

results by Mahshid et al.25 who had observed rutile formation starting above 500 °C in the absence of any ligand. Interestingly, H1 produced both anatase and rutile phases, as can be seen from Figure 3. These results suggest that, most likely, small anatase particles formed in the presence of H1 served as seeds for the growing and stabilization of this phase, whereas the rutile phase derived probably from amorphous regions. Similar results regarding the stabilization of one phase over another, due to the influence of the peptide sequence, were reported by Nonoyama et al.,26 who designed a peptide [(LK)8] that stabilizes the rutile phase at temperatures as low as 400 °C, whereas temperatures as high as 900 °C are usually necessary. In accordance with these results, the specific sequence plays an important role in the nucleation and growth process of oxotitanate clusters, thus directing the formation of distinct crystalline arrays. 3.3. Formation of Anatase Nanoparticles. Because of the unique properties of the H1 peptide to induce anatase crystallization at room temperature and its capability to stabilize this phase even at temperatures as high as 700 °C, we hypothesize that this peptide must be able to induce complete crystallization under the appropriate conditions. As a consequence, experiments of TiO2 biomineralization were conducted in aqueous solution; no other ions were introduced to prevent other nonspecific interactions with the peptide. The [Ti:peptide] molar ratio is a key parameter in achieving a real template effect of the peptide. When the ratio was [1000:1], we obtained only a few regions constituted by a crystalline core covered by a shell of amorphous material (see Figure 2a). The formation of MTSALs can explain the core−shell structures (for more details see refs 17 and 18); however, if the amount of peptide molecules is small, they cannot stabilize the amorphous oxide producing the resulting network or aggregates observed in Figure 2. The use of a lower [Ti:peptide] molar ratio of [100:1] resulted in the formation of a sol that was very stable. To catalyze the hydrolysis−condensation reactions, a small amount of HCl was added to adjust the pH to approximately 2; after several hours, a colloidal system was formed, which evolved into a white solid at the bottom of the tube. TEM analysis showed the formation of clearly crystalline anatase nanoparticles with a size of approximately 5 nm (see Figure 4a), as is evident in the HR-TEM image of Figure 4b; the lattice fringes at 3.5 and 2.4 Å correspond to the (101) and (103) 4088

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Figure 4. (a) TEM images of TiO2 biomineralized using the peptide H1 and a [Ti:peptide] molar ratio of [100:1]. (b) HR-TEM image of the anatase particles; the arrows show the fringes, and the selected area analyzed by electron diffraction is shown in the inset. (c) XRD pattern of the anatase nanoparticles.

Figure 5. (a) Development of average particle size of the micelles of titanium oxide formed in the presence of peptide molecules over time, as determined by DLS measurements. (b) Size distribution of the final particles obtained with H1 and L1.

Scheme 1. Proposed Mechanism of a Sol-Gel Reaction for the Synthesis of TiO2 Using the Peptides H1 and L1 as a Ligand Capable of Biomineralizing Titanium Oxidea

a

Drawing is not to scale.

(200), (105), and (204) planes of the anatase structure. To the best of our knowledge, the assembly of anatase nanoparticles at room temperature using an alkoxide precursor mediated by

planes of anatase. This result was confirmed by XRD. The Xray pattern (Figure 4c) showed reflections at 25.2, 37.8, 48.0, 53.9, and 62.6° 2θ, which correspond to the (101), (004), 4089

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Figure 6. TEM images of SiO2 biomineralized using the peptide H1 in the (a) Tris and (b) sodium borate buffers and using the peptides (c) R1 and (d) H5, both in the Tris buffer.

began to appear turbid with a concomitant increase in the size of the particles, as determined by DLS measurements. It should be noted that the particles reached a maximum size and then began to decrease their size, reaching an average size of 80 nm after 48 h (see Figure 5a). After 4 days, a peak around 5.6 nm was visible from DLS recordings, and additional peaks corresponding to bigger aggregates were also observed. In accordance with these results, we propose a tentative mechanism that is depicted in Scheme 1. As we mentioned, clusters of oxotitanates are previously formed in the solution. When water (blank) or water containing peptides are added to the solution the clusters serve as nucleation centers (core) and react with the water, increasing their size as was proven by DLS; however, the surrounding network (shell) created is amorphous and evolved to form precipitates. On the contrary, in the presence of H1, the micelles grew but were stabilized by the peptide ligand, forming micelles templated by self-assembly of ligands (MTSALs) (pathways 1a and 1b, respectively).17,18 However, when acid was added, the size of the micelle increased, probably due to proton-catalyzed hydrolysis− condensation reactions that lead to a thicker shell; the micelle was, however, stabilized by the protonated groups of the peptide (see Figure S2 of the Supporting Information). Furthermore, because it has been reported that ligands such as citrates and lactates are capable of dissolving amorphous titanium oxide,17,29 the C-terminal end of the peptide likely solubilizes the outer shell assisted by the acid addition, allowing the peptide to direct the crystallization of anatase nanoparticles. As we described before, the sequence of the peptide plays a role in the formation of the ordered structures. Therefore, L1 was also used (at the same molar ratio as that of H1) to follow the size of clusters and MTSALs. With this peptide we

MTSALs stabilized by specific heptapeptide ligands has not been previously reported. Accordingly, we propose that a low [Ti:peptide] ratio results in an optimal increase in the formation and stabilization of the MTSALs, which leads to the formation of a crystalline anatase final product. Recently, Perry’s group27 prepared crystalline TiO2 anatase nanoparticles using two dodecapeptides and TiBALDH as a titanium precursor. As previously noted, TiBALDH and the alkoxide precursor we used exhibit important differences in their behavior in water, suggesting that the reaction mechanisms leading to the crystalline final products might differ. Puddu et al.27 observed that amorphous nanoparticles were obtained in the presence of PB, whereas crystalline nanoparticles were obtained in water alone. Thus, in order to gain insight into the mechanism of formation of TiO2 nanoparticles, the size of the particles at different stages was determined from experiments in which a [100:1] molar ratio was used. The solution of titanium alkoxide and its parental alcohol was prepared at environmental conditions, producing a transparent solution. Because no water-free conditions were used, microhydrolysis reactions proceeded rapidly and produced clusters of oxotitanates.28 This was confirmed by DLS experiments, which demonstrated the existence of oligonuclear species with a size of ∼3 nm, coexisting with bigger aggregates of several hundreds of nanometers, on alkoxide-alcohol mixtures prepared immediately before size determinations were carried out. When this mixture was added to the H1 peptide solution, the sol kept stable and the size of the particles reached a size of around 80 nm (even after 3 months) with no sign of precipitation. TiO2 NPs were produced when the acid was added to the alkoxide/alcohol/ peptide mixture; after 24 h, the original transparent solution 4090

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networks’ ordered structures. 27 The formation of SiO 2 nanoparticles was induced only when the pH was slightly basic (8.0 and 9.3). This can be partially explained by the charge of the H1 peptide, which is greater at low pH levels (+4.5) and decreases to approximately +1.75, when the pH value is increased (see Figure 1). From these results, we observe that the charge of the peptide as well as the buffer system play important roles in the biomineralization process. One possible explanation for the formation of SiO2 NPs in TB is that the interactions are optimized in the presence of Tris molecules and do not compete with TEOS molecules for the peptide. Notably, TB was the buffer used for the biopanning screening experiments, and the oxide−peptide interaction was maximized under this condition. Thus, the ions added from the buffer during the biopanning technique can influence the emerging sequence and the biomineralization process; we are, therefore, currently conducting biopanning experiments in different buffers in our laboratory. Finally, the biotemplating activity toward SiO2 formation was tested with all of the proposed mutants. For mutants L1, H2, H3, and the blank (see Figure S4 of the Supporting Information), no change occurred that was observable with the naked eye (i.e., they did not exhibit any templating effect). In the case of the R1 instead of the H1 sequence, the substitution of a basic residue with another of a similar nature promoted the formation of a precipitate (see Figure 6c). In contrast, the mutant L1, which contains an aliphatic residue has no effect on the reaction. The replacement of serine with histidine in the fifth position (H5 mutant) appears to not affect SiO2 formation, according to the results in Figure 6d. Only the peptides with a large positive charge were able to induce the formation of SiO2; thus, the basic nature of the residue controls this aspect of biomineralization of SiO2 (see Table 3).

observed a different behavior from that with H1: the micelles size increased during the first 24 h and began to decrease after two days, forming particles between 120 and 180 nm (see Figure 5). At the end of the reaction, these particles coalesced, forming bigger aggregates; moreover, TEM images of this sample did not show crystalline structures (see Figure S3 of the Supporting Information). Most likely, the L1 peptide can stabilize the MTSALs, but it cannot direct the size and crystallinity of the final oxide. This also can partially explain why, when the concentration of the peptides is less than a molar ratio [1000:1], there are not enough peptide molecules to stabilize the particles and further dissolve them and the formed product is a network of interconnected particles (Figure 2a). It should be noted that no acid was added in these reactions, which probably has an effect in the final crystallinity but is beyond the scope of this article, and more experiments are in progress to clarify this issue. 3.4. Biomineralization of SiO2. With respect to the biomineralization of SiO2, Chen and co-workers13 claim that the H1 peptide can also bind to SiO2. In fact, they observed subtle differences between the binding affinities of linear and cyclic peptide structures to TiO2 and SiO216 (i.e., the binding affinity is stronger in the case of linear peptides). Because of their flexibility, linear peptides can bind to the two surfaces in various conformations. In addition, the linear peptides prefer SiO2 to TiO2 in terms of binding affinity. Consequently, we attempted the biomineralization of SiO2 using the H1 peptide. In this case, we used TEOS as a precursor and kept the [TEOS:peptide] molar ratio constant at [1:50]. Because this reaction is slow, we could test the influence of several buffers on the biomineralization process. In the presence of phosphates (PB), a white precipitate is formed; however, no morphology is apparent by TEM analysis (data not shown). Interestingly, the use of a Tris buffer (TB) leads to the formation of a colloidal solution. Figure 6a shows the formation of quasi-spherical particles with a size of approximately 30 nm. Similar results were obtained in the presence of borate buffer (Figure 6b), although the particles were smaller (∼20 nm). Table 2

Table 3. Biomineralization of SiO2 Using All of the Investigated Peptides in the 50 mM Tris Buffer

Table 2. Reactions Using the H1 Peptide in Different Buffers with a [Peptide:TEOS] Molar Ratio of [1:50]a buffer

negative control

H1 peptide

pH

MES sodium citrate sodium acetate barium acetate PB TB sodium borate

gel gel gel gel precipitates X X

X gel gel gel precipitates colloid colloid

3.3 4.3 4.4 4.6 6.5 8.0 9.3

a

peptide

result

chargea22

R1 H1 H5 L1 H2 H3 negative control

precipitate colloid colloid and precipitate X X X X

3.5+ 2.5+ 2.5+ 2.5+ 1.5+ 1.5+ -

Calculed at pH = 8. X = without change.

4. CONCLUSION In this study, we synthesized titanium dioxide via a biomimetic strategy using Ti alkoxides instead of the more common TiBALDH as the Ti source. Several mutants of the H1 peptide were observed to influence the characteristics of the final material during the TiO2 biomineralization process. A mechanism of formation of TiO2 was suggested in terms of the formation of MTSALs that are stabilized by the peptides. Apparently, the micelle core−shell structure is capped by peptide molecules with high positive charge, which stabilizes the micelles producing nanostructured networks of particles smaller than 50 nm. In contrast, less positive peptide molecules are unable to stabilize the micelle-producing aggregates. However, we demonstrated that biomineralization is a complex mechanism that also depends on other factors, such as the

a X: without change; MES: 2-(N-morpholino)ethanesulfonic acid; PB: phosphate buffer; and TB: Tris buffer.

summarizes the results of syntheses performed using various buffer systems; at low pH levels (approximately 3), neither a colloidal solution nor a precipitate were formed. When the pH was increased to approximately 4.4, we observed only gel formation. The use of phosphates at pH 6.5 produced, in our case and in the cases of other groups, only precipitates. Others have argued that these ions are necessary to induce the precipitation of TiO2 and SiO2; however, these anions become trapped in the metal oxide networks, deteriorating the 4091

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nature and sequence of the amino acid, as indicated by the fact that peptides with the same composition and same properties but with different sequences resulted in products with dissimilar morphologies. Remarkably, we established the optimal conditions under our strategy to obtain, for the first time, anatase nanoparticles from heptapeptides and Ti alkoxides. However, additional research is needed to fully understand the interplay of all factors involved in the formation of crystalline structures of TiO2. In addition, we showed that the H1 peptide is a versatile peptide capable of inducing crystallization of SiO2. We investigated the effect of pH and found that pH values ≥6.5 are necessary to induce biomineralization. The added ions also affect the characteristics of the material, whereas phosphate resulted in a precipitate and the Tris and borate ions allowed the formation of colloids that consisted of 30 and 20 nm nanoparticles, respectively. Overall, we showed that subtle differences in the sequence of the peptide can direct the structure, size, and morphology of the resultant material. However, other factors, such as pH and the presence of external ions, can modify the interaction between the precursor and peptide, and these modifications must be taken into account during biomineralization experiments.

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ASSOCIATED CONTENT

* Supporting Information Methodology of the molecular dynamics simulations. TEM images of the micelles, final materials with titanium alkoxides with different peptides and the blank, and the blank for the TEOS precurors in TBS are included. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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REFERENCES

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ACKNOWLEDGMENTS

A.H.G. thanks Consejo Nacional de Ciencia y Tecnologı ́a (CONACYT) from Mexico for the Ph.D. scholarship. This work was mainly supported by SEP-PROMEP “Apoyo a la incorporación de Nuevos Profesores de Tiempo Completo” (Grant UAM-PTC-369 SEP-Promep). We thank Patricia Castillo (Laboratorio Central de Microscopı ́a Electrónica UAM-I) for the TEM and HR-TEM images, the electron diffraction experiments, and helpful suggestions. We thank Dr. Jaime Vernon for allowing us the use of his DLS equipment. The authors want to thank CONACyT-Mexico for providing financial support under Project INFR-2011-1-163250. Also special thanks to LDRX (T-128) UAM-I for XRD measurements. We must also express our sincere thanks to the reviewers for their helpful suggestions. 4092

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