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Biomineralization of CaCO3 through the Cooperative Interactions between Multiple Additives and Self-Assembled Monolayers Xiaoqiang An and Chuanbao Cao* Research Center of Materials Science, Beijing Institute of Technology, Beijing 100081, People’s Republic of China ReceiVed: NoVember 20, 2007; In Final Form: January 18, 2008
In this paper, the biomineralization of calcium carbonate through the cooperative interactions between selfassembled monolayers (SAMs) and multiple soluble additives was first investigated. The polymorphs and morphologies of products were well controlled by the existence of ordered surface and the co-effect of silk fibroin and magnesium ions. It was found that polymorphs and morphologies of products were determined by silk fibroin, magnesium ions had a promotional effect at lower concentration of magnesium ions, while at higher concentration of magnesium ions, the growth of vaterite crystals was stabilized. This work avails us to clarify the exact role of silk fibroin and inorganic ions in the biomineralization mechanism under a mimicking condition approaching the natural environment.
1. Introduction Biomineralization has recently received much attention from material scientists, because these biomaterials form inorganicorganic hybrid composites with controlled hierarchical structures, which show significant properties.1 It is well-known that biomacromolecules such as proteins and polysaccharides play key roles during the production process of inorganic substrates by living organisms. Active biomacromolecules here can be divided into two classes: (i) soluble, hydrophilic proteins and (ii) an insoluble matrix which serve as a template for the mineral crystallization.2 The crystal habit, morphology, as well as the size and shape are well controlled by interactions between them (lattice matching, charge matching, stereochemistry complementarity, spatial localization, etc.).3 Elucidation of the biomineralization mechanism is essential for the design of new materials. Thus series of in vivo and in vitro experiments have been carried out to mimic this process. Soluble additives (such as protein, synthetic block copolymers, and dendrimers) or highly ordered surfaces (such as Langmuir and SAMs) have been employed as synthetic models, acting as the soluble or insoluble matrix.4-7 Recently, mineralization through the cooperative interactions between SAMs and soluble additives offers us an ideal tool to research the interfacial molecular recognition.8,9 In our earlier work, the calciteamorphous-aragonite polymorph diversification was achieved by these interactions between silk fibroin molecules and SAMs.10 However, the natural environment during the biomineralization process is complex and the influence of inorganic ions should be considered, especially for Mg2+, which is known to exert a significant effect on precipitation.11 Research on the cooperative interactions between SAMs and multiple additives may give us more useful information. In this paper, biomineralization of CaCO3 by the cooperative interactions between soluble multiple additives (silk fibroin and Mg2+) and SAMs was first investigated. The polymorph and morphology of * Corresponding author. E-mail:
[email protected]. Phone: +86 10 6891 3792. Fax: +86 10 6891 2001.
CaCO3 were well controlled through the co-effect and the possible mechanism was put forward. 2. Experimental Section 2.1. Preparation of Regenerated Bombyxmori Silk Fibroin Solution. Raw silk was degummed with 0.5% (w/w) Na2CO3 solution at 100 °C for 60 min and then washed with distilled water. Degummed silk was dissolved in a solvent system of CaCl2/H2O/EtOH solution (1/8/2 in mole ratio) for 40 min at 80 °C and dialyzed to remove salts in a cellulose tube against distilled water for 3 days at room temperature. Then the SF solution was filtered.12 2.2. Preparation of SAMs on Substrates. Carefully cleaned glass slides were oxidized by immersing in a piranha solution (70:30 concentrated H2SO4:H2O2 (30% v/v)) for 0.5 h at 80 °C. Then they were immersed in NH3:H2O2:H2O (1:1:5 (v/v)) solution for 12 h to achieve hydroxylation, blow-dried with nitrogen, and placed in the solutions of 3-aminopropyltriethoxysilane (APTS, NH2(CH2)3Si(OC2H5)3, Geel Chemicals, Belglum)/benzene at 1 mM silane concentration. They were kept immersed in the solution for 24 h at room temperature. Then these substrates were washed in benzene, acetone, and distilled water and dried in vacuum at 120 °C to remove the excess reactant. 2.3. Growth of CaCO3 on the SAMs Surfaces. The SAMs modified substrate was placed vertically in the calcium chloride solution in which a certain amount of magnesium chloride and silk fibroin solution were added. The beaker was placed in the desiccator containing ammonium carbonate. After reacted for 24 h at 60 °C, the final substrate was rinsed with distilled water and dried for characterization. 2.4. Characterization of Products. The formation of uniform SAMs on the substrate was confirmed by the contact angles measurement system (JY-82, Chengde Dahua Testing Machine Ltd., China) and atomic force microscopy measurements (AFM, Nano Scope IIIa, Digital Instruments Inc.). A scanning electron microscope (SEM, Hitachi TM-1000) was used to observe the morphologies of the products. The structure of the products was
10.1021/jp711044s CCC: $40.75 © 2008 American Chemical Society Published on Web 04/02/2008
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Figure 1. AFM images of (a) hydroxylated substrate and (b) SAMs modified substrate.
Figure 3. (a-c) SEM images of CaCO3 products grown when 10 mL of silk fibroin and 10 (a), 20 (b), and 50 mM Mg2+ (c) are added. (d-f) SEM images of CaCO3 products grown when 20 mL of silk fibroin and 10 (d), 20 (e), and 50 mM Mg2+ (f) are added.
Figure 2. SEM images of CaCO3 grown on (a) hydroxylated substrate and (b) APTS SAMs. SEM images of CaCO3 grown on SAMs in the presence of (c) 10 mM Mg2+, (d) 20 mM Mg2+, (e) 10 mL of silk fibroin, and (f) 20 mL of silk fibroin.
examined by X-ray diffraction analysis (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. 3. Results and Discussion After treatment by NH3/H2O2/H2O, the measured contact angles of the glass substrate for water are close to 0°, which demonstrate good hydroxylation. And the average contact angles of APTS SAMs modified glass change to 45°. The AFM images of the hydroxylated and SAMs modified substrates are shown in Figure 1. Contrasted to the bare glass, uniform nanoparticles are seen in Figure 1b, showing evidence of the formation of uniform organic film on the substrate. Panels a and b of Figure 2 are the SEM images of CaCO3 grown on the hydroxylated and APTS SAMs modified substrates. There is no obvious difference between the morphologies of rhombohedral calcite crystals, indicating that the single SAMs have no influence on the product. Panels c-f of Figure 2 are the CaCO3 products on SAMs when 10 mM Mg2+, 20 mM Mg2+, 10 mL of silk fibroin, and 20 mL of silk fibroin are used as single additives, respectively. When 10 mM Mg2+ is added in Figure 2c, irregular calcite crystals appear. Further morphol-
ogy diversification is seen when the Mg2+/Ca2+ ratio increases to 2; the elongated crystals indicate the promoted structure transition to aragonite in Figure 2d. This promotional effect is deficient because of the existence of plenty of rhombohedral crystals. This is consistent with the fact that the reported optimal Mg2+/Ca2+ ratio for aragonite growth is 4.13 The absolute calcite-aragonite transition is successfully achieved through the cooperative interactions between protein and SAMs. Only needle-like aragonite crystals are seen in Figure 2e. The concentration of silk fibroin is crucial for the polymorph transition because only amorphous CaCO3 are obtained when 20 mL of silk fibroin is added into the reaction solution (Figure 2f). This is ascribed to the unreachability of Silk I to Silk II transition and the inhibitive growth of CaCO3 in the protein matrix, which has been well discussed in another paper. On the basis of the above results, the coexistence of SAMs and additives shows cooperative interactions to the polymorph of CaCO3. In addition, different morphologies are seen when protein and magnesium ions are used respectively. Their influence on the biomineralization is further researched in Figure 3. Panels a-c of Figure 3 are the products grown when 10, 20, and 50 mM Mg2+ are added, respectively, accompanied by 10 mL of silk fibroin solution. Compared to the needle-like structures in Figure 2e, the addition of 10 mM Mg2+ leads to the appearance of cluster-like aragonite crystals in Figure 3a. Growth of separate aragonite nanorods is greatly promoted when the Mg2+/Ca2+ ratio increases to 2. The appearance of nanorods with the diameter of 500 nm and the length of several microns in Figure 3b indicates their preferential c-axis growth. A further increase of Mg2+/Ca2+ ratio to 5 results in the formation of vaterite spheres in Figure 3c, the thermodynamics most unstable polymorph of CaCO3. Although different concentrations of Mg2+ are tried, aragonite crystals cannot be achieved when the concentration of silk fibroin increases to 20 mL, because of the stronger inhibitive effect of protein. When 20 mL of silk fibroin
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Figure 4. XRD patterns of aragonite nanorods (a), discus-like amorphous CaCO3 (b), and vaterite film (c) in our work.
companied by 10 mM Mg2+ are added, as-obtained CaCO3 product is still an amorphous structure. What is different from those in Figure 2f is that the splitting of the discus-like amorphous CaCO3 into nanosheets is obviously seen (Figure 3d). Addition of a higher concentration of Mg2+ also results in the formation of vaterite structures, indicating that multiple additives are favorable for the stabilization of this unstable phase. On the basis of the higher magnification image in Figure 3e, we can know that the flower-like vaterite crystals are attributed to the aggregated growth of nanosheets under 20 mM Mg2+. They further aggregate into vaterite film when the Mg2+ concentration increase to 50 mM. The structure diversification of CaCO3 product in our work is determined by XRD measurement. Figure 4a shows the XRD pattern of aragonite nanorods, in which all the peaks can be index to the standard aragonite pattern (JCPDS Card No: 752230). The amorphous nature of the splited discus-like CaCO3 (20 mL of silk fibroin and 10 mM Mg2+) is easily deduced from the broadened diffraction peak around 23° in Figure 3b. Their polymorph transition to the vaterite phase under higher Mg2+ concentration is well confirmed through the pattern shown in Figure 3c, in which all the peaks can be index to the standard vaterite phase (JCPDS Card No: 74-1867). On the basis of our above results, we think silk fibroin plays an important role in the biomineralization of CaCO3. The molecule structures of protein hybridized in the product are studied by IR measurement, as shown in Figure 5. As we know, the amide I stretching band of silk fibroin is located at 16601640 cm-1 for the Silk I structure, while it is located at 16401620 cm-1 for the Silk II structure.14 For the cluster-like aragonite crystals and aragonite nanorods in Figure 5a,b, the amide I bands are located at 1630 and 1620 cm-1, respectively, indicating their accomplishment of transition to the Silk II
Figure 5. IR spectra of CaCO3 products: (a) cluster-like aragonite crystals; (b) aragonite nanorods; (c) discurs-like CaCO3; (d) flowerlike CaCO3; and (e) vaterite film.
Figure 6. SEM images of amorphous CaCO3 precipitation in the solution of Figure 3b (a) and Figure 3e (b).
structure. A different structure is observed for the silk fibroin molecules hybridized in amorphous CaCO3, flower-like vaterite crystals, and vaterite film. Silk I structure is easily confirmed through the amide I peak located at around 1640 cm-1 in Figure 5c-e. The formation of vaterite structures under higher Mg2+ concentration is also proved by the splitting of the strong peak around 1450 cm-1 and the appearance of a characteristic peak located at about 875 cm-1.15
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Figure 7. XPS spectra of silk fibroin-SAMs modified substrates: (a) C1s and (b) N1s.
SCHEME 1: Supposed Growth Mechanism of Aragonite Crystals on the SAMs, Using Silk Fibroin and Magnesium Ions as Additives
To investigate the possible growth mechanism of CaCO3, the precipitations obtained in solutions from Figure 3b,e are also characterized, and their SEM images are shown in Figure 6. Different from the aragonite and vaterite crystals on the SAMs in panels b and e of Figure 3, only amorphous spherical particles are observed, indicating the failure to achieve the polymorph transition in the soluble additive matrix. This also proves that the cooperative interactions between the soluble additives and SAMs are essential for the polymorph adjustment of CaCO3 in the mineralization process. Here, as there are plenty of carboxyl functional groups in silk fibroin, electrostatic interactions between the amido of SAMs and the carboxyl of protein molecules might dominate these cooperative interactions.16 This interaction is further confirmed by the XPS characterization of silk fibroin-SAMs modified substrates in Figure 7, during which a SAMs modified substrate is dipped into silk fibroin solution for 10 min. Binding energies corresponding to CsC/ CsH, CsO, CdO, and -COO are seen in the C1s spectrum (Figure 7a). The peaks in Figure 7b are ascribed to N-H and -C(dO)sN- of N1s, indicating the linkage of silk fibroin molecules to SAMs. In our mimicking strategy, the possible growth mechanism is supposed as below: silk fibroin molecules are first absorbed on SAMs through electrostatic interactions, followed by a hydrophobic structure adjustment. Then mineral nuclei are formed among the matrix. Finally, the simultaneous addition of Mg2+ also shows a co-effect on the growth of nuclei and morphology of CaCO3. These processes are well illustrated in Scheme 1, which is the proposed growth mechanism of aragonite crystals. As seen in Figure 3, this growth is affected by the ratio of protein and Mg2+ ions. At the lower concentration of Mg2+, polymorphs of CaCO3 are mainly determined by the structure of silk fibroin. The growth of CaCO3 is strongly inhibited in the Silk I matrix, which is rather favorable for the growth of amorphous CaCO3.17 Aragonite crystals can only be obtained when the structure of silk fibroin successfully transited from Silk I to Silk II. Addition of Mg2+ shows the promotional effect on the c-axis growth of CaCO3. There may be two possible reasons: First, the structure transition of silk fibroin to Silk II is promoted with the addition of Mg2+ (the red shift value increases along with the increase of the ratio of Mg2+/
Ca2+). Second, magnesium ions themselves also have the effects of inhibiting calcite formation and promoting c-axis growth because of their specific absorption on the planes parallel to the c-axis.18 It is believed that there is a competition between the inhibited growth in the protein matrix and promoted c-axis growth by Mg2+, which leads to the formation of the aragonite nanorods and the splitting of the discus-like amorphous product. At the higher concentration of Mg2+, the surface free energy of product is decreased and growth of vaterite crystals is stabilized, which is the most unstable phase of CaCO3 in nature.19 4. Conclusion In summary, the cooperative interactions between SAMs and multiple additives were first used to mimic the mineralization of CaCO3. Addition of soluble silk fibroin molecules and magnesium ions showed a co-effect on the morphology of products. Polymorph and morphology diversifications were achieved under different protein concentrations and Mg2+/Ca2+ ratios. At lower concentration of Mg2+, the final polymorph was determined by the structure of silk fibroin. Mg2+ showed the promotional effect on the c-axis growth of CaCO3, which led to the formation of aragonite nanorods and the splitting of amorphous CaCO3 into nanosheets, while at a higher concentration of Mg2+, the growth of CaCO3 was further inhibited and the soluble multiple additives showed the perfect stabilizing effect on the vaterite products. Acknowledgment. This work was supported by the National Science Foundation of China via Grant No. 20471007. 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) Ferster D. Science 2004, 303, 1618. (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) Volkmer D.; Harms M.; Gower L.; Ziegler A. Angew. Chem., Int. Ed. 2005, 44, 639. (7) Aizenberg J.; Black A. J.; Whitesides G. M. Nature 1999, 398, 495.
6530 J. Phys. Chem. C, Vol. 112, No. 16, 2008 (8) Han Y. J.; Aizenberg J. J. Am Chem. Soc. 2003, 125, 4032. (9) 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. (10) An X.; Cao C.; Zhu H. Biomineralization of CaCO3 by the cooperative interaction of SAMs and silk fibroin molecules. Cryst. Growth Des. Submitted for publication. (11) Falini G.; Albeck S.; Weiner S.; Addadi L. Science 1996, 271, 67. (12) Ma X. L.; Cao C. B.; Zhu H. S. J. Biomed. Mater. Res. B 2006, 78B, 89. (13) Mann, S.; Dldymus, J. M.; Sanderson, N. P.; Heywood, B. R. J. Chem. Soc., Faraday Trans. 1990, 86, 1873.
An et al. (14) Lv, Q.; Cao, C. B.; Zhang, Y.; Ma, X. L.; Zhu, H. S. J. Appl. Polym. Sci. 2005, 96, 2169. (15) Zhan, J. H.; Lin, H. P.; Mou, C. Y. AdV. Mater. 2003, 15, 621. (16) Wang, X.; Kim, H. J.; Xu, P.; Matsumoto, A.; Kaplan, D. L. Langmuir 2005, 21, 11335. (17) Jiao, Y.; Feng, Q.; Li, X. Mater. Sci. Eng. C 2006, 26, 648. (18) Zhu, L.; Zhao, Q.; Zheng, X.; Xie, Y. J. Solid State Chem. 2006, 179, 1247. (19) Addadi, L.; Joester, D.; Nudelman, F.; Werner, S. Chem. Eur. J. 2006, 12, 980.
