Novel Morphology of Calcium Carbonate Controlled by Poly(l-lysine)

Jul 15, 2009 - The novel calcium carbonate (CaCO3) morphology, twin-sphere with an equatorial girdle, has been obtained under the control of poly(L-ly...
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Novel Morphology of Calcium Carbonate Controlled by Poly(L-lysine) Yuan Yao,† Wenyong Dong,† Shenmin Zhu,‡ Xinhai Yu,§ and Deyue Yan*,† †

School of Chemistry and Chemical Engineering and ‡State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P.R. China, and §School of Mechanical and Power Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, P.R. China Received May 28, 2009. Revised Manuscript Received June 30, 2009 The novel calcium carbonate (CaCO3) morphology, twin-sphere with an equatorial girdle, has been obtained under the control of poly(L-lysine) (PLys) through gas-diffusion method. The effect of the concentration of calcium cation and PLys, the reaction time, and the initial pH value are investigated, and various interesting morphologies, including twinsphere, discus-like, hexagonal plate, and hallow structure are observed by using scanning electronic microscopy. Laser microscopic Raman spectroscopy studies indicated that all these CaCO3 are vaterite. A possible mechanism is suggested to explain the formation of the twin-sphere based morphologies according to the results. It is proven that alkaline polypeptides can control the mineralization of CaCO3 precisely as the reported acidic polypeptides and double hydrophilic block copolymers.

Introduction Biomineralization, a very common and important phenomenon in nature, has received intensive concern for decades. It is extremely interesting to mimic the biomineralization processes and explore new bioinspired materials with well-controlled morphologies, structure, and functions by using different templates including small molecules, natural products, homopolymers, random copolymers, amphiphilic block copolymers, double hydrophilic block copolymers, graft copolymers, dendrimers, supramolecular matrixes, and patterned surfaces.1 Some significant reviews focused on the latest advances of controlled mineralization have been published.1,2 Polypeptide/protein-template synthesis of inorganic crystals and inorganic/organic hybrid structures has been highlighted in recent years. One of the most widely studied systems is calcium carbonate (CaCO3). For example, poly(aspartic acid) (PAsp) was used to prepare the hollow helical superstructure of CaCO3,3 and the PAsp section containing block copolymers and PAsp/polymer complexes could control the mineralization of CaCO3.4 Similar to PAsp, poly(glutamate) (PGlu) is another widely adopted polypeptide template section.4c For instance, poly(ethylene glycol)PGlu block copolymer (PEG110-PGlu6) can generate highly monodisperse vaterite CaCO3 microspheres in the mixed solvent of ethanol and water, whereas under the control of another PEGPGlu block copolymer (PEG=1680 g/mol, PGlu=2060 g/mol), *To whom correspondence should be addressed.Telephone: þ86-2154742664. Fax: 54741297. E-mail: [email protected]. (1) (a) C€offen, H Top. Curr. Chem. 2007, 271, 1. (b) Yu, S.-H. Top. Curr. Chem. 2007, 271, 79. (c) Naka, K. Top. Curr. Chem. 2007, 271, 119. (d) Ueyama, N.; Takahashi, K.; Onoda, A.; Okamura, T.; Yamamoto, H. Top. Curr. Chem. 2007, 271, 155. (2) (a) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128. (b) C€offen, H.; Mann, S. Angew. Chem., Int. Ed. 2003, 42, 2350. (c) B€auerlein, E. Angew. Chem., Int. Ed. 2003, 42, 614. (d) Yu, S. H.; C€offen, H. J. Mater. Chem 2004, 14, 2124. (3) (a) Gower, L. A.; Tirrell, D. A. J. Cryst. Growth 1998, 191, 153. (b) Gower, L. B.; Odom, D. J. J. Cryst. Growth 2000, 210, 719. (4) (a) Liu, X.-R.; Liu, B.-H.; Wang, Z.-Y.; Zhang, B.-C.; Zhang, Z.-P. J. Phys. Chem. C 2008, 112, 9632. (b) Zhang, Z.-P.; Gao, D.-A.; Zhao, H.; Xie, C.-G.; Guan, G.-J.; Wang, D.-P.; Yu, S.-H. J. Phys. Chem. B 2006, 110, 8613. (c) Kaparova, P.; Antonietti, M.; C€offen, H. J. Colloid Interface Sci. 2004, 250, 153. (d) Chen, X.-G.; Varona, P. L.; Olszta, M. J. J. Cryst. Growth 2007, 307, 395.

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CaCO3 mineralizes into different structures in dimethyl formamide depending on crystallization temperature and the copolymer concentration.5 Recently, a series of polypeptides and peptide-peptide copolymers have been used to direct the formation of CaCO3 crystals. The resulting morphologies include irregular, rhombohedra, and spherical shapes.6 Copoly[L-(phosphorylated)serine75block-aspartic acid25] P(Ser(P)75-b-Asp25) can direct CaCO3 to generate the helical calcite superstructures, which are corresponding to the enantiomer of the copolymer.7 Natural proteins, such as nacre-associated oyster shell protein, mollusk shell protein, collagen, spider silk, and viruses, were also employed as the template to control CaCO3 crystallization.8,1b It is well-known that hydrophilic acidic polypeptides and proteins play a vital role in the mineralization of CaCO3 in nature,1,9 and most polymers capable of controlling the CaCO3 mineralization have the acidic section. Only a few works involved the cationcontaining polyelectrolyte templates in the mineralization of CaCO3.6a,10 As far as poly(L-lysine) (PLys) is concerned, only its salt, that is, hydrobromide, has been utilized to control the CaCO3 mineralization,6a but only one concentration of the template (0.125 mg/mL of PLys 3 HBr) was investigated, and only rhombohedra calcites were observed. In this paper, the formation of various CaCO3 morphologies under the control of PLys with higher concentration, instead of its salt, is investigated. Some interesting morphologies, including the twin-sphere with an (5) (a) Gao, X.-H.; Yu, S.-H.; Cai, G.-B. Angew. Chem., Int. Ed. 2006, 45, 3977. (b) Guo, X.-H.; Xu, A.-W.; Yu, S.-H. Cryst. Growth Des. 2008, 8, 1233. (6) (a) Euliss, L. E.; Bartl, M. H.; Stucky, G. D. J. Cryst. Growth 2006, 286, 424. (b) Euliss, L. E.; Trnka, T. M.; Deming, T. J.; Stucky, G. D. Chem. Commun. 2004, 1736. (7) Sugawara, T.; Suwa, Y.; Ohkawa, K.; Yamamoto, H. Macromol. Rapid Commun. 2003, 24, 847. (8) (a) Deshpande, A. S.; Benish, E. Cryst. Growth Des. 2008, 8, 3084. (b) Cheng, C.; Shao, Z.-Z.; Vollrath, F. Adv. Funct. Mater. 2008, 18, 2172. (c) Kim, W.II; DiMasi, E.; Evans, J. S. Cryst. Growth Des. 2004, 4, 1113. (d) Collino, S.; Kim, W.II; Evans, J. S. Cryst. Growth Des. 2006, 6, 839. (e) Falini, G.; Fermani, S.; Gazzano, M.; Ripamonti, A. Chem.;Eur. J. 1998, 4, 1048. (9) (a) Berman, A.; hanson, J.; Leiserowitz, L.; Koetzle, T. F.; Weiner, S.; Addadi, L. J. Phys. Chem. 1993, 97, 5162. (b) Gotliv, B.; Kessler, N.; Sumerel, J. L.; Morse, D. E.; Tuross, N.; Adadi, L. ChemBioChem 2005, 6, 304. (10) Nassif, N.; Gehrke, N.; Pinna, N.; Shirshova, N.; Tauer, K.; Antonietti, M.; C€olfen, H. Angew. Chem., Int. Ed. 2005, 44, 6004.

Published on Web 07/15/2009

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equatorial girdle, the peanut-like twin-sphere, the hexagonal plate, and the hollow oblate/micro-ring have been observed by high resolution scanning electronic microscope (HRSEM), among which the peanut like twin-sphere was scarcely found, especially the twin-sphere with an equatorial girdle has not been reported yet in the past. Laser microscopic Raman spectrum (LM-Raman) has been used as a powerful solution to distinguish the crystallinity of different morphologies at high lateral resolution. The influences of the concentration of Ca2þ and PLys, the initial pH value, and the reaction time are further discussed. In addition, a reasonable explanation is provided to demonstrate the formation of the twin-sphere with equatorial girdle under the direction of PLys.

Experimental Section Preparation of PLys and CaCO3 Crystals. PLys was prepared according to the previous references.11 The ε-carbobenzoxy-L-lysine N-carboxyanhydride (Z-Lys-NCA) was synthesized first, and then the ring-opening polymerization of it was initiated by 1-amantdiane in anhydrous chloroform for 72 h. After being precipitated in ethyl ether and dried under vacuum, poly(ε-carbobenzoxy-L-lysine) (PZlys) was obtained. The molecular weight of the resulting PZlys was 2.52  104 by GPC. The PZlys was deprotected by 33% HBr/acetic acid in the mixture of trifluoroacetate/dichloroacetate and then neutralized by NaHCO3. After being dialyzed in deionized water and freeze-dried, the pure PLys was obtained. CaCO3 crystals were obtained via the gas-diffusion method. A certain amount of PLys was dissolved in 10 mL of ultrapure water in a 25 mL bottle. Then the solution was adjusted to the desired pH value by the HCl or KOH aqueous solution. After the corresponding volume of 1 M CaCl2 was injected into the bottle under stirring, some clean glass wafers were also put into the bottle to collect the CaCO3 crystals. The bottle was covered by the pierced Parafilm and stood in a closed 300 mm desiccator with NH4HCO3 for a certain time at 25 °C. Finally, the wafers were taken out, washed by ultrapure water and acetone several times, and then dried under vacuum. The other CaCO3 crystal samples were prepared by the same procedure. Measurement and Characterization. The morphologies of the resulting CaCO3 crystals were characterized by the JEOL7401F field emission SEM at 5 kV. LM-Raman spectra were measured by the RENISHAW inVia Raman Microscope with 514.5 nm laser source, and the photomicrographs were taken by the 50, NA = 0.75 lens. XRD was recorded on the D/MAX2550/PC spectrometer with Cu KR radiation. The TEM image and SAED spectrum were taken by the JEOL-2100F HRTEM instrument. The CaCO3 crystals on the glass wafer were collected carefully and grinded gently. Then they were dispersed in ethanol by 2 min of supersonic treatment. The ethanol solution of the cracked CaCO3 crystals was dropped on the ultrathin carbon film plated copper mesh and dried at room temperature. CD spectra were recorded by the Jasco-J815 CD spectrometer.

Results and Discussion Morphologies Controlled by the PLys Concentration. To study the influence of PLys concentration, the concentration of Ca2þ is kept constant at 1.00 mM, the initial pH in the range of 7.20-7.48, and the reaction is stopped after 48 h. The PLys concentration ranges from 0.1 mg/mL to 10 mg/mL. Figure 1 shows the morphologies of mineralized CaCO3 with different PLys concentrations. There are two kinds of twin-sphere (11) (a) Yao, Y.; Li, W.-W.; Wang, S.-B.; Chen, X. -S.; Yan, D. Y. Macromol. Rapid Commun. 2006, 27, 2019. (b) Sun, J.; Chen, X.-S.; Deng, C.; Yu, H.-J.; Xie, Z.-G.; Jing, X. B. Langmuir 2007, 23, 8308. (c) Deng, C.; Chen, X.-S.; Yu, H.-J.; Sun, J.; Lu, T.-C.; Jing, X. B. Polymer 2007, 48, 139.

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Figure 1. Morphologies of mineralized CaCO3 with different PLys concentrations: (A) 0.10 mg/mL; (B) 2.00 mg/mL; (C) 5.00 mg/mL; and (D) 10.0 mg/mL.

Figure 2. Twin-sphere with equatorial girdle morphology. (A) single twin-sphere; (B) enlarged equator girdle; (C) enlarged surface of the girdle; and (D) enlarged surface of twin-sphere.

based morphologies at lower and medium PLys concentrations. The peanut-like twin-spheres without girdle and rhombohedra crystals are predominant under the lower PLys concentration (Figure 1A), while the twin-spheres with an equatorial girdle are prevailing in a medium PLys concentration system (Figure 1B). Under higher PLys concentration, the ball-like and the discus-like oblate CaCO3 morphologies are observed (see Figure 1C,D, respectively). The morphologies seemed like the polar axis (the axis vertical to the twin-sphere’s equator) compressed twinsphere. Further Investigation of Twin-Sphere with Equatorial Girdle. The SEM images in Figure 2 show the twin-sphere with an equatorial girdle of the CaCO3 macrocrystal, the enlarged equator girdle, and other part of the sphere surface. The length ratio of the polar axis to the equatorial axis is approximately 1.20-1.42:1 (Figure 1B), and the width of the equatorial girdle is about 2.13-2.99 μm (Figure 2B). Both the equatorial girdle and other parts of the sphere surface consist of crystal pieces; DOI: 10.1021/la901913d

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Figure 3. (A) LM-Raman spectrum of a CaCO3 twin-sphere with equatorial girdle. (B) LM-Raman spectrum of the rhombohedral crystal. The inset pictures are the photos taken by microscope, and the cross-bars show the positions that the laser beam irradiated. (C) HRTEM photos of a fragment of the twin-sphere with equatorial girdle. The inset photo is the corresponding SAED image.

Figure 4. Surface morphologies of CaCO3 particles at different mineralization times: (A) 8 h; (B) 16 h; (C) 24 h; (D) 32 h; (E) 40 h; and (F) 48 h. [Ca2þ] = 1.00 mM, [PLys] = 2.00 mg/mL. In this process, pH values of the resulting solution change from 7.53 to 9.48. The inset photo is the panorama of the corresponding CaCO3 particle.

however, the crystal pieces in the equator girdle are vertical to the spherical surface (Figure 2A,B) while those in the spherical surface grow somewhat along the tangential direction of the spherical surface (Figure 2A,B, Supporting Information S1). It is interesting that every crystal piece seems to be in a fractal shape (Figure 2D). Figure 3A shows the LM-Raman spectrum of the twin-sphere with equatorial girdle CaCO3 macrocrystal. The peaks at 1090 and 1075 cm-1 are attributed to ν1 symmetric stretching vibration of the vaterite C-O bond. The weak peaks at 740-750 cm-1 are ν4 in-plane bending vibration of vaterite O-C-O.12 The LMRaman spectra of the ball-like and discus-like oblate CaCO3 crystals have been determined, which also show the characteristics of vaterite (see Supporting Information S2). In comparison, the corresponding peaks of the rhombohedra crystal are located at 1086 cm-1 and 712 cm-1, which are the characteristic peaks of calcite.12 The WAXRD spectrum (Supporting Information S3) indicates that there are two kinds of CaCO3 crystals in the resulting products, of which one is vaterite and the other one is calcite. The data measured from LM-Raman spectra (12) (a) Gabrielli, C.; Jaouhari, R.; Joiret, S.; Maurin, G. J. Raman Spectrosc. 2000, 31, 497. (b) Tlili, M. M.; Amor, M. B.; Gabrielli, C.; Joiret, S.; Maurin, G.; Rousseau, P. J. Raman Spectrosc. 2001, 33, 10.

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Figure 5. CaCO3 morphologies formed under different initial pH values. [Ca2þ] = 1.00 mM, [PLys] = 2.00 mg/mL, reaction time = 48 h. Initial pH values: (A) 2.18, (B) 4.51, (C) 8.97, and (D) 10.66. The inset photo is the panorama of the corresponding CaCO3 particle. Langmuir 2009, 25(22), 13238–13243

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Figure 6. LM-Raman spectra of CaCO3 formed under different initial pH values. [Ca2þ] = 1.00 mM, [PLys] = 2.00 mg/mL, reaction time = 48 h. Initial pH values: (A) 2.18, (B) 4.51, (C) 8.97, and (D) 10.66. The inset pictures are the photos taken by microscope, and the cross-bars show the positions that the laser beam irradiated.

and WAXRD are in agreement with each other. Figure 3D shows the TEM image of a fragment of the twin-sphere with equatorial girdle. SAED indicates that the fragment of the twin-sphere with equatorial girdle is composed of polycrystals (Figure 3C). Evolution of the Twin-Sphere with Equatorial Girdle. To investigate the evolution of the twin-sphere with equatorial girdle, six 25 mL bottles filled with 10.0 mL of aqueous solution ([Ca2þ]=1.00 mM, [PLys]=2.00 mg/mL) and glass wafers were covered by the pierced Parafilm, respectively, and then stood in a closed 300 mm desiccator with NH4HCO3 at 25 °C. By the end of each 8 h, one of the bottles was taken out, and then the wafers in it were treated as mentioned in the Experimental Section for measurements. Figure 4 shows a series of CaCO3 particle morphologies taken at different mineralization times. After 8 h, no crystal could be observed in the scene (Figure 4A). After 16 h, the twin-sphere morphology appeared on the plate (the inset photo of Figure 4B), and the sphere surface was composed of small grainlike particles (Figure 4B). At 24 h, both the twin-spheres and the grain-like particles on the surface became somewhat larger (Figure 4C) than those in Figure 4B. At 32 h, the equatorial girdle emerged (Figure 4D). After 40 h (Figure 4E), the equatorial girdle grows up, and the CaCO3 crystal pieces start to appear on the sphere surface. Finally, at 48 h, a remarkable equatorial girdle was formed, and the sphere surface is covered by the CaCO3 crystal pieces (Figure 4F). The LM-Raman spectra confirm that all the twin-spheres at various periods only consist of vaterite crystals, and there has not been crystalline transformation during the formation of the twin-sphere with equatorial girdle CaCO3 macrocrystals (see Supporting Information S4). Morphologies Controlled by the Initial pH Value. The initial pH value can affect the morphology of CaCO3 particles Langmuir 2009, 25(22), 13238–13243

Figure 7. CD spectra of the gas-diffusion system with different reaction times during the mineralization (initial 2 mg/mL PLys, 1 mM Ca2þ aqueous solution, the path length of the cuvette is 0.2 mm).

as well. Figure 5 shows the respective surfaces and the appearances of CaCO3 crystals generated from different initial pH values. When the initial pH equals 2.18, most of CaCO3 exists as the rhombohedra crystals, and only a few spheres can be obtained. The sphere surface is made of a lot of thin pieces which are vertical to the surface and parallel with one another within a small domain (Figure 5A). LM-Raman spectrum of the spheres shows obvious calcite features (Figure 6A). If the initial pH is 4.51, some twin-spheres without the equatorial girdle are observed, but their surface consists of grain-like small crystals DOI: 10.1021/la901913d

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Figure 8. Hexagonal (A, B, and C) and hollow structures (D, E, and F) CaCO3 formed at higher Ca2þ concentration and longer reaction time. Scheme 1. Formation Illustration of Various CaCO3 Morphologies Directed by PLys

(Figure 5B). Under neutral (see Figure 2) or weak alkaline conditions (Figure 5C), twin-spheres predominately with equatorial girdle are detected, and the surfaces are covered with small crystal pieces. At alkaline conditions (pH=10.66), discus-like morphology is formed, and the crystal pieces on the surface are larger than those in other particles (Figure 5D). LM-Raman spectra demonstrate that all of these twin-spheres are vaterite (Figure 6B-D). The XRD spectra with a subtle difference for the four kinds of morphologies are provided in Supporting Information S5. It is suggested that the pH value affects the mineralization in the following three aspects. First, PLys possesses the R-helix 13242 DOI: 10.1021/la901913d

conformation under alkaline conditions,13 which is a suitable template for CaCO3 mineralization. Second, CaCO3 is easier to deposit in an alkaline environment; hence, the crystal pieces tend to grow larger and larger. Third, the acidic environment may inhibit the coordination between PLys and Ca2þ because the ε-amino group exists as an ammonium cation NH3þ without the lone electron pair, and it repulses Ca2þ cations with the same charge. Under the alkaline environment, the ε-amino groups are (13) Shibata, A.; Yamamoto, M.; Yamashita, T.; Chiou, J.-S.; Kamaya, H.; Ueda, I. Biochemistry 1992, 31, 5728.

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Figure 9. LM-Raman spectra of morphologies corresponding to parts A (A) and D (B) of Figure 8A. The inset pictures are the photos taken by microscope, and the cross-bars show the positions that the laser beam irradiated.

free, which can combine with Ca2þ via weak interaction. These are the reasons why the rhombohedra crystal is the primary product at acidic initial condition, and the alkaline environment is prone to generate larger crystal pieces and girdles. Possible Formation Mechanism of the Twin-Sphere with Equatorial Girdle. Evidently, the conformation of the template molecules plays an important role in the PLys controlled mineralization of CaCO3. Circular dichroism (CD) spectra were used to investigate the conformation of PLys chains (Figure 7). In solution the PLys chain is of partially R-helix and partially random coil,14 and the content of the R-helix increases with increasing pH value or reaction time during the mineralization. A possible formation mechanism of the twin-sphere with equatorial girdle is shown in scheme 1. At the beginning, the interconnected PLys chains coordinate with Ca2þ and direct the formation of anisotropic vaterite nanocrystals. Some nanocrystals aggregate to form a nuclear plate, and then the nanocrystals assemble onto both sides of the nuclear plate. However, the growth along the equator is districted somewhat by the higher growth energy. So the twin-sphere morphology is formed and grows larger and larger. With the environmental pH value increasing to stronger alkaline, the PLys chains with more R-helix lead to the tangential oriented growth on the sphere surface and vertical oriented growth on the equatorial zone. Finally, the tangential oriented crystal tiles and the equatorial girdle lamellae are formed. Morphologies Controlled by the Ca2þ Concentration. Under higher concentration of Ca2þ and longer reaction time, different morphologies were observed as shown in Figure 8. When the concentrations of Ca2þ and PLys are 5.00 mM and 2.00 mg/mL, respectively, hexagonal discuses can be obtained after 15 days. When the PLys concentration increased to 10.00 mg/mL, hollow rings were formed predominantly. The results indicate that both the PLys and Ca2þ concentrations are significant for controlling the morphology of CaCO3. The LMRaman spectra show that both the hexagonal discus and the hollow micro-ring morphologies are composed of vaterite crystals (Figure 9). (14) Gao, Y.-X.; Yu, S.-H.; Cong, H.; Jiang, J.; Xu, A.-W.; Dong, W.-F.; C€olfen, H. J. Phys. Chem B 2006, 110, 6432.

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Conclusions The polymeric template for the mineralization of CaCO3 usually contains carboxyl groups. Interestingly, this work found that PLys with side amine groups can control the mineralization of CaCO3 just like the acidic polypeptides. The novel morphology, twin-sphere with equatorial girdle, has been formed using a simple gas-diffusion method, which is determined to be vaterite by using accurate LM-Raman spectrum. A reasonable process for forming the morphology is discussed preliminarily. At first, the anisotropic vaterite nanocrystals are formed under the direction of the PLys, then the nanocrystals assemble to form a nuclear plate, and the growth from both sides of the nuclear plate results in the twinsphere. With the environmental pH value increasing to stronger alkaline, the conformation change of PLys chains leads to the tangential oriented growth on the sphere surface and vertical oriented growth on the equatorial zone. Finally, the twin-sphere with equatorial girdle morphology is generated. Other morphologies, including twin-sphere, discus-like, hexagonal plate, and hallow structure are observed as well under different conditions. The crystal structure of them is vaterite. It suggests that alkaline polypeptide or protein may also play a suitable role in the biomineralization process in nature. Acknowledgment. This work was subsidized by the National Natural Science Foundation of China (20874060, 50503012, 50772067, 20606011), National Basic Research Program (2007CB808000), the Basic Research Foundation (07DJ14004) of Shanghai Science and Technique Committee, and Shanghai Leading Academic Discipline Project (B202). Supporting Information Available: SEM images of the side surface of the twin sphere with equatorial girdle morphology shown in Figure 2, the LM-Raman spectra of CaCO3 crystals corresponding to Figure 1C,D, the WAXRD spectrum of twin-sphere with equatorial girdle CaCO3 crystals, the LMRaman spectra of CaCO3 crystals obtained at different reaction times, the XRD spectra of CaCO3 formed under different initial pH values, and XRD spectra of morphologies of the hexagonal discuses shown in Figure 8A and hollow micro-ring shown in Figure 8D. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la901913d

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