Coeffect of Silk Fibroin and Self-Assembly Monolayers on the

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J. Phys. Chem. C 2008, 112, 15844–15849

Coeffect of Silk Fibroin and Self-Assembly Monolayers on the Biomineralization of Calcium Carbonate Xiaoqiang An and Chuanbao Cao* Research Center of Materials Science, Beijing Institute of Technology, Beijing 100081, People’s Republic of China ReceiVed: June 2, 2008; ReVised Manuscript ReceiVed: July 11, 2008

As is well-known, the formation of nacre is governed by the cooperation of soluble macromolecules and insoluble predeposited matrix which is composed of β-chitin and silk fibroin-like protein. However, little is known about the influence of silk fibroin on the biomineralization of CaCO3. In this paper, we described an effective mimic strategy in which growth of CaCO3 was well controlled by the cooperative interaction of silk fibroin molecules and short-chain silane self-assembled monolayers (SAMs). The diversification from calcite crystals, amorphous CaCO3 to aragonite crystals, accompanied with the structure transformation of silk fibroin molecules was first observed. We think these results offer useful and important information, which avails us to clarify the specific role of pure silk fibroin in the biomineralization process. 1. Introduction Nature has ingeniously succeeded in producing an impressive variety of inorganic functional structures with designed shapes and sizes on specific sites through a biologically controlled mineralization process. And recently, much effort has gone into this field to apply this strategy in the bioinspired preparation of advanced inorganic materials.1 There are at least three construction processes for biomineralization: supramolecular preorganization, interfacial molecular recognition, and assembly. Previous researches indicated that biological macromolecules were intimately associated with the mineralization process. Active biomacromolecules here can be divided into two classes, (i) soluble, hydrophilic polymers (mostly proteins/glycoproteins or polysaccharides) and (ii) insoluble matrix such as collagen and chitin which serves as a template for the mineral crystallization.2 However, due to the complexity in chemical composition, structure of a biological system, and complex interactions between them, the specific role of biomacromolecules is not clear. Furthermore, the detailed formation mechanism is still very difficult to elucidate. Mimicking experiments are essential to clarify the exact role of these biomolecules in the biomineralization mechanism. Polymer dispersions, synthetic block copolymers, proteins, micellar solutions, vesicles, and dendrimers were extensively used in the in vitro studies.3-7 Especially the recent promising methods such as Langmuir-Blodgett and self-assembled monolayer (SAM) techniques, which involve the interfacial molecular recognition at the organic-inorganic interfaces, have been employed as synthetic models of the biomacromolecules.8,9 The oriented growth and structure diversification of CaCO3 polymorphs were well controlled on these surfaces.10-12 The SAM technique offers us an ideal tool to research the molecule interactions in the mineralization process. But reports on the cooperative interactions between SAMs and solution additives are quite limited, which is a crucial aspect in the biomineralization.13,14 Especially the controllable growth of CaCO3 through * Corresponding author. E-mail: [email protected]. Tel.: +86 10 6891 3792. Fax: +86 10 6891 2001.

the interactions between SAMs and biomacromolecules is seldom researched. In our earlier work, the coeffect of multiple additives (silk fibroin molecules and magnesium ions) and SAMs on biomineralization was first researched.15 To elucidate the observed polymorph transitions clearly, research on the role of silk fibroin is urgently needed. In this paper, the coeffect of SAMs and silk fibroin molecules on the biomineralization of CaCO3 was well studied for the first time. The existence of SAMs availed the structure transition from silk I to silk II of biomolecules, and polymorph diversifications were easily accomplished through those cooperative interactions. As silk fibroin-like protein plays an important role in the formation of nacre, our result offers an effective way to find out the exact interaction mechanism in the biomineralization.16 2. Experimental Section The regenerated Bombyx mori silk fibroin solution and NH2terminated SAMs were prepared by the method described in our earlier paper.15 COOH-terminated SAMs were prepared through placing the glass substrate in the solutions of vinyltriethoxysilane (VTES, Tianjin Special type Chemical Reagents Development Center, China)/chloroform at 1 mM silane concentration. The terminal ethylenic double bonds were converted to -COOH groups through oxidation just as reference.17 For CaCO3 growth, the SAMs substrate was placed vertically in the 10 mM calcium chloride solution in which a certain amount of silk fibroin solution was added (with a concentration of 30 mg/mL). During those, the volumes of reaction solution were kept at 60 mL. Then it was placed in the desiccator containing ammonium carbonate. After being reacted for 24 h at 60 °C, the final substrate was rinsed with distilled water and dried for characterization. Precipitations of calcium carbonate in our experiments were resulted from the diffusion of carbon dioxide vapor into the CaCl2 solution, according to the following reactions:18

10.1021/jp804848q CCC: $40.75  2008 American Chemical Society Published on Web 09/12/2008

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(NH4)2CO3(s) f 2NH3(g) + CO2(g) + H2O CO2 + Ca2+ + H2O f CaCO3(s) + 2H+ A scanning electron microscope (SEM, Hitachi TM-1000) was used to observe the morphologies of the products. For “offline” SEM observation, a series of SAMs-modified substrates were displayed in the same reaction solution (10 mL of silk fibroin solution was used). They were taken out in sequence with different intervals and were directly analyzed by SEM and X-ray diffraction (XRD X Pert Pro MPD) using Cu KR radiation (λ ) 0.15418 nm). Fourier transform infrared (FT-IR, Digilab FTS-3100) spectroscopy was used to study the structure of the silk fibroin molecules hybridized in the film by a horizontal total-reflection accessory at room temperature. Photoluminescence spectroscopy (PL, Hitachi F-4500) was employed to study the structure transition of silk fibroin molecules. PL measurements were all performed at room temperature. The exciting wavelengths were 274 and 295 nm for tyrosine and tryptophan residues. 3. Results and Discussion The morphology of CaCO3 products was observed by SEM, and Figure 1 shows the results. Calcite crystals with a typical rhombohedral morphology were generally seen when no additive was added (Figure 1a). For the amorphous product in the 1 mL silk fibroin added solution (in a concentration of 0.5 mg/mL), compressed calcium carbonate that lay on the substrate was obtained (Figure 1b). When the amount of silk fibroin solution increased to 10 mL (in a concentration of 5 mg/mL), aggregates of needle-like crystals uniformly grew on the SAMs surfaces (Figure 1c). They were exactly wire bundles with a length of 10 µm, as could be seen in the higher magnification image shown in Figure 1d. The morphology of the precipitated products in the 10 mL protein added solution was also characterized, as shown in Figure 1e. Different from the needlelike crystals on the substrate, only some spherical particles were obtained. The product collected from the air-liquid interface was also characterized in Figure 1f. Thick film composed of spherical particles was obviously observed. The structures of as-synthesized products in our experiments were characterized by XRD. Typical calcite phase was confirmed in Figure 2a when no additive was introduced. The strong diffraction peak could be index to typical (104) plane of calcite (JCPDS card no: 86-1650). But when 1 mL of silk fibroin solution was added, only a broad peak centered at 25° is seen in Figure 2b, indicating the amorphous nature of the compressed CaCO3 in Figure 1b. For the needle-like product, further polymorph diversification was obviously seen when 10 mL of biomacromolecule solution was introduced. Except the peak located at about 29.8°, all of the other peaks were well consistent with those of the standard aragonite pattern (JCPDS card no: 75-2230). The peak marked with a / could be ascribed to the existence of a small amount of calcite crystals in the product. Aragonite phase is not the most thermodynamically stable state of CaCO3 aggregates. The wide existence of this structure in the natural minerals is believed to be related with the stabilization effect of biomacromolecules in the mineralization process. Thus, regulation effect of silk fibroin was deduced because of the growth of aragonite in our work. Figure 2d is the XRD pattern of the precipitation in Figure 1e, from which their amorphous nature was easily deduced. Similarly, the product collected from the air-liquid interface in Figure 2e was also amorphous. On the basis of the above polymorph diversifications

Figure 1. SEM images of CaCO3 products grown in the different systems: (a) typical calcite crystals on the SAMs (no silk fibroin added); (b) CaCO3 grown on the SAMs (1 mL of protein, in a final concentration of 0.5 mg/mL); (c and d) needle-like crystals (10 mL of protein, in a final concentration of 5 mg/mL); (e) precipitation collected from the bottom of the beaker (10 mL of protein); (f) CaCO3 film collected from the air-liquid interface.

of CaCO3, the existence of ordered organic surface and silk fibroin matrix both showed great influence to the growth of CaCO3. To find out the possible growth mechanism of CaCO3 in our system, contrast experiments were carried out by adjusting the reaction conditions, and those results are shown in Figure 3. In Figure 3, parts a and b, NH2-terminated SAMs were substituted by the untreated glasses and COOH-terminated SAMs, keeping the other condition as same as that in Figure 1c. It was found that the functional group of SAMs was essential for the controllable biomineralization of aragonite because only mixed phases of CaCO3 were obtained on untreated glasses and COOH-terminated SAMs. Some special reaction around the NH2-terminated SAM was reasonably deduced, availing the growth of aragonite in Figure 1c. Some other factors are also essential for the biomineralization of aragonite, including the concentration of silk fibroin, temperature, and the concentration of calcium ions. No aragonite crystals were achieved when any of them was unreachable. First, the regulated growth of aragonite could only be achieved at a certain macromolecule concentration. The decrease of silk fibroin solution from 10 mL resulted in the incomplete transition from amorphous to aragonite phase, whereas under higher protein concentration (15 mL of protein solution, in a final concentration of 7.5 mg/mL), the growth of aragonite was strongly inhibited and amorphous CaCO3 was achieved, as shown in Figure 3c. It was found that enhanced temperature availed the transition except the denaturalization of silk fibroin above about 100 °C. Mainly amorphous product was obtained in the parallel experiment carried out at room temperature even though a different volume of silk fibroin was added. Figure 3d was just the product when 10 mL of protein was used. The promotive effect at higher temperature was ascribed to the structure transition caused by the temperature, which was discussed in the IR and PL measurements below. Finally, the concentration of Ca2+ was also important for the

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Figure 3. (a) CaCO3 grown on untreated glasses (10 mL of protein); (b) CaCO3 grown on COOH-terminated SAMs (10 mL of protein); (c) amorphous CaCO3 when 15 mL of protein was used (in a final concentration of 7.5 mg/mL); (d) amorphous CaCO3 grown at room temperature (10 mL of protein); (e) CaCO3 grown when 5 mM CaCl2 and 10 mL of silk fibroin were used; (f) CaCO3 grown when 20 mM CaCl2 and 10 mL of silk fibroin were used. Figure 2. XRD patterns of CaCO3 products: (a) no biomolecule was added; (b) 1 mL of silk fibroin solution was used; (c) 10 mL of protein solution was added; (d) precipitation in the solution; (e) CaCO3 film collected from the air-liquid interface.

biomineralization. The decrease of calcium ions led to the appearance of several irregular calcite crystals, as seen in Figure 3e, whereas polyhedral calcite was obtained when 30 mM CaCl2 was used, in Figure 3f. Here, we thought different Ca2+ concentration resulted in different bulk supersaturation, which influenced the final polymorph of CaCO3. On the basis of these, the polymorph choice of CaCO3 was the integrative result of the above factors. The relationship between the final polymorph and these factors is well illustrated in Scheme 1. To clarify the configuration of silk fibroin molecules in the organic-inorganic hybrids, the horizontal total-reflection infrared spectrum was used to characterize these products. Figure 4 displays the IR spectra of the silk fibroin on the untreated glass substrates (Figure 4a), silk fibroin on the NH2-terminated SAMs (Figure 4b), CaCO3 precipitations in the solution (Figure 4c), amorphous CaCO3 in Figure 1b (Figure 4d), and aragonite crystals in Figure 1c (Figure 4e). In the spectrum in Figure 4a, the characteristic peaks at 1658 and 1535 cm-1 were assigned to the amide I and amide II stretching bands of the silk I structure.19 These peaks shifted to 1643 and 1529 cm-1 in Figure 4b, approaching those of the silk II structure, which were located at 1640-1620 and 1512-1530 cm-1. This indicated that the ordered surface availed the structural transition of silk fibroin molecules, toward silk II structure. Similar shifts of the amide I stretching band were also observed in the spectra on Figure 4, parts c and d. They were located at 1645 and 1642 cm-1, respectively. The accomplishment of transition from silk I to silk II for the silk fibroin hybridized in the aragonite minerals was proved by the peak at 1630 cm-1.20 On the basis of these

SCHEME 1: Formation of CaCO3 Products in Different Experimental Conditions

IR results, we can know that the diversification of CaCO3 was intimately associated with the protein structure. More details about silk fibroin molecules in the products were studied by the PL measurements. Fluorescence of Trp and Tyr residues are often used as intrinsic probes of protein hydrophobic or hydrophilic interactions.21 And this hydrophobic and hydrophilic environment is directly related with the structure transition of protein. Figure 5 showed the PL spectra of the products. The spectra a, b, and c were corresponding to the CaCO3 precipitation in the solution, the amorphous CaCO3, and the aragonite crystals on the SAMs. For our regenerated silk fibroin solution, the emission at 350 nm with an exciting wavelength of 295 nm contained a distinct contribution from the Trp residues. The measured emission at 310 nm with an exciting wavelength of

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Figure 5. PL spectra of the Tyr (a) and Trp (b) residues. Here, a, b, and c are corresponding to the CaCO3 precipitation in the solution, the amorphous CaCO3, and the aragonite crystals grown on the SAMs.

Figure 4. IR spectra of the products at room temperature: (a) silk fibroin molecules on the untreated glass substrate; (b) silk fibroin molecules on the NH2-terminated SAMs; (c) CaCO3 precipitation in the solution; (d) amorphous CaCO3 in Figure 1b; (e) aragonite crystals in Figure 1c. The strong peak at about 1385 cm-1 in spectra c-e is the asymmetric C-O stretching vibration in CaCO3.

274 nm was originated from the Tyr residues. For the Trp residues in Figure 5a, emission peaks located at 364 and 362 nm were detected for spectra a and b. A more hydrophilic situation was deduced from the red shift compared to the characteristic 350 nm emission of the regenerated SF solution. Oppositely, this peak position has blue-shifted to 325 nm for the aragonite product, indicating a more hydrophobic environment. Similar changes had also been observed. In Figure 5b. The Tyr emission peaks were located at 340, 330, and 308 nm, respectively for the samples a, b, and c. Here, we believed that the protein molecules in the amorphous CaCO3 were in an intermediate state during the structural transition, which was also observed in the other reports.22 In these partially changed silk I structures, Tyr and Trp residues were exposed in the hydrophilic environment because of the so-called hydrophobic collapse process.23 But when the structure transformation completed in spectrum c, silk fibroin was in a β-pleated silk II structure. Tyr and Trp residues were well hidden in the hydrophobic core because of the hydrophobic interactions. Thus, higher exciting energy was needed for this structure, which resulted in the blue shift in spectrum c. In the earlier reports, 2-D ordered, anionic SAMs could serve as an active interface for heterogeous nucleation so that the

Figure 6. “Offline” SEM images of aragonite growth after different intervals: (a) 1, (b) 3, (c) 6, (d) 9, (e) 12, and (f) 15 h.

energetic barrier to nucleation and the threshold saturation required for nucleation of CaCO3 were reduced.24 However, NH2-terminated SAMs were much more favorable for the nucleation of CaCO3 than the COOH-terminated ones in our work. In addition, formation of aragonite crystals could only be achieved on the NH2-SAMs through specific interactions. All of those indicated that using protein changed the mineralization characteristic around the SAMs surface, just similar as the in vitro interactions between chitin and silk fibroin studied by Falini et al.25 Approved by the X-ray photoelectron spectroscopy (XPS) result, we assumed a cooperative interaction between SAMs and protein here.15 It was possible that carboxyl in silk

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SCHEME 2: Proposed Growth Mechanism of CaCO3 on the Biomacromolecule-Modified SAMs at Lower (a) and Higher (b) Silk Fibroin Concentrationa

a

60 °C.

fibroin molecules was much more active during that structure. Thus, the formation of the CO-NH bond was more favorable on the NH2-terminated SAMs, resulting in the different product on COOH-SAMs in Figure 3b. In fact, silklike protein was an important biomolecule in the mineralization process. It was assumed that the silklike insoluble molecules in a β-pleated structure offered the insoluble matrix for the CaCO3 film deposition.26,27 But its exact role in the nacre formation was still unknown. Recently, a hydrated gel phase of silk fibroin was detected by Kalisman et al., which was essential for the nucleation and crystal growth in mollusc shell formation.28 In our work, although the gel was not formed, the concentration of silk fibroin solution (2.9 wt %) was just similar with that in the nacreous matrix of Atrina (2.5 wt %).29 Thus, similar structural transition from silk I to silk II was expected during the reaction process, which was proved by the IR and PL measurements. The successful transition to β-sheet structure could be achieved under certain conditions (such as concentration, temperature, the existence of SAMs in our work).30 As reported, this preorganization of macromolecules played an important role in the natural mineralization. Thus, the sequent structural readjustment and reorganization around the SAMs surface in our experiments is crucial. In addition, aragonite crystals were obtained neither in the silk I solution nor on the silk II film. Also no aragonite was observed during biomineralization at room temperature, using 60 °C pretreated silk fibroin solutions. We assumed that the following two processes were necessary for the formation of aragonite: the former is the inhibited growth of amorphous CaCO3 in silk I matrix, the latter is their amorphous-aragonite diversification together with the structural transition of biomolecules. Having known these, it is necessary to compare our results with the natural minerals. Is there also a similar growth mechanism of nacreous matrix upon aging of silk fibroin molecules? Does silk fibroin play some additional roles in the mineralization of CaCO3, not only as insoluble matrix for deposition? We believe these results might serve some useful information, which need more future research. On the basis of the above characterizations, the proposed growth mechanism of CaCO3 on the self-assembled surface is well illuminated in Scheme 2. First, in the low silk fibroin concentration (Scheme 2a), intermediate-state related structure was achieved at elevated temperature. Then, the hydrophobic collapse process formed abundant exposed polar groups, which generated strong electrostatic interactions with the calcium cations (Scheme 2a-I). Growth of CaCO3 was highly restrained in this protein matrix with partial structural change. Thus, only amorphous CaCO3 products (Figure 3b) were formed because of this macromolecule restrictive effect (Scheme 2a-II).31 Here, we thought the distribution of silk fibroin molecules around the

substrate surface led to the appearance of compressed CaCO3, compared to the spherical particles in the solution. Second, when the certain situation was satisfied in Scheme 2b (higher temperature, enough concentration, and ordered organic surface in the contrast experiments), further transformation to β-pleated protein occurred on the SAMs (Scheme 2b-I). The functional groups of macromolecules incorporated into the CaCO3 products and changed the kinetics of growth.32 Accompanied by the secondary structure transition of silk fibroin (Scheme 2, parts b-II and b-III), the amorphous-aragonite diversification of CaCO3 gradually completed (see Figure 3d).7,33 To validate the above transformation mechanism of CaCO3, we conducted an “offline” SEM analysis to track the mineralization process of aragonite in silk fibroin matrix. A series of SAMmodified substrates were dipped into the same reaction solution. They were taken out with different intervals for direct SEM observation. Figure 6 is the representative SEM images of CaCO3 after 1, 3, 6, 9, 12, and 15 h of reaction. It was observed that stubbed amorphous CaCO3 formed on the substrate at the early stage of reaction (Figure 6, parts a and b). The transition from egglike amorphous particles to spindle-shaped aragonite crystals gradually began after 6 h of reaction, which could be easily seen in Figure 6c-e. From the image of product with 15 h of reaction in Figure 6f, we could clarify that CaCO3 had already possessed the final bundle-needle morphology because of the structure splitting of aragonite. These results obtained from the “offline” SEM observations are in good agreement with the mechanism illustrated in Scheme 2. 4. Conclusion Biomineralization of CaCO3 by the cooperative interactions between silk fibroin molecules and short-chain silane SAMs was first investigated. The appearances of amorphous and aragonite phase CaCO3 was found to be associated with the structure transition of silk fibroin molecules. An intermediate-state related silk I structure was assigned to the inhibited growth of amorphous CaCO3. And the further transformation from silk I to silk II β-pleated structure was essential to the growth of aragonite crystals. Is there a resemblance between this amorphous-aragonite transition and the formation of nacre in the nature? Of course, the above results might offer some essential and important information for further in vitro or in vivo work. Acknowledgment. This work was supported by the National Science Foundation of China via Grant No. 20471007. Supporting Information Available: XRD patterns of products in Figure 3, parts c and d, and Figure 6, parts a and b.

Biomineralization of Calcium Carbonate This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Gao, Y.; Koumoto, K. Cryst. Growth Des. 2005, 5, 1983. (2) Albeck, S.; Aizenberg, J.; Addadi, L.; Weiner, S. J. Am. Chem. Soc. 1993, 115, 11691. (3) Kim, I. W.; Robertson, R. E.; Zand, R. Cryst. Growth Des. 2005, 5, 513. (4) Sugawara, A.; Kato, T. Chem. Commun. 2000, 6, 487. (5) Lee, I.; Han, S. W.; Lee, S. J.; Choi, H. J.; Kim, K. AdV. Mater. 2002, 14, 1640. (6) Naka, K.; Tanaka, Y.; Chujo, Y.; Ito, Y. Chem. Commun. 1999, 19, 1931. (7) Volkmer, D.; Harms, M.; Gower, L.; Ziegler, A. Angew. Chem., Int. Ed. 2005, 44, 639. (8) Aizenberg, J.; Black, A. J.; Whitesides, G. M. J. Am. Chem. Soc. 1999, 121, 4500. (9) Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature 1999, 398, 495. (10) Lee, I.; Han, S. W.; Choi, H. J.; Kim, K. AdV. Mater. 2001, 13, 1617. (11) Travaille, A. M.; Kaptijn, L.; Verwer, P.; Hulsken, B.; Elemans, J. A. A. W.; Nolte, R. J. M.; van Kempen, H. J. Am. Chem. Soc. 2003, 125, 11571. (12) Wei, H.; Ma, N.; Shi, F.; Wang, Z.; Zhang, X. Chem. Mater. 2007, 19, 1974. (13) Han, Y. J.; Aizenberg, J. J. Am. Chem. Soc. 2003, 125, 4032. (14) Balz, M.; Therese, H. A.; Li, J.; Gutmann, J. S.; Kappl, M.; Nasdala, L.; Hofmeister, W.; Butt, H.; Tremet, W. AdV. Funct. Mater. 2005, 15, 683. (15) An, X. Q.; Cao, C. B. J. Phys. Chem. C 2008, 112, 6526.

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