CRYSTAL GROWTH & DESIGN
Preparation of Calcite and Aragonite Complex Layer Materials Inspired from Biomineralization Rui Liu,† Xurong Xu,*,† Yurong Cai,§ Anhua Cai,† Haihua Pan,† Ruikang Tang,*,† and Kilwon Cho‡
2009 VOL. 9, NO. 7 3095–3099
Department of Chemistry and Centre for Biomaterials and Biopathways, Zhejiang UniVersity, Hangzhou, 310027, China, Department of Chemical Engineering, Polymer Research Institute, Pohang UniVersity of Science and Technology, Pohang, 790-784, Korea, and College of Materials and Textile, Zhejiang Sci-Tech UniVersity, Hangzhou, 310018, China ReceiVed August 8, 2008; ReVised Manuscript ReceiVed April 29, 2009
ABSTRACT: Abalone shell is natural inorganic/organic hybrid material, which is biologically constructed by using two calcium carbonates, aragonite and calcite. Aragonite can provide an enhanced mechanical support for the shell, and the stable phase, calcite, acts as the outer layer. In the current study, large-area aragonite film is deposited onto a calcite film in the presence of magnesium ion to biomimetically construct an aragonite-calcite complex structure. A calcite film is fabricated on a silicon wafer by a controlled phase transformation of amorphous calcium carbonate film at 120 °C, which is used as a substrate to induce the deposition of the aragonite layer with the addition of magnesium ion. We demonstrated the importance of the transition process from calcite to aragonite during the formation of the aragonite-calcite complex layer. This study could be used to mimic a sharp transition process of calcite to aragonite in vitro without any organic macromolecules. Introduction Calcium carbonate is one of the most extensively studied biominerals and is found to exist widely in living organisms such as sea shell, coral, sea urchin spine, etc. Calcium carbonate has different crystallized phases in nature. Thermodynamically, calcite is the most stable phase of calcium carbonate in aqueous solutions, and it is frequently observed in calcium biominerals. However, aragonite, a metastable phase of calcium carbonate, is also often found in mollusk shells. Interestingly, the precipitated aragonite can be stabilized in living systems. Generally, biominerals, natural inorganic/organic hybrid materials, are formed through a cooperation of inorganic materials with organic macromolecules, where the macromolecules control the nucleation, growth, morphology, structure, and crystal orientation of the inorganic components. It is noted that biominerals are hierarchically constructed under mild solution conditions.1 It is believed that different biomacromolecules play a key role in the biomineralization of calcium carbonate. Calcite and aragonite crystallites can be in vivo induced by calcite-related and aragonite-related proteins, respectively.2 Belcher, et al. have confirmed that soluble macromolecules can control the crystal phases of calcium carbonate so that the thermodynamically metastable aragonite is precipitated.3 It is well-known that the abalone shell has layer structure of aragonite (brick) and organic macromolecules (mortar), which results in extremely strong mechanical strength. This characteristic structure is very attractive since it can be used to achieve new materials with improved functions. In biomineralization, there are two kinds of organic macromolecules: insoluble and soluble macromolecules, which play an important role in deposition of inorganic minerals. Various organic templates including Langmuir monolayers4 and self-assembled monolayers (SAMs)5 have been used to induce the crystallization of CaCO3. Aizenberg, et al.6 have used SAMs to mimic and demonstrate * To whom correspondence should be addressed. Fax: 86-571-87953736; tel: 86-571-87953736; e-mail:
[email protected] (X.X.);
[email protected] (R.T.). † Zhejiang University. § Zhejiang Sci-Tech University. ‡ Pohang University of Science and Technology.
the effect of insoluble matrix on nucleation of inorganic crystals. Co¨lfen et al.7 have used double-hydrophilic block copolymers to biomimetically show the effect of soluble macromolecules on adjusting morphology and crystal phase. From the mechanism of the nacre deposition suggested by Addadi et al.,8 the mineral phase is formed within the organic matrix and the nucleation of the aragonite is induced by the acidic proteins. Different CaCO3 thin films have been produced using the cooperation between insoluble matrices and soluble macromolecules.9 However, it is more difficult to obtain the pure aragonite film than the stable calcite film. Recently, Kato et al.10 biomimetically fabricate aragonite thin film in the presence of chitosan matrices, poly(aspartate) and Mg2+. Litvin et al.11 demonstrate that aragonite crystals can be induced under a Langmuir monolayer of 5-hexadecyloxyisophthalic acid (C16ISA). Although aragonite is the main component of abalone shell due to its distinct mechanical properties, a layer of calcite actually locates in the outer portion of the shell, which is often ignored in the study of biomineralization. Actually, biomineralization of the shell generally starts from the outer calcite layer, and the shell has a complex structure of calcite-aragonite. An abrupt transition from calcite to aragonite has been observed when inorganic substrates are inserted into abalone shell.12-14 It means that living organisms can induce the growth of metastable aragonite phase onto a stable substrate of calcite. Here we show a process to biomimetically construct calcite and aragonite complex layer materials without any organic macromolecules. Experimental Section Substrate. Silicon wafers were cleaned by 70% (v/v) H2SO4 solution at temperature of 100 °C, and then were rinsed thoroughly with distilled water and were dried by nitrogen gas. Calcite Film.17,18 Six grams of ammonium carbonate powder was placed at the bottom of a desiccator. A vial containing 50 mM calcium chloride (CaCl2) solution was placed in the desiccator (L ) 20 cm). The cleaned silicon wafer was inverted and placed above the CaCl2 solution. A CaCO3 thin film was induced on the cleaned substrate via slow diffusion of CO2, produced by the decomposition of ammonium carbonate at room temperature (50 min). The film was rinsed with ethanol and was dried by nitrogen gas. The formed amorphous calcium
10.1021/cg800872j CCC: $40.75 2009 American Chemical Society Published on Web 05/15/2009
3096
Crystal Growth & Design, Vol. 9, No. 7, 2009
Liu et al.
Figure 1. Calcite substrate: (a) cross polarized microphotograph and (b) SEM image.
Figure 2. SEM images of the resulting multilayer films after 24 h deposition: (a) low magnification; (b) high magnification. carbonate (ACC) film was heated at 120 °C for 48 h and the calcite film was obtained. Aragonite Film. Five grams of ammonium carbonate powder was used as the resource of carbon dioxide. A 25 mL vial with mixed solution containing 10 mM magnesium chloride (MgCl2) and 10 mM CaCl2 was put into the desiccator. The calcite film was inverted and placed above the mixed solution. Different deposition times were applied in the experiments, and the reaction temperature was 25 °C. The resulting film was rinsed with ethanol and was dried by nitrogen gas. Bare silicon wafer was also used as a substrate to repeat the experiment. The control experiment was performed without Mg ion in solution. Film Characterization. The CaCO3 thin films on the silicon wafers were observed using an optical microscope equipped with a cross polarizer in a reflective mode (Zeiss, Germany). High-resolution images of the films (Pt-coated prior to examination) were examined using a field emission scanning electron microscope (FESEM, Hitachi S-4200, Japan) at an operating voltage of 8 kV. IR analysis was performed by a Nexus 670 (Nicolet, USA), and the cleaned silicon wafer was used as the reference. X-ray diffraction (XRD) was studied by a D/max2550PC (Rigaku, Japan).
Results and Discussion Amorphous calcium carbonate (ACC) is a kind of calcium carbonate phase, which has high solubility. Usually, ACC is unstable in aqueous solution, and its presence during the formation of calcium carbonate minerals is always ignored. However, Addadi et al.15 have revealed an important role of ACC in biomineralization, which acts as a precursor during the calcium carbonate crystallization. Recently, the studies of ACC have received extensive attention.16 Previously, we suggested a simple method to prepare ACC film and studied the phase transformation of the ACC film.17 Calcite film would be obtained when the ACC film was heated at a temperature of >100 °C.18 Inspired from nature that the aragonite layers were deposited onto the existing calcite outer layer during the biological formation of the shells, calcite film was used as the
substrate to induce the deposition of aragonite so that a multilayer calcium carbonate film with different phases could be biomimetically synthesized. When ACC film was heated at 120 °C for 48 h, a crystallized film was obtained, which showed typical crystal character under cross-polarized light (Figure 1a). The surface of the film was flat (Figure 1b), which was similar to the ACC film.18 The crystal phase of the film was determined as calcite by X-ray diffraction (XRD).18 It was noted that the thickness of calcite film on the silicon was also about 400 nm. The presence of magnesium could induce the transition from calcite to aragonite during the crystallization onto the existed calcite film. When the initial molar ratio of Mg/Ca ) 1 in the solution, the deposition of the second layer film was observed at 24 h (Figure 2). It could be seen that the thickness of the new formed layer was increased to ∼5 µm, implying a growth of calcium carbonate. The side view (Figure 2b) showed that the new layer had a different structure from the original calcite film, which was composed of bundles of needle-like crystals. The phase of resulting film was characterized by XRD, and the formation of aragonite was confirmed (Figure 3). It was found that the original calcite film remained during the deposition of aragonite. The boundary between two calcium carbonate phases could be distinguished, which indicated the abrupt transition from calcite to aragonite. This experiment demonstrated that the abalone shell-liked structure of the calcite-aragonite film complex was achieved in laboratory. In order to investigate the evolution from calcite to aragonite, the deposition process was followed by XRD at different time periods (Figure 3a). At the beginning of the experiment, the film was pure calcite, and the obvious formation of aragonite was observed at t ) 2 h. The intensity of aragonite diffraction peaks increased with time, and at t ) 24 h, the diffraction of calcite could not be observed since it was covered by the thick
Calcite and Aragonite Complex Layer Materials
Crystal Growth & Design, Vol. 9, No. 7, 2009 3097
Figure 3. (a) XRD data and (b) IR spectra of the deposition processes at different times.
aragonite layer. It should be emphasized that the diffraction intensity of (012) aragonite crystal plane was relatively great. This result showed that an oriented aggregation of the formed needle-like crystallites was formed in the film as they preferred to pack along the [012] direction. It was interesting to note that the predominantly [012] orientation of the aragonite assemblies was also observed in fresh nacre.19 The deposition of the unstable aragonite phase on the stable calcite layer was also confirmed by the IR examinations. The calcite substrate had the characteristic adsorption bands at 712 and 872 cm-1. The bands at 856, 712, and 700 cm-1 indicated that the second layer was aragonite at t ) 24 h (Figure 3b). The IR results were in good agreement with those of XRD, which confirmed the nucleation and growth of aragonite on calcite. It was unexpected that the thermodynamically stable phase of calcite was not epitaxially crystallized on the existing calcite phase, which was against the classical understanding of crystal growth. It was suggested that magnesium ions played a key role to induce the precipitation of aragonite. The films were also examined under SEM at different experimental periods. Figure 4 showed the crystallization of aragonite on calcite substrate clearly. At 10 min, a new layer of calcium carbonate was overgrown from calcite substrate as the surface was different from the original calcite substrate. Although the growth of calcium carbonate on the original calcite substrate was obvious (Figure 4a), both XRD and IR did not indicate any formation of aragonite at this stage. Only calcite phase was detected. This experimental result implied that the overgrowth of calcite was induced. It was reasonable that calcite could epitaxially grow on the calcite substrate. However, the typical morphology of calcite single crystal, rhombohedra, was not observed in the newly formed layer. It had been revealed that magnesium could participate in the precipitation of calcite to result in the formation of magnesium-doped calcite, which had the different crystal habits.19,20 At t ) 0.5 h, it could be seen that some prismatic aggregates randomly distributed on the calcite film. The individual needle-like crystals could be identified in the aggregates (Figure 4b). At this stage, the diffraction peaks appeared as a small shift due to magnesium ion entering crystal lattice, and the main crystal phase was still calcite, which indicated by the XRD pattern (Figure 3a). However some diffraction peaks derived from aragonite also could be observed. It might suggest that the formed prismatic aggregates were aragonite, but their amount was relatively low. We should emphasize that the previously deposited magnesiummodified calcite film actually acted as the transition layer between the aragonite and original calcite substrate. At t ) 2 h, lots of prismatic aggregates were observed, and most of them
stood upright (Figure 4c). The original calcite film could be still observed. XRD and IR results clearly showed that aragonite was produced, and the coexistence of calcite phase was also confirmed. At t ) 4 h, more aragonite aggregates were deposited (Figure 4d), and they began to pack closely to form a continuous film structure. At t ) 12 h (Figure 4e), the growth of aragonite resulted in a complete film structure. At this stage, the signals of calcite were too low to be detected by XRD or FT-IR. This phenomenon could be understood because the calcite layer was covered fully by a relatively thick aragonite layer. The suggested oriented assembly of the needle-like aragonite single crystals was also confirmed by Figure 4f. Clearly, our experiments demonstrated that aragonite could deposit on an existing calcite substrate. The suggested steps of the formation of the abalone shell-liked structure were (i) the formation of a layer of magnesium-modified calcite on the pure calcite substrate; (ii) the sequent deposition of aragonite onto the magnesium-modified calcite layer. It was important that the formed aragonite crystallites in this pathway were ordered assembled along their long axis. In the transition from calcite to aragonite, it was believed that the involvement of magnesium might play a key role. In this study, we noticed that the phase transition was related to the presence of calcite substrate and magnesium ions. It was well-known that there was no deposited aragonite film, and only randomly distributed crystals were obtained on bare silicon wafer under the same experimental conditions because silicon wafer could not induce the oriented growth of calcium carbonate crystals. The formed crystals were randomly aggregated, and the film structure could not be constructed. Figure 5a showed the result that the deposition process was performed in the absence of magnesium ion. The resulting film on the calcite substrate showed characteristics of the rhombohedral crystal morphology of calcite, which was confirmed by XRD results (Figure 5b). Thus, it could be considered as epitaxial crystal growth of calcite onto the calcite substrate in the absence of magnesium ion. This control experiment confirmed the function of magnesium during the formation of the aragonite layer. It was generally considered that the inhibition effect of magnesium on the calcite crystallization contributed to the smaller dimension, higher charge density, and greater hydration energy of Mg2+, which impeded calcite nucleation and growth until the dehydration of Mg2+ occurred.21 Checa et al. studied the growth of calcitic bivalves in magnesium-rich marine water (Mg/Ca ratio 8.3-9.2)22 and found the formation of aragonite on the calcite surfaces. It was revealed that high magnesium ion concentration assisted the formation of aragonite. The molar ratio of Mg/Ca in the solution was only 1 in the current
3098
Crystal Growth & Design, Vol. 9, No. 7, 2009
Liu et al.
Figure 4. SEM images of deposition process: (a) after 10 min; (b) after 30 min; (c) after 2 h; (d) after 4 h; (e, f) after 12 h.
Figure 5. (a) SEM image and (b) XRD result of control experiment: the deposition film onto calcite substrate in the absence of Mg ion after 12 h.
experiments, but it was found that the molar ratio of Mg/Ca in solution increased dramatically with the deposition time. It could be understood by that calcium was consumed, and the additive, magnesium, was not precipitated at a large scale. The higher molar ratio of Mg/Ca in the solution would promote the formation of the aragonite layer.
Many kinds of seashells contain not only aragonite but also calcite. However, the inorganic minerals are spatially separated in different parts of the shell. The growth of shell usually starts from outer calcite layer, and then, the crystallization of aragonite occurs successively on the calcite substrate.1b Fine “brick and mortar” structure of nacreous layer of abalone shell is composed
Calcite and Aragonite Complex Layer Materials
of aragonite and biomacromolecules, but it is covered by one layer calcite in its outer surface. Stucky et al. used inorganic substrate to implant an abalone shell and revealed that mineralization is initiated by the deposition of calcite on the organic layer, followed by the growth of aragonite, and observed the critical transition of biofabrication of abalone shell in detail and proved that the transition from calcite to aragonite was induced by soluble proteins isolated from nacre.13 Thompson et al.14 directly observed the transition of calcite-to-aragonite in the presence of protein by AFM. Our result was very interesting since the organized calcium carbonate film with a two layer structure, aragonite layer on calcite film, was achieved experimentally. The film showed (002) diffraction peak of aragonite and predominantly (012) orientation, which was generally found in biogenic aragonite. Additionally, it is generally known that biomacromolecules play an important role in the nucleation of aragonite. Herein, we demonstrated the nucleation of aragonite on calcite could be only controlled by magnesium ion. The formation of magnesium-modified calcite layer was important to induce the transition from pure calcite to aragonite under different Mg/Ca ratios. In the current work, we mimicked the biomineralization process of abalone shell in vitro. First, we prepared large-area ACC film on silicon wafer and controlled it to transform into calcite film by heating at 120 °C for 48 h, which mimicked the initial stage that a prismatic calcite layer is first formed on nucleating matrix in biomineralization of the abalone shell. Then one layer of aragonite film was deposited on calcite film in the presence of magnesium ion, which mimicked the stage that an abrupt transition from calcite to aragonite is switched by mollusk-shell protein.
Crystal Growth & Design, Vol. 9, No. 7, 2009 3099
(2) (3) (4)
(5)
(6) (7) (8) (9)
(10) (11) (12) (13)
Conclusion In summary, we succeeded in preparing a large-area abalone shell-like film with an aragonite layer on calcite film in vitro. The mechanism of the transition process from calcite to aragonite by the control of magnesium was also discussed. Furthermore, the oriented assembly of aragonite crystals could be constructed in the presence of magnesium and calcite substrate. This strategy can provide a simple pathway for the fabrication of the calcium carbonate complex film with abalone shell-like structure. Acknowledgment. The work was supported by National Science Foundation of China (20601023) and the Projectsponsored by SRF for ROCS, SEM. K.C. thanks the support from “Center for Nanostructured Materials Technology” under “the 21st Century Frontier R&D Programs” of the Ministry of Science and Technology of Korea (07K1501-01010).
References (1) (a) Mann, S.; Webb, J.; Williams R. J. P. Biomineralization: Chemical and Biochemical PerspectiVes; VCH Publishers: New York, 1989. (b)
(14) (15) (16)
(17) (18) (19) (20) (21) (22)
Mann, S. Biomineralization Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: New York, 2001. Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271, 67. Belcher, A. M.; Wu, X. H.; Christensen, R. J.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. Nature 1996, 381, 56. (a) Mann, S.; Heywood, B. R.; Rajam, S.; Birchall, J. D. Nature 1988, 334, 692. (b) Heywood, B. R.; Mann, S. AdV. Mater. 1994, 6, 9. (c) Heywood, B. R.; Rajam, S.; Mann, S. J. Chem. Soc., Faraday Trans. 1991, 87, 735. (d) Xu, G. F.; Yao, N.; Aksay, I. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 11977. (a) Ku¨ther, J.; Seshadri, R.; Knoll, W.; Tremel, W. J. Mater. Chem 1998, 8, 641. (b) Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature 1998, 394, 868. (c) Aizenberg, A. J.; Black, G. M. Nature 1999, 398, 495. (d) Aizenberg, J.; Black, A. J.; Whitesides, G. M. J. Am. Chem. Soc. 1999, 121, 4500. (e) Han, Y. J.; Aizenberg, J. Angew. Chem., Int. Ed. 2003, 42, 3668. Aizenberg, J. AdV. Mater. 2004, 16, 1295. Yu, S. H.; Colfen, H. J. Mater. Chem. 2005, 14, 2124. Addadi, L.; Joester, D.; Nudelman, F.; Weiner, S. Chem.sEur. J. 2006, 12, 980. (a) Kato, T.; Suzuki, T.; Amamiya, T.; Irie, T.; Komiyama, M.; Yui, H. Supramol. Sci. 1998, 5, 411. (b) Kato, T.; Sugawara, A.; Hosoda, N. AdV. Mater. 2002, 14, 869. (c) Hosoda, N.; Kato, T. Chem. Mater. 2001, 13, 688. (d) Sugawara, A.; Ishii, T.; Kato, T. Angew. Chem., Int. Ed. 2003, 42, 5299. (e) Zhang, S. K.; Gonsalves, K. E. Mater. Sci. Eng. 1995, C3, 117. (f) Zhang, S. K.; Gonsalves, K. E. Langmuir 1998, 14, 6761. (g) Ajikumar, P. K.; Lakshminarayanan, R.; Valiyaveettil, S. Cryst. Growth Des. 2004, 4, 331–335. (h) Lakshminarayanan, R.; Valiyaveettil, S.; Loy, G. L. Cryst. Growth Des. 2003, 3, 953. (i) Falini, G.; Fermani, S.; Gazzano, M.; Ripamonti, A. Chem.sEur. J. 1997, 3, 1807. Sugawara, A.; Kato, T. Chem. Commun. 2000, 487. Litvin, A. L.; Valiyaveettil, S.; Kaplan, D. L.; Mann, S. AdV. Mater. 1996, 9, 124. Fritz, M.; Belcher, A. M.; Radmacher, M.; Walters, D. A.; Hansma, P. K.; Stucky, G. D.; Morse, D. E.; Mann, S. Nature 1994, 371, 49. (a) Zaremba, C. M.; Belcher, A. M.; Fritz, M.; Li, Y. L.; Mann, S.; Hansma, P. K.; Morse, D. E.; Speck, J. S.; Stucky, G. D. Chem. Mater. 1996, 8, 679. (b) Su, X. W.; Belcher, A. M.; Zaremba, C. M.; Morse, D. E.; Stucky, G. D.; Heuer, A. H. Chem. Mater. 2002, 14, 3106. Thompson, J. B.; Paloczi, G. T.; Kindt, J. H.; Michenfelder, M.; Smith, B. L.; Stucky, G.; Morse, D. E.; Hansma, P. K. Biophys. J. 2000, 79, 3307. Addadi, L.; Raz, S.; Weiner, S. AdV. Mater. 2003, 15, 959. (a) Nassif, N.; Pinna, N.; Gehrke, N.; Antonietti, M.; Ja¨ger, C.; Co¨lfen, H. Proc. Natl. Acad. Sci. 2005, 102, 12653. (b) Politi, Y.; Arad, T.; Klein, E.; Weiner, S.; Addadi, L. Science 2004, 306, 1161. (c) Faatz, M.; Gro¨hn, F.; Wegner, G. AdV. Mater. 2004, 16, 996. (d) Raz, S; Hamilton, P. C.; Wilt, F. H.; Weiner, S.; Addadi, L. AdV. Funct. Mater 2003, 13, 480. (e) Xu, A. W.; Yu, Q.; Dong, W. F.; Antonietti, M.; Co¨lfen, H. AdV. Mater. 2005, 17, 2217. Xu, X. R.; Han, J. T.; Cho, K. Chem. Mater. 2004, 16, 1740. Xu, X. R.; Han, J. T.; Kim, D. H.; Cho, K. J. Phys. Chem. B 2006, 110, 2674. Raz, S.; Weiner, S.; Addadi, L. AdV. Mater. 2000, 12, 38. (a) Han, Y. J.; Aizenberg, J. J. Am. Chem. Soc. 2003, 125, 4032. (b) Han, Y. J.; Wysocki, L. M.; Thanawala, M. S.; Siegrist, T.; Aizenberg, J. Angew. Chem. Int. Ed 2005, 44, 2386. Lippmann, F. Sedimentary Carbonate Minerals; Springer, Berlin, 1973. Checa, A. G.; Jime´nez-Lo´pez, C.; Rodrı´guez-Navarro, A; Machado, J. P. Mar. Biol. 2007, 150, 819.
CG800872J
