CRYSTAL GROWTH & DESIGN
Synthesis and Structure of Hollow Calcite Particles Michio Suzuki,† Hiromichi Nagasawa,† and Toshihiro Kogure*,‡ Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The UniVersity of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan, and Department of Earth and Planetary Science, Graduate School of Science, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
2006 VOL. 6, NO. 9 2004-2006
ReceiVed May 18, 2006
ABSTRACT: Hollow spherical particles of calcite with a diameter of 2-10 µm were synthesized on a polyvinyliden difluoride (PVDF) membrane blotted with crude organic matrices prepared from the pearl oyster shell in a supersaturated solution of calcium carbonate. Examination of the produced particles as a function of the reaction time showed that the surface topography of the particles changed from a smooth surface to a ragged one with fine {104} facets of calcite crystals. Cross-sections and thin specimens of the particles were prepared by using the focused ion beam technique to observe the inside and determine the crystalline phases, respectively. Transmission electron microscopy (TEM) analysis showed that the particles with a short reaction time consist of vaterite and amorphous calcium carbonate (ACC). The ragged particles are polycrystalline calcite with a hollow structure (especially in relatively smaller particles) or a void space inside the particles. Large calcite crystals form a crust of the sphere, and minute calcite crystallites exist inside with the void space. Such a hollow structure can be explained by the solid-to-solid transformation from vaterite or ACC to calcite initiating from the surface and proceeding toward the inside of the sphere, with volume reduction by the phase transition. Calcium carbonate is a material that receives much attention in various fields. In geoscience, calcium carbonate constitutes limestone, coral reefs, etc. Those are the major solid reservoirs of carbon dioxides when considering the global carbon cycle. Calcium carbonate is also an important biomaterial for hard tissues in many organisms including mollusk shells, crustacean exoskeletons, and eggshells.1 Furthermore, calcium carbonate is widely used in industry as a raw material for cement, paper coating, medicine, etc. It is known that calcium carbonate adopts three anhydrate polymorphs: calcite, aragonite, and vaterite. Only calcite is the stable phase in the ambient temperature and pressure. However, the other two phases also frequently appear naturally and artificially. Amorphous calcium carbonate (ACC) that contains a certain amount of water molecules receives much attention too.2 Control of the polymorphs, morphologies, and textures of calcium carbonate is an interesting topic for the basic understanding and application of calcium carbonate. Organic matrices are thought to regulate the nucleation of calcium carbonate crystals3,4 and to control their morphology and polymorph.5-7 Some organic matrices from the pearl oyster shell change the morphology of the calcite.8,9 In particular, it is thought that acidic proteins in the mollusk shells play important roles in controlling calcite/aragonite switching in the shells,10,11 but the detailed processes of the control have not been clarified yet. In a series of the experiments to elucidate the mechanism of biomineralization, the effects of organic matrices on in vitro calcium carbonate formation have been examined. During such experiments using crude organic matrices of the nacreous layer of the pearl oyster, Pinctada fucata, spherical particles of calcium carbonate were obtained. They were formed only on polyvinyliden difluoride (PVDF) membranes (ATTO, Tokyo) blotted with acid-insoluble, but SDS (sodium dodecyl sulfate)-soluble organic matrices in a supersaturated solution of calcium carbonate. Scanning electron micrographs indicated that these spherical particles could have a hollow structure. This paper describes the fine structure and probable formation mechanism of the hollow particles, which may open a new insight of the interaction of calcium carbonate and organic matrices, as well as new potential applications of the material. * To whom correspondence should be addressed. Fax: +81-3-58414555. Tel: +81-3-5841-4548. E-mail:
[email protected] † Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences. ‡ Department of Earth and Planetary Science, Graduate School of Science.
Figure 1. (a) An SEM image of the spherical particles formed by the reaction of a CaCl2 solution including the organic matrices from the pearl oyster shell with sublimated (NH4)2CO3 for 3 h. The surface morphology varied among the individual particles. The surfaces indicated by the arrows probably consist of many {104} facets of rhombohedral calcite. (b) An SEM image showing the particles with a hollow structure.
Figure 1a shows the spherical particles precipitated 3 h after the reaction in the presence of organic matrices prepared from pearl oyster shell.12 Surface morphology of the particles varied among individual particles. In the same experiment with acid-soluble organic matrices derived from the whole shell including both nacreous and prismatic layers or without organic matrices, such spherical particles were not observed (data not shown). There is also no relationship between the particle size and surface morphology. The surface of the particles indicated by white arrows clearly consists of tiny {104} facets of rhombohedral calcite, suggesting that each particle is an aggregate of calcite crystals. Scanning electron microscopy (SEM) observations of the particles occasionally showed that the particles have a hollow structure, as shown in Figure 1b. Probably these particles grew attaching to a base like the inner wall of the vessel. However, it was not certain whether such a hollow structure is common for all particles in the specimen. To reveal the structure inside the spherical particles, we used the focused ion beam (FIB) technique, by which the specimen can be cut arbitrarily by a focused gallium ion beam.13 Figure 2 shows
10.1021/cg0602921 CCC: $33.50 © 2006 American Chemical Society Published on Web 08/04/2006
Communications
Crystal Growth & Design, Vol. 6, No. 9, 2006 2005
Figure 2. SEM images of the cross sections of the spherical particles, a hemisphere of which was eroded using an FIB system (FEI Quanta 200 3D).
Figure 4. (a) An SEM image of the particles precipitated in a short reaction time (∼5 min). (b) A bright-field TEM image from a thin section of one of the particles. The opaque material around the particle is Pt-Pd coating deposited for SEM observation. (c) Diffraction pattern of the inside of the particle in (b). The top-right inset in (c) is the calculated Debye-Scherrer rings for vaterite. The white arrowhead indicates the halo ring from amorphous material, probably ACC.
Figure 3. (a) A bright-field STEM image of a thin section of the spherical particle, prepared using an FIB system (Hitachi FB-2100). The opaque material around the particle is tungsten coating deposited in the FIB process. (b, c) Selected-area electron diffraction patterns from (b) the outer region and (c) inner fine crystallites. Both patterns can be interpreted as those from calcite. The top-right inset in (c) is the calculated Debye-Scherrer rings for calcite.
two examples. In both cases, the smaller particles have a hollow structure. Examination of about 10 particles indicated that such a hollow structure was formed in the particles with a diameter of 3-5 µm. The thickness of the crust is about 1 µm in most particles. Generally, the larger particles are not hollow at the center, although void space is often found at a certain depth from the surface (Figure 2b). Next, thin specimens for TEM examination were fabricated, leaving a great circle of the sphere. The thickness of the specimens was about 200 nm. Figure 3a represents a bright-field STEM image of the hollow particle with a diameter of about 4 µm. The outer crust consists of relatively large crystals, whereas very fine crystals are found inside the sphere. Electron diffraction patterns (Figure 3b,c) indicate that both crystals, large and fine ones, are calcite. It is difficult to imagine that such hollow particles were precipitated directly from solution. One explanation is that some precursor with lower density than that of calcite must be formed initially and then transformed to calcite, starting from the outer surface. To find the precursor, precipitated particles in a short reaction time (about 5 min) were investigated. Figure 4a is an SEM micrograph of the particles. The particles are far smoother than those in Figure 1a. Although very fine cracks are found on the surface in Figure 4a, other particles showed no surface texture with the resolution of SEM. Figure 4b,c is a bright-field TEM image of
a particle that was cut by FIB and its diffraction pattern, respectively. All the reflections in the pattern can be explained by vaterite but not by calcite. A strong halo ring is observed as indicated by the arrowhead in Figure 4c, suggesting an amorphous phase, probably ACC, in the specimen. It is known that ACC is precipitated from a highly supersaturated solution and rapidly crystallizes (in a few minutes) to vaterite or calcite at room temperature.14 Although we have not obtained the direct evidence, it is likely that the initial phase formed in this experiment is also ACC. It was reported that ACC contains 1.01.5 water molecules per CaCO3 unit.2 The precise density of ACC is not known, but probably the volume decreases if ACC transforms to vaterite or calcite by the release of water. The surface crack in Figure 4a and micro-voids in Figure 4b may reflect such volume reduction. On the other hand, the densities of vaterite and calcite are 2.54 and 2.71, respectively. This means that the transformation of vaterite to calcite accompanies the volume reduction of 6.3%. The origin of the hollow structure is probably due to such a volume decrease during the transition sequence of ACC to calcite through vaterite, and the apparent volume of the particle was retained as the transition to calcite initiating from the surface of the particle. There is discussion about whether the phase transition of calcium carbonate in a solution occurs through dissolution and reprecipitation or solid-to-solid transformation.14 The present result evidently indicates the latter. On the other hand, large calcite crystals with a rhombohedral form, which are commonly formed, were observed without the organic matrices. This is probably because calcite was nucleated and grew after or during the dissolution of vaterite. It is inferred that the organic matrices incorporated in the vaterite crystals resist or prolong the dissolution of vaterite and calcite starts to nucleate directly on the surface of vaterite. Besides the insight of the interaction of calcium carbonate with organic matrices, the
2006 Crystal Growth & Design, Vol. 6, No. 9, 2006 hollow structure of calcium carbonate particles is also attractive for industrial applications of this material.15 Adjustment of experimental conditions may give and control more varied hollow particle structures. At present, it is not known how the organic matrices are associated with the formation of spherical particles. SDS-PAGE analysis of the organic matrices showed that they were comprised of various protein components, in which N1616 was an extremely major one (data not shown). In addition, some acidic proteins were detected by Stains-all staining and might be associated with the formation of spherical particles because it is generally thought that acidic macromolecules play important roles in CaCO3 crystallization.11 Purification of the principle for spherical particle formation is now in progress. Acknowledgment. This work was supported by a Grant-in-Aid for Creative Scientific Research (No. 17GS0311) from Japan Society for the Promotion of Science. M.S. was supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists.
References (1) Mann, S. Biomineralization Principles and Concepts in Bioinorganic Materials Chemistry; Compton, R. G., Davies, S. G., Evans, J., Eds.; Oxford University Press: Oxford, 2001. (2) Addadi, L.; Raz, S.; Weiner, S. AdV. Mater. 2003, 15, 959-970. (3) Kato, T.; Sugawara, A.; Hosoda, N. AdV. Mater. 2002, 14, 869877. (4) Colfen, H.; Mann, S. Angrew. Chem. Int. Ed. 2003, 42, 2350-2365. (5) Lakshminarayanan, R.; Valiyaveettil, S.; Loy, G. L. Cryst. Growth Des. 2003, 3, 953-958. (6) Schlesinger, H. D. H.; Hay, D. I. J. Biol. Chem. 1977, 252, 16891695. (7) Wheeler, A. P.; George, J. W.; Evans, C. A. Science 1981, 212, 1397-1398.
Communications (8) Suzuki, M.; Murayama, E.; Inoue, H.; Ozaki, N.; Tohse, H.; Kogure, T.; Nagasawa, H. Biochem. J. 2004, 382, 205-213. (9) Valiyaveettil, G. Fu, S.; Wopenka, B.; Morse, D. E. Biomacromolecules 2005, 6, 1289-1298. (10) Addadi, L.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 41104114. (11) Weiner, S.; Addadi, L. Trends Biochem. Sci. 1991, 16, 252-256. (12) Shells of the Japanese pearl oyster (Pinctada fucata) were decalcified with 1 M acetic acid. The acidic water-insoluble material originating from the nacreous layer was collected. This insoluble material was washed with distilled water and treated with 1% SDS/10 mM dithiothreitol/50 mM Tris/HCl (pH 8.0) at 100 °C for 10 min. After concentration and desalting by ultrafiltration (Ultrafree M. W. 10, 000 cutoff, Millipore), the resulting solution containing high molecular weight compounds was used for the following experiment. A solution (200 µL) of 20 mM CaCl2 was added to each well of a 96-well plate. Next, a PVDF membrane (5 × 5 mm, blotted uniformly with the above-mensioned solution (4.0 µg protein equivalents) was added to each well. This plate was placed in a 6-L desiccator that also contained solid (NH4)2CO3 crystals. Then the desiccator was vacuumed by an aspirator for a few minutes. Sublimation of (NH4)2CO3 yielded an oversaturated calcium carbonate solution. (13) The precipitated particles on the PVDF membrane were coated with a Pt-Pd film for SEM examination. A Hitachi S-4500 SEM with a field-emission gun was used for observation. The FIB systems (Hitachi FB-2100 and FEI Quanta 200 3D) were used to observe the cross-section of the particles and to make thin specimens for TEM/ STEM observation. The thin specimens were examined by using a JEOL JEM-2010 TEM or a Hitachi HD-2300 STEM. (14) Ogino, T.; Suzuki, T.; Sawada, K. Geochim. Cosmochim. Acta 1987, 51, 2757-2767. (15) Walsh, D.; Mann, S. Nature 1995, 377, 320-323. (16) Samata, T.; Hayashi, N.; Kono, M.; Hasegawa, K.; Horita, C.; Akera, S. FEBS Lett. 1999, 462, 225-229.
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