Article pubs.acs.org/Langmuir
Mineralization of Calcium Carbonate on Multifunctional Peptide Assembly Acting as Mineral Source Supplier and Template Kazuki Murai,*,† Takatoshi Kinoshita,§ Kenji Nagata,‡ and Masahiro Higuchi*,‡ †
Department of Materials Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan ‡ Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan § Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan S Supporting Information *
ABSTRACT: Crystal phase and morphology of biominerals may be precisely regulated by controlled nucleation and selective crystal growth through biomineralization on organic templates such as a protein. We herein propose new control factors of selective crystal growth by the biomineralization process. In this study, a designed β-sheet Ac-VHVEVSCONH2 peptide was used as a multifunctional template that acted as mineral source supplier and having crystal phase control ability of calcium carbonate (CaCO3) during a selfsupplied mineralization. The peptides formed three-dimensional nanofiber networks composed of assembled bilayer βsheets. The assembly hydrolyzed urea molecules to one carbonate anion and two ammonium cations owing to a charge relay effect between His and Ser residues under mild conditions. CaCO3 was selectively mineralized on the peptide assembly using the generated carbonate anions on the template. Morphology of the obtained CaCO3 was fiber-like structure, similar to that of the peptide template. The mineralized CaCO3 on the peptide template had aragonite phase. This implies that CaCO3 nuclei, generated using the carbonate anions produced by the hydrolysis of urea on the surface of the peptide assembly, preferentially grew into aragonite phase, the growth axis of which aligned parallel to the direction of the β-sheet fiber axis.
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INTRODUCTION Biomineralization yields functional organic−inorganic hybrid materials called biominerals (e.g., seashells, bones, and teeth). Biominerals have a wide variety of functions owing to their hierarchic organic−inorganic nanohybrid structures formed through exquisite self-organization. In recent years, formation of functional organic−inorganic nanohybrid materials inspired by biomineralization has gathered interest as a novel process for fabricating next-generation functional materials under mild conditions. However, biomineralization processes, which involve transport of mineral source ions1,2 and selective crystal growth of inorganic material,3−9 have not been sufficiently elucidated. In fact, inorganic crystals appear in different crystal phases in different parts of a biomineral. For example, calcium carbonate (CaCO3), as a chief component in seashells, takes on different crystal phases in the prismatic layer (calcite, a stable phase) and the nacreous layer (aragonite, a metastable phase).10−18 A solution for this “calcite−aragonite problem” has been sought in biomineralization research.19 Miyashita et al. reported that nacrein existing in the nacreous layer has two functional domains: one is a carbonic anhydrase domain that acts as a mineral source supplier, and another is Gly-Xaa-Asn (Xaa = Asp, Asn, or Glu) repeat domain. It has been assumed © XXXX American Chemical Society
that the Gly-Xaa-Asn repeat domain in nacrein controls the nucleation and crystal growth of CaCO 3 during the biomineralization process.20 Our research group has reported that selective formation of rutile crystal phase of titanium dioxide can be achieved by exploiting the epitaxial relationship between the organic and the inorganic interfaces.21 Recently, we reported the primary results concerning the selective mineralization of aragonite phase using a multifunctional βsheet peptide template.22 We designed the multifunctional βsheet peptide to act as a mineral source supplier and template for the CaCO3 biomimetic mineralization. The peptide spontaneously self-assembled to form a bilayered nanofiber owing to the hydrophobic interaction between the hydrophobic faces of β-sheet with exposed Val side chains and the bilayered nanofibers assembled to form three-dimensional peptide nanonetworks. Crystal phase and morphology of the CaCO3 obtained via the biomimetic mineralization were the metastable aragonite crystal phase and fiber-like structure similar to that of the peptide assembly, respectively. These results indicated that Received: July 1, 2016 Revised: August 23, 2016
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DOI: 10.1021/acs.langmuir.6b02439 Langmuir XXXX, XXX, XXX−XXX
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Figure 1. TM-FTIR spectra of the (a) VHVEVS, (b) VAVEVS, and (c) VHVEVA peptides. Dotted lines show the peak deconvolution of the amide I band from antiparallel β-sheet, random coil, α-helical, and β-sheet conformations. the VHVEVS peptide aqueous solution (1.0 mM, 0.5 mL) was added to a mixture composed of urea (50 mM, 2 mL) and calcium acetate (50 mM, 2 mL) aqueous solutions, and then the mixture (pH 7.2) was stirred at 20 °C for 21 days. Biomimetic Mineralization of CaCO3. The biomimetic mineralization of CaCO3 using the VHVEVS peptide as the template was carried out using calcium acetate as calcium cation source and urea as carbonate anion precursor. In this mineralization, carbonate anions were self-supplied by the hydrolysis reaction of urea on the VHVEVS peptide surface. The aqueous VHVEVS peptide solution (1.0 mM, 0.5 mL) was added to a mixture containing calcium acetate (50 mM, 2 mL) and urea (50 mM, 2 mL) aqueous solutions, and the mixture (pH 7.2) was stirred at 20 °C for 21 days. We call this CaCO 3 mineralization system “self-supplied mineralization”. For comparison, another mineralization method using an aqueous ammonium hydrogen carbonate solution as carbonate anion source was carried out. This mineralization was carried out as follows: 0.5 mL of the aqueous VHVEVS peptide solution (1.0 mM) was added to a mixture containing 2 mL of calcium acetate (50 mM) and 2 mL of ammonium hydrogen carbonate aqueous solutions. Three concentrations of ammonium hydrogen carbonate solution were used. The final concentrations of added carbonate anion were 0.13, 0.29, and 0.61 mM, respectively. The reaction solution (pH 7.2) was stirred at 20 °C for 21 days. We call this mineralization system with ammonium hydrogen carbonate as the carbonate anion source “externally supplied mineralization”. Spectroscopic Measurements and TEM Observations. To obtain second-order structure of the peptide templates before and after mineralization, transmission Fourier transform infrared (TM-FTIR) measurement (PerkinElmer Spectra 2000, wavenumber range 1800− 1550 cm−1, resolution 4 cm−1, number of scans 64) was carried out. Second-order structure of the peptide templates before mineralization was determined using the KBr pellet method. The peptides were dissolved in pure water ([peptide] = 0.10 mM, pH 7.2) and incubated for 7 days at room temperature. For TM-FTIR measurement, the suspension of the peptide assembly was lyophilized to obtain a measurement sample. Sample pellets were prepared by mixing the peptide (1 wt %) with KBr. Also, the CaCO3−VHVEVS peptide hybrid material obtained after mineralization was deposited on CaF2 plate and then washed in water and dried under ambient conditions for TM-FTIR measurement. The TM-FTIR chamber was purged with dried N2 before and during measurement. The 1800−1550 cm−1 regions of spectra were analyzed as a sum of Gaussian/Lorentzian (9/ 1) composition of the individual bands. The sum of the calculated individual spectra was best fit to the experimental spectra. The ratio of the integrated peak intensities assigned to individual secondary structures, which were obtained by peak deconvolution of the amide I band, gave the percentage of each conformation of the peptide. Morphology and crystal phase of the mineralized CaCO3 were determined using a transmission electron microscope (TEM, JEMz2500, JEOL). For TEM observation, a small aliquot was taken from the reaction solution and placed on an elastic carbon-coated STEM grid, and the CaCO3−VHVEVS peptide hybrids were allowed time to adsorb onto its surface. After adsorption, the excess solution was removed by absorption onto filter paper, and the grid was rinsed with
the mineralized CaCO3 was controlled by specific structures on the β-sheet peptide template. However, we could not discuss the mechanisms of the mineral source supply and the selective crystal growth process in sufficient details. In particular, formation of the aragonite phase cannot be explained only by the epitaxial relationship between the organic and the inorganic interfaces, because the epitaxial relationships between the aragonite−peptide and calcite−peptide interfaces had the same mismatches, respectively. In this paper, we attempt to clarity the mechanism of aragonite phase formation on the multifunctional β-sheet peptide template. We closely investigated the crystal growth process of CaCO3 by biomimetic mineralization on the multifunctional peptide template. We propose new control factors of selective crystal growth during the biomineralization process.
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EXPERIMENTAL METHODS
Peptide Syntheses. We synthesized the Ac-Val-His-Val-Glu-ValSer-CONH2 (VHVEVS), and the two kinds of analogous peptide, the hydrophilic amino acid (His or Ser) of the VHVEVS was replaced by Ala, respectively, Ac-Val-Ala-Val-Glu-Val-Ser-CONH2 (VAVEVS) and Ac-Val-His-Val-Glu-Val-Ala-CONH2 (VHVEVA). The 6-mer peptides used in this study were prepared by solid-phase peptide synthesis on CLEAR-amide resin (CLEAR, cross-linked ethoxylate acrylate resin) using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Fmoc-protected amino acids were polymerized in the order of the sequence on the resin. To cap the N-terminal amino group of the synthesized peptide resin with acetyl group, the peptide resin was stirred in a mixture of acetic anhydride and pyridine (v/v 1/2) for 4 h. To remove all sidechain protecting groups and detach the peptide molecule from the resin, the peptide resin was added to 95 vol % trifluoroacetic acid (TFA) aqueous solution. The peptide-containing TFA cocktail was then dropped into diethyl ether, and the precipitated peptide was recovered and lyophilized. The resulting peptide was identified by matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF-MS) spectroscopy on a JEM-S3000 system (JEOL). Hydrolysis of Urea by the Peptides. The urea hydrolysis reactions were carried out as follows: the three aqueous peptide solutions (1.0 mM, 0.5 mL) were added to a mixture of aqueous urea solution (50 mM, 2 mL) and H2O (2 mL), and then the mixtures (pH 7.2) were stirred at 20 °C for 21 days. The hydrolysis activity of the peptides toward urea was measured by indophenol method using a urease activity kit (BUN Kinos, Kinos Laboratories, Japan) following the procedure from the supplier. We determined the concentration of ammonium cations from the UV−vis absorption spectral changes caused by the indophenol generated by the reactions. UV−vis measurements were performed using a quartz cell with a 1.0 mm path length over the range of 500−800 nm. The concentration of the generated carbonate anions was determined by halving the value of concentration of ammonium cations. The measurements were performed 3 times. The margins of error were less than ±10%. In the mineralization solution system, the generated carbonate anion concentration was determined using the same protocol. In this case, B
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Langmuir water to remove unreacted urea and calcium acetate. Crystal phase of the mineralized CaCO3 was determined by selected-area electron diffraction (SAED).
changes in carbonate anion concentration in the control system (peptide free), in the peptide solution systems (VHVEVS, VAVEVS, and VHVEVA), and in the mineralization system with the VHVEVS peptide. The hydrolysis of urea did not occur within 21 days in the control system (Figure 2a, green line). On the other hand, the VHVEVS peptide containing the all-hydrophilic amino acids (His, Glu, and Ser) generated 0.61 mM carbonate anions after 21 days (Figure 2a, blue line). The hydrolysis activity of the analogous peptide having Ala that was replaced in substitution for the His or Ser residue of the VHVEVS peptide (VAVEVS, Figure 2b; VHVEVA, Figure 2c) was lower than that of the VHVEVS peptide. Final concentration of the generated carbonate anions by the VAVEVS peptide after 21 days was 0.11 mM. Particularly, the VHVEVA peptide that the Ser residue of the VHVEVS was replaced to Ala did not show the obvious hydrolysis activity for urea. These results imply that the His and Ser residues, particularly the Ser residue, are required for the hydrolysis activity of the peptide toward urea. We propose the hydrolysis mechanism of urea by the VHVEVS peptides as follows. The imidazole group of the His residue activated the hydroxyl group of the Ser residue on the β-sheet (Scheme 1a) or between the bilayered β-sheets (Scheme 1b) by taking a proton. This interaction between the His and the Ser residues is well known as a “charge relay effect” that is shown in serine protease. The activated hydroxyl group of the Ser residue hydrolyzes a urea molecule to one carbonate anion and two ammonium cations. Next, we investigated the kinetics of the urea hydrolysis reaction induced by the VHVEVS peptide and by thermal reaction in the control system. Figure S3 shows concentration changes of the generated carbonate anions in the VHVEVS peptide system (Figure S3a) and control system that is in the absence of the peptide (Figure S3b) at each reaction temperature (10, 15, 20, and 30 °C). In the control system, we cannot observe the generation of carbonate anion by hydrolysis of urea at the low-reaction temperature ranges (10, 15, and 20 °C). However, we observed that the urea molecule was thermally hydrolyzed to carbonate anion and ammonium cations increase with increase in reaction time at 30 °C. On the other hand, the concentration of the generated carbonate anions in the VHVEVS peptide system was increased by the hydrolysis of urea at all reaction temperatures (10, 15, 20, and 30 °C). Figure 3a shows the relationship between the kinetic constant and reaction temperature. The kinetic constant was calculated from eq 1
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RESULTS AND DISCUSSION Molecular Design and Structural Analyses of the Peptides. In our previous study, we reported the multifunctional β-sheet peptide (VHVEVS), which acts as a mineral source supplier through the hydrolysis of urea and an organic template for the self-supplied mineralization of CaCO3.22 However, we could not discuss the mechanisms of the urea hydrolysis and selective CaCO3 growth process on the peptide template surface in sufficient detail. To investigate the urea hydrolysis mechanisms by the peptide assembly, we designed new two analogous peptides (VAVEVS and VHVEVA). The designed peptides had Ala that was replaced in substitution for the hydrophilic amino acid (His or Ser) of the VHVEVS. Molecular weights of the synthesized VHVEVS, VAVEVS, and VHVEVA peptides were obtained to be 710.6, 666.4, and 716.4 from the MALDI-TOF-MS spectra analysis, respectively. Their values were in fair agreement with the calculated values of 709.4, 643.4, and 693.4 (Figure S1). From MALDI-TOF-MS study, we obtained evidence indicating the successful solidphase peptide synthesis of the designed peptides. We investigated second-order structure of the synthesized peptides by the TM-FTIR spectral analysis. Figure 1 shows the TMFTIR spectra of the peptide assemblies. The TM-FTIR spectra showed the characteristic absorptions of the β-sheet amine I bands (1630 cm−1), antiparallel β-sheet (1698 cm−1), α-helical (1650 cm −1 ), and random coil conformations (1675 cm−1).23−26 Ratios of the integrated peak intensities assigned to individual second-order structure, which were obtained by peak deconvolution of the amide I band, and the results are summarized in Table 1. The TM-FTIR results imply that all Table 1. Fraction of Second-Order Structure of the Peptides conformation (%) peptide
α-helix
β-sheet
random coil
VHVEVS VAVEVS VHVEVA
0 5 0
85 76 77
15 19 23
peptides took mainly antiparallel β-sheet conformation in aqueous solution at pH 7.2. Additionally, TEM observations revealed that the VHVEVS, VAVEVS, and VHVEVA peptides had a three-dimensional nanonetwork morphology consisting of nanofibers (Figure S2). Isopropyl groups of the Val side chains were positioned on one surface of the β-sheet peptide. To avoid exposure of the hydrophobic side chains to the aqueous phase, the β-sheet peptide spontaneously selfassembled to form bilayered nanofibers owing to the hydrophobic interaction between individual hydrophobic side chains. Furthermore, the peptide nanofibers became hierarchically tangled into the three-dimensional nanonetworks.27,28 Hydrolysis Activity of the Peptide Assembly toward Urea. To demonstrate the mechanisms of the urea hydrolysis by the peptide assemblies, we investigated the hydrolysis activity of the VHVEVS, VAVEVS, and VHVEVA peptide assemblies toward urea. Concentrations of the added peptides and urea were set at 0.10 and 22 mM, respectively. For comparison, the hydrolysis of urea was also performed in the absence of the peptide system (control system). Figure 2 shows
x = a exp( −kt )
(1)
where x is concentration of the generated carbonate anion (mol/L), a is initial concentration of urea (mol/L), k is kinetic constant (s−1), and t is reaction time (s). The kinetic constant, k, of the VHVEVS peptide solution system was composed of two kinetic constants: one is the kinetic constant based on the catalytic reaction induced by the VHVEVS peptide (kcat) and another is that based on the pyrolytic reaction (ktherm). Hence, the kinetic constant, k, is denoted by k = kcat + k therm
(2)
On the other hand, in the control system, the kinetic constant was only ktherm based on pyrolytic reaction. We calculated k and ktherm by the curve fitting of Figure S3a and S3b and using eq 1. The kcat value was calculated by eq 2 with k and ktherm. Generically, the kinetic constant increases with increasing reaction temperature. In the VHVEVS peptide system, C
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Figure 2. Changes in concentration of the carbonate anion generated by the hydrolysis of urea in the (a) VHVEVS peptide system (blue line) and control system (green line), (b) VAVEVS, (c) VHVEVA, and (d) mineralization system containing the VHVEVS peptide and calcium acetate.
Scheme 1. Schematic Image of Mechanisms of the Urea Hydrolysis (a) on the Bilayered β-Sheet and (b) between the Bilayered β-Sheets of the VHVEVS Peptide Assembly
that the VHVEVS peptide changed to a random coil from a βsheet conformation by thermal stimuli. Therefore, the denaturation of the VHVEVS peptide assembly should inhibit the occurrence of the charge relay effect for the urea hydrolysis reaction. In fact, the drastic conformational transition of the peptide induced by the thermal stimuli occurred at 30 °C, and the hydrolysis activity of the denaturated VHVEVS peptide assembly obviously decreased. Thus, we chose 20 °C as the reaction temperature for the self-supplied mineralization. Under this condition the pyrolytic reaction did not occur, and the VHVEVS peptide assembly had a relatively high hydrolysis activity for urea.
increasing rate of the kcat above 20 °C was decreased from that at lower reaction temperatures. As a point of focus, pyrolytic reaction of urea did not occur at less than 20 °C. We presumed that inactivation of the VHVEVS peptide by thermal stimuli would be induced by conformational transition. To demonstrate inactivation of the peptide assembly induced by denaturation, we investigated the second-order structural changes of the VHVEVS peptide induced by thermal stimuli by CD measurement (Figure S4). From the CD spectral analysis we found that the negative molecular ellipticity value, [θ]218, at a wavelength of 218 nm decreased with a rise in measurement temperature (Figure 3b). This result indicates D
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Figure 3. Temperature dependence of (a) kinetic constants, kcat and ktherm, and (b) molar ellipticity at 218 nm.
band revealed that the second-order structure of the VHVEVS peptide in the hybrid material was mainly the β-sheet conformation (95% β-sheet and 5% random coil). Thus, the content of the β-sheet conformation in the peptide template increased by CaCO3 mineralization. This implies that the CaCO3 mineralization on the peptide template surface induced stabilization of β-sheet conformation as a result of the coating of the peptide template surface by inorganic material. Mechanism of the CaCO3 Mineralization on the Peptide Template Surface. We reported in the previous study that the CaCO 3 obtained by the self-supplied mineralization method formed only a fiber-like morphology consisting of aragonite crystals, which was similar to the structure of the peptide three-dimensional nanonetwork.22 Two different mineralization methods (self-supplied mineralization and externally supplied mineralization) were carried out to investigate the template effect of the VHVEVS assembly for CaCO3 mineralization. In both systems the initial concentration of calcium cation was set to 22 mM. In the self-supplied mineralization, the concentration of the carbonate anion gradually increases because of generation of carbonate anion by the VHVEVS peptide assembly. On the other hand, in the externally supplied mineralization system concentrations of the selected carbonate anion were chosen to be 0.13, 0.29, and 0.61 mM that corresponded with that in several stages of the selfsupplied system. The low and middle concentrations (0.13 and 0.29 mM) agree with values of the generated carbonate anions after 7 and 21 days in the self-supplied mineralization system, respectively (Figure 2d). To investigate the early-stage crystal growth of the process, we studied time-dependent crystal phase and morphological changes in the CaCO3 generated by the self-supplied mineralization and externally supplied mineralization. For the externally supplied mineralization, the concentration of the added carbonate anions was fixed at 0.29 mM. Figure 5 shows TEM images and SAED patterns of the mineralized CaCO3 after 3 and 7 days. At the early stage (3 days) of the selfsupplied mineralization, three-dimensional peptide nanonetworks were observed but the obvious characteristic SAED patterns such as hollows and spots could not be seen (Figure 5a). In contrast, aggregates composed of the nanocubic and film-like structures were observed for the externally supplied mineralization after 3 days (Figure 5c), and characteristic SAED patterns were also observed (Figure 5c, inset). The SAED
In addition, we carried out also the urea hydrolysis in the mineralization system containing not only the VHVEVS peptide and urea but also calcium acetate. Under the mineralization system, concentrations of the peptide, calcium acetate, and urea were set at 0.10, 22, and 22 mM, respectively. We carried out the urea hydrolysis at 20 °C. The urea hydrolysis was observed also in the mineralization system. Concentration of the generated carbonate anions increased with increasing reaction time (Figure 2d). However, the rate of urea hydrolysis by the VHVEVS peptide in the mineralization system gradually decreased in this system compared with the VHVEVS peptide system after 7 days. The final concentration of the generated carbonate anions was 0.29 mM after 21 days. This phenomenon was caused by the covering of the VHVEVS peptide assembly surface by the mineralized CaCO3, because the active site for the urea hydrolysis existed only on the peptide assembly. Interestingly, we found that this covering of the peptide template by the mineralized CaCO3 induced not only a decrease in hydrolysis activity but also stabilization of the second-order structure of the peptide template. The conformation of the peptide in the peptide−CaCO3 hybrids produced by the self-supplied mineralization was analyzed by TM-FTIR measurement. Figure 4 shows the TM-FTIR spectrum of the mineralized CaCO3−VHVEVS peptide hybrids. The spectrum exhibited an amide I band assigned to the VHVEVS peptide template. Deconvolution of the amide I
Figure 4. TM-FTIR spectrum of the CaCO3−VHVEVS peptide hybrid material obtained by the self-supplied mineralization after 21 days. Dotted lines show the peak deconvolution of the amide I band from antiparallel β-sheet, random coil, and β-sheet conformations. E
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the peptide template surface had a critical mineral source concentration. After 3 days, the concentration of the carbonate anions generated by the VHVEVS assembly was likely lower than the critical concentration for CaCO3 mineralization. In fact, no CaCO3 mineralization was observed in the externally supplied system when the carbonate anion concentration was 0.053 mM, which corresponds to the concentrations generated at the early stage (3 days) in the self-supplied mineralization system. This result suggests that the template effect is dependent on the concentration of mineral source, carbonate anion, and the peptide assembly. The influence of carbonate anion concentration on the externally supplied mineralization was further investigated. Figure 6 shows TEM images of the CaCO3 obtained by the externally supplied mineralization at each stage (3, 7, and 21 days) under the lower (0.13 mM) and higher (0.61 mM) carbonate anion concentration conditions, respectively. At the higher carbonate anion concentration, 0.61 mM, the morphology of the mineralized CaCO3 was observed to be aggregates composed of cubic nanoparticles after 3 days. The crystal phase of the obtained CaCO3 was a mixture of calcite and aragonite phases (Figure 6a). This result is the same as that observed for the externally supplied mineralization at an initial carbonate anion concentration of 0.29 mM after 3 days (Figure 5c). It is well known that calcite, the most stable crystal phase, is generated when nucleation and crystal growth occurs in bulk solution.29−32 In fact, only calcite phase was mineralized in the solution without the VHVEVS assembly. In addition, Guenoun et al. in the previous report showed that the acidic β-sheet peptide arrangement could not act as an efficient template for mineralization on the air−water interface using supersaturated CaCO3 solution.33 This implies that formation of calcite phase occurred preferentially at the early stage in bulk solution at the higher carbonate anion concentration. In contrast, at the lower initial carbonate anion concentration, 0.13 mM, a fiber-like structure containing cubic nanoparticles that were a mixture of calcite and aragonite phases was clearly observed (Figure 6d). The CaCO3 mineralization by the externally supplied system
Figure 5. TEM images of the CaCO3−VHVEVS peptide generated by the (a and b) self-supplied mineralization and (c and d) externally supplied mineralization. Concentration of carbonate anion in the externally supplied system was 0.29 mM. TEM images of the CaCO3 obtained (a and c) after the 3 days’ mineralization and (b and d) after the 7 days’ mineralization.
patterns corresponded to calcite (104) (d = 3.04 Å) and aragonite (112) (d = 2.37 Å) phases. After the 7 days’ externally supplied mineralization, no obvious changes were observed in the morphology or crystal phase of the obtained CaCO3 compared with those observed after 3 days (Figure 5d). In contrast, after the 7 days’ self-supplied mineralization, structural changes were observed in the generated CaCO3 (Figure 5b). The obtained CaCO3 formed a large fiber-like structure composed of aragonite phase, assigned from the d = 2.37 Å corresponding to aragonite (112) observed in the SAED pattern. We next focused on the change in concentration of the carbonate anions generated by the VHVEVS assembly in the self-supplied mineralization system. The concentration of the generated carbonate anions in the self-supplied mineralization system was 0.053 and 0.13 mM after 3 and 7 days, respectively (Figure 2d). This suggests that the mineralization of CaCO3 on
Figure 6. TEM images of the CaCO3 generated by the externally supplied mineralization at selected initial carbonate anion concentrations. The concentration of carbonate anion were (a−c) 0.61 and (d−f) 0.13 mM. Incubation times of a and d, b and e, and c and f were 3, 7, and 21 days, respectively. F
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Figure 7. (a) Functional group position on the surface of the β-sheet peptide template. Ca-atom position on the a face of (b) aragonite phase and (c) calcite phase, superimposed on a. Red, yellow, and blue circles in a represent the positions of imidazole, carboxyl, and hydroxyl groups, respectively. The relative diameter of the circles for imidazole (red) and carboxyl (yellow) groups was estimated by assuming the free rotation of the α-carbon−β-carbon bond of Glu and His, respectively.
formation by the self-supplied mineralization, we focused on the epitaxial relationship between the organic and the inorganic interfaces. In our previous study we reported that the crystal phase of the inorganic material produced by mineralization could be controlled by the epitaxial relationship between the organic and the inorganic interfaces. In the initial investigation the interfacial Ca-atom arrangement of the mineralized CaCO3 was studied by high-resolution TEM (HR-TEM). Lattice fringes arising from the aragonite (002) phase were observed, the d spacing of which was 3.08 Å. These results indicated that the aragonite a face of CaCO3 was in contact with the VHVEVS peptide template surface.22 To elucidate the selective formation of aragonite phase, the positional relationships between the functional groups of the peptide template surface and the interfacial Ca atoms on the a face of aragonite and calcite phases were investigated in this work. Figure 7 shows the position of the functional groups (hydroxyl, blue circle; carboxyl, yellow circle; imidazole groups, red circle) on the peptide template surface and interfacial Ca atoms on the a face of aragonite and calcite phases. The position of the functional groups of the His and Glu residues is very important because the imidazole and carboxyl groups can capture calcium cations. In particular, the Glu residue is the main binding site for the Ca atom in the VHVEVS peptide. The distances between the αcarbons of Glu (carboxyl group) and His (imidazole group) along the long and short axes of the fibers in the β-sheet conformation are 4.7 and 7.0 Å, respectively (Figure 7a). Figure 7b and 7c shows the positional relationship between the functional groups on the peptide template surface and the interfacial Ca atoms of the a face of aragonite and calcite phases, respectively. The interfacial Ca-atom positions of aragonite35 and calcite36 crystal were overlapped with the positions of the functional groups on the β-sheet peptide assembly according to previous reports.21 However, the positional relationship between the functional groups and the Ca atoms of both crystal phases (Figure 7b, aragonite−peptide; Figure 7c, calcite−peptide) had the same mismatch. Therefore, selective formation of aragonite phase in the self-supplied mineralization cannot be explained only on the basis of this epitaxial relationship. Next, we discuss the formation mechanism using a new approach. In the present case, the selective nucleation of aragonite or calcite on the template was not explained in the early stage owing to the same mismatches of the aragonite− peptide and calcite−peptide epitaxial relationships. Mineralization on the template occurred via an Ostwald ripening.37 We
occurred on the peptide surface and in bulk solution, because the mineral source, carbonate anion and calcium cation existed. The added carbonate anion reacted with calcium cation that the captured calcium cation on the VHVEVS peptide template surface to form CaCO3. At the lower carbonate anion concentration, the mineralization of CaCO3 in bulk solution was relatively inhibited, because of the lower concentration of the mineral source, carbonate anion, in bulk solution, that is, the mineralization on the peptide surface was caused with precedence in comparison with that in the bulk at the lower carbonate anion concentration. This suggests that the template effect functioned effectively under the lower carbonate anion concentration. After 7 days, no drastic change in the morphology and crystal phase of the mineralized CaCO3 was observed at the higher carbonate anion concentration (0.61 mM; Figure 6b). In contrast, growth of cubic nanoparticles was observed at the lower concentration (0.13 mM; Figure 6e). After 21 days, the distinction was clear between the morphology and the crystal phase of the CaCO3 obtained at the higher and lower carbonate anionic concentrations. At the higher initial carbonate anion concentration (0.61 mM), the mineralized CaCO3 appeared as aggregates of the cubic nanoparticles and the SAED pattern of the aggregates showed only the diffraction spots of calcite (104) phase (Figure 6c). The result indicates that the most stable calcite crystal phase preferentially grew at the high carbonate anion concentration. In contrast, at the lower carbonate anion concentration (0.13 mM), the fiber-like structure consisting of aragonite phase was maintained, although growth of calcite phase cubic nanoparticles was observed after 21 days (Figure 6f). This implies that aragonite phase grew on the peptide template, while calcite phase grew in bulk solution. Particularly, the mineralization of the more stable calcite phase occurred in bulk solution without being affected by the template at the higher carbonate anion concentration. These results suggest that in the self-supplied system the gradually generated carbonate anions reacted with the captured calcium cations on the peptide template surface and the metastable aragonite phase of CaCO3 was formed under the influence of the template effect. Crystal Phase Control of the CaCO3 by the SelfSupplied Mineralization. Mann et al. reported that aragonite crystal was produced by the template-mediated transformation and crystallization of the amorphous CaCO3 precursor nanoparticles.34 However, we cannot observe any nanoparticles containing amorphous and crystalline CaCO3 at the early stage by TEM. To explain the mechanism of the aragonite phase G
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Langmuir
morphology and calcite phase. In the self-supplied mineralization, the multifunctional β-sheet peptide acted as the mineral source supplier and mineralization template that regulated the morphology and crystal phase of the mineralized CaCO3. The local concentration of carbonate anions on the peptide template was gradually increased by the hydrolysis of urea. The generated carbonate anions reacted with calcium cations that were captured by imidazole and carboxyl groups on the template surface. We suggest that the selective formation of aragonite phase by the self-supplied mineralization was achieved by two kinds of template effect: (1) selective formation of nuclei on the surface of the peptide template and (2) selective crystal phase growth of CaCO3 along the βsheet peptide template morphology. The obtained knowledge of selective crystal phase growth during CaCO3 mineralization by a biomimetic process is expected to promote our understanding of biomineralization processes and could lead to the creation of many other functional organic−inorganic hybrid materials having nanometer-scaled biomimetic structures.
focus on the growth direction of the nucleus during the Ostwald ripening on the peptide template. It is known that the aragonite phase grows preferentially along the c axis relative to other crystallographic directions. As the above HR-TEM results showed, the a face of aragonite phase contacted the VHVEVS peptide template. This means that the c axis of aragonite phase was matched to the long axis of the fibers of the β-sheet peptide template. We summarize the relationship in Scheme 2, right Scheme 2. Mechanisms of the Selective Formation and Growth of Aragonite Nuclei on the VHVEVS Peptide Templatea
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b02439. MALDI-TOF-MS spectra and TEM images of the synthesized peptides and changes in concentration of the CO32− generated by the hydrolysis of urea in the VHVEVS peptide and control systems at various temperature conditions (PDF)
a
Red, yellow, and blue circles show imidazole, carboxyl, and hydroxyl side chains on the template, respectively. Red cube and white cuboid show the nucleus of calcite and aragonite, respectively.
image. It is thought that the nucleation of aragonite and calcite in the self-supplied mineralization system occurred on the template surface at the same time. It is thought that the aragonite nuclei, the growth direction of which is the c axis, was aligned parallel to the direction of the β-sheet fiber axis, absorbed calcite nuclei during the Ostwald ripening, and grew into the observed fiber-like aragonite crystals (Scheme 2). We suggest that the selective formation of aragonite phase during the self-supplied mineralization was achieved by two kinds of template effect, which were the selective formation of nuclei on the surface of the β-sheet peptide template and the effects of the relationship between the fiber long axis of the peptide template and the growth direction of the CaCO3 crystal. In the self-supplied mineralization system, the selective nucleation and crystal growth of CaCO3 occurred only on the peptide template surface owing to the gradual supply of carbonate anions by the hydrolysis of urea by the VHVEVS assembly, namely, the CaCO3 crystal obtained by the self-supplied mineralization was strongly affected by the templating effect of the peptide. Therefore, in the self-supplied mineralization system, the mineralized CaCO3 crystal on the peptide template surface took aragonite phase as a result of not only selective formation of its nuclei on the template but also regulation of the growth direction by the morphology of the peptide template.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions
K.M., T.K., and M.H. designed this study and developed the hypothesis. K.M. performed sample characterization. K.M. and K.N. performed TEM observations. All authors discussed the results. K.M. and M.H. prepared the manuscript. All authors have read and approved the submitted manuscript. Notes
The authors declare no competing financial interest.
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REFERENCES
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CONCLUSION We investigated the crystal growth mechanism of CaCO3 formed by self-supplied mineralization using the multifunctional β-sheet peptide template. The β-sheet peptide having His and Ser residues acted as a mineral source supplier that hydrolyzed urea to generate carbonate anion owing to the charge relay effect between the His and the Ser residues. The obtained CaCO3 had a fiber-like structure and aragonite metastable crystal phase. In contrast, the CaCO3 obtained by the externally supplied mineralization took on a nanocubic H
DOI: 10.1021/acs.langmuir.6b02439 Langmuir XXXX, XXX, XXX−XXX
Article
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DOI: 10.1021/acs.langmuir.6b02439 Langmuir XXXX, XXX, XXX−XXX
