Dopamine-Induced Mineralization of Calcium Carbonate Vaterite

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Dopamine-Induced Mineralization of Calcium Carbonate Vaterite Microspheres Sungjin Kim and Chan Beum Park* Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), 335 Science Road, Daejeon 305-701, Republic of Korea Received May 19, 2010. Revised Manuscript Received August 13, 2010 Two biogenic materials from mussels are attracting attention from scientists: calcium carbonate (CaCO3), the most widely studied biomineral that composes the shell, or nacre, of mussels, and dopamine, a small catechol-containing biomimetic molecule of adhesive foot proteins secreted by mussels. We have incorporated these two materials into the biomimetic mineralization process to produce stable vaterite microspheres, which are the most unstable crystalline phase of CaCO3. Spherical vaterite crystals were readily formed within two minutes in the presence of dopamine undergoing polymerization and were preserved for over two months in aqueous solution. The microspheres consisted of nanoparticles smaller than 100 nm and exhibited porous and spherulitic cross sections. The prolonged maintenance of spherical structure is attributed to the affinitive interaction between calcium in the vaterite microspheres and catechols from dopamine retarding the dissolution of vaterite and the growth of calcite crystals. The mussel-inspired inducement of a stable vaterite phase suggests a facile route for the synthesis of complex organic-inorganic hybrid materials utilizing biogenic systems.

Introduction Natural biogenic systems can synthesize highly complicated structures of biominerals that exhibit fascinating morphologies, outstanding mechanical/optical properties, and unique biological functions such as navigation, storage, and homeostasis.1-5 Attempts at mimicking the natural routes for biomineralization for the fabrication of noble inorganic-organic hybrid materials have been made by many researchers.6 Calcium carbonate (CaCO3) is one of the most widely studied biominerals in nature7-9 and is often found in the exoskeletons of algae, egg shells, mussel shells, and sea urchin spines.10,11 Vaterite is the most unstable phase among three different types of crystalline polymorphs of CaCO3 (aragonite, calcite, and vaterite) that are crystallized from amorphous calcium carbonate (ACC).8,16 The inherent energetic instability of vaterite results in the phase transformation to other stable crystalline phases in aqueous solution,1 which occurs via a dissolution and recrystallization process (i.e., the dissolution of metastable phase and the growth of stable phase).12-14 Stabilizing the vaterite phase has been one of *To whom correspondence should be addressed. Tel.: þ82 42 350 3340. Fax: þ82 42 350 3310. E-mail address: [email protected].

(1) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: Oxford, U.K., 2001. (2) Kr€oger, N.; Sandhage, K. H. MRS Bull. 2010, 35, 122–126. (3) Smith, B. L.; Sch€affer, T. E.; Viani, M.; Thompson, J. B.; Frederick, N. A.; Kindt, J.; Belcher, A.; Stucky, G. D.; Morse, D. E.; Hansma, P. K. Nature 1999, 399, 761–763. (4) Aizenberg, J.; Weaver, J. C.; Thanawala, M. S.; Sundar, V. C.; Morse, D. E.; Fratzl, P. Science 2005, 309, 275–278. (5) Addadi, L.; Weiner, S. Angew. Chem., Int. Ed. Engl. 1992, 31, 153–169. (6) Mann, S. Angew. Chem., Int. Ed. 2000, 39, 3392–3406. (7) Sommerdijk, N. A. J. M.; de With, G. Chem. Rev. 2008, 108, 4499–4550. (8) Pouget, E. M.; Bomans, P. H. H.; Goos, J. A. C. M.; Frederik, P. M.; de With, G.; Sommerdjik, N. A. J. M. Science 2009, 323, 1455–1458. (9) Kato, T.; Sakamoto, T.; Nishimura, T. MRS Bull. 2010, 35, 127–132. (10) Naka, K.; Chujo, Y. Chem. Mater. 2001, 13, 3245–3259. (11) Politi, Y.; Arad, T.; Klein, E.; Weiner, S.; Addadi, L. Science 2004, 306, 1161–1164. (12) Ogino, T.; Suzuki, T.; Sawada, K. Geochim. Cosmochim. Acta 1987, 51, 2757–2767. (13) Sawada, K. Pure Appl. Chem. 1997, 69, 921–928. (14) Spanos, N.; Koutsoukos, P. G. J. Cryst. Growth 1998, 191, 783–790.

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major issues in biomineralization studies, not only because of its rareness due to its intrinsic instability, but also because of its potential for biomedical and industrial applications since vaterite exhibits unique properties such as high surface area, solubility, dispersion, and a smaller specific gravity than calcite or aragonite.15,16 Recent findings on biogenic CaCO3 demonstrates that vaterite phases exist in pearls found in a particular genus of mussels (hyriopsis),17-19 which suggests that certain biogenic materials secreted from mussels may induce or stabilize the vaterite phase. On the basis of this phenomenon, we attempted to stabilize the vaterite phase using dopamine, which is a mimic of 3,4-dihydroxyL-phenylalanine (DOPA) that is found in the adhesive protein Mefp-5 (Mytilus edulis foot protein 5) secreted from mussels; the oxidative polymerization of dopamine in aqueous solution spontaneously forms polydopamine that exhibits a strong adhesive property to a variety of substrates under wet conditions20-22 (Figure 1a). We anticipated that the universal adhesive property of catechol group in dopamine might play a certain role in the mineralization of CaCO3. According to our results, spherical vaterite crystals are produced in the presence of dopamine and could be preserved for over two months. The mussel-inspired approach to stabilizing vaterite microspheres may provide a new horizon for the biogenic inducement of vaterite phases and their future applications.

Experimental Section Materials. Granular anhydrous calcium chloride and dopamine hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO) (15) Naka, K.; Tanaka, Y.; Chujo, Y. Langmuir 2002, 18, 3655–3658. (16) Naka, K. Top. Curr. Chem. 2007, 271, 119–154. (17) Ma, H. Y.; Lee, I. S. Mater. Sci. Eng., C 2006, 26, 721–723. (18) Qiao, L.; Feng, Q. -L.; Li, Z. Cryst. Growth Des. 2007, 7, 275–279. (19) Soldati, A. L.; Jacob, D. E.; Wehrmeister, U.; Hofmeister, W. Mineral. Mag. 2008, 72, 579–592. (20) Lee, H.; Scherer, N. F.; Messersmith, P. B. Proc. Natl. Acad. Sci. U.S.A 2006, 103, 12999–13003. (21) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Science 2007, 318, 426–430. (22) Waite, J. H. Nat. Mater. 2008, 7, 8–9.

Published on Web 08/26/2010

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Figure 1. (a) Photograph of mussels binding onto a rock using an adhesive foot protein rich of DOPA. Dopamine is the mimicry catecholamine product of DOPA, and CaCO3 is the major inorganic component of mussel shells. (b-h) Electron microscopic images of CaCO3 crystals formed by incubation with or without dopamine (2 mg/mL) for 4 days at 25 °C and 1 atm. (b) Rhombohedral crystals formed in the absence of dopamine. (c) Spherical CaCO3 microspheres formed in the presence of dopamine. (d) Single CaCO3 microsphere composed of CaCO3 nanoparticles, which can be confirmed by (e) a TEM image and (f) a close-up SEM image of CaCO3 nanoparticles existing on the surface of microsphere. (g) A cross-section of a CaCO3 microsphere revealing its porous structure and (h) spherulitic orientation by CaCO3 nanoparticles. and sodium carbonate was purchased from Junsei (Tokyo, Japan). As a solvent for dopamine hydrochloride, 10 mM Tris buffer (pH 8.5) was prepared by mixing Tris-base and Tris-HCl purchased from Sigma-Aldrich (St. Louis, MO) in deionized water (distilled at 18.2 MΩ cm-1) at room temperature. Dopamine-Induced Mineralization of CaCO3. For the mineralization of CaCO3, 0.33 M of Na2CO3 solution and 0.033 M of CaCl2 solution were prepared by dissolving the corresponding reagents in deionized water at room temperature. Dopamine hydrochloride was dissolved in a 10 mM Tris solution buffered to a typical marine environmental pH of 8.5 to obtain an aqueous dopamine solution with a concentration of 2 mg/mL. Langmuir 2010, 26(18), 14730–14736

Subsequently, a time interval of 2 min was provided to let the dopamine monomers initiate polymerization before participating in a mineralization process. The mineralization process was initiated by directly mixing the three aqueous solutions (i.e., 0.33 M Na2CO3, 0.033 M CaCl2, and 2 mg/mL dopamine solution) with a 1:1:1 volume ratio, followed by vigorous stirring. The mixture was then continuously stirred at 100 rpm and 25 °C for further reaction in a shaking incubator for predetermined time intervals (2 min, 10 min, 1 h, 1 day, and 4 days). The precipitated products were obtained by filtration through a 0.2 μm nylon membrane filter (Whatman, England) after each time interval, washed with deionized water, and dried at ambient temperatures before DOI: 10.1021/la1027509

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Figure 2. XRD patterns for CaCO3 crystals formed in the (a) absence of dopamine and the (b) presence of dopamine (2 mg/mL). C and V designate peaks for calcite and vaterite, respectively. Note that the intensity of highest calcite peak in pattern (b) is dramatically reduced compared to that in pattern (a). characterization. The mineralization of CaCO3 in the absence of dopamine was conducted using the same method described above, by mixing an equal volume ratio of 0.033 M CaCl2, 0.33 M Na2CO3, and 10 mM Tris buffer (pH 8.5), excluding the addition of dopamine hydrochloride. Characterization. To characterize morphologies and sizes of CaCO3 precipitates, scanning electron microscopy (SEM) images were obtained using a S-4800 field-emission scanning electron microscope (Hitachi High-Technologies Co., Japan) at an acceleration voltage ranging from 10 to 15 kV after sputtering the precipitates with platinum using a SCD005 Pt-coater (Bal-Tec AG., Liechtenstein). For the transmission electron microscopy (TEM) (JEM-3010, JEOL Co., Japan) analysis, samples were prepared by placing a droplet of aqueous solution containing CaCO3 precipitates onto carbon-coated copper grids, followed by immediate drying in a vacuum for 3 h. To identify the crystallographic structure of the CaCO3 precipitates, X-ray diffraction (XRD) patterns were obtained using a D/MAX-RC thin-film X-ray diffractometer (Rigaku Co., Japan) equipped with a nickel filter. The analysis was conducted under following conditions: scan speed of 3°/min, Cu KR radiation with λ=1.5418 A˚, and 2θ range from 20 to 70° at ambient temperatures. To substantiate the timewise polymerization of dopamine in aqueous solution, UV-visible absorption spectra were obtained using Biospec Mini (Shimadzu Co., Japan). To verify the existence of polydopamine in the CaCO3 precipitates, high-resolution dispersive-Raman spectra were measured using LabRAM HR (Horiba-Jobin-Yivon Co., France) and thermogravimetric analyses were performed using TG 209 F3 (Netzsch GmbH & Co., Germany). For the analysis of the phase transition of CaCO3 during the mineralization process, infrared spectra were obtained using a FT-IR IFS66 V/S (Bruker Optics Co., Germany) with a germanium single-crystal in an attenuated total reflection (ATR) mode at 4 cm-1 resolution.

Results and Discussion We prepared CaCO3 precipitates by mixing CaCl2 and Na2CO3 in the presence of dopamine. CaCO3 precipitates were collected after four days of incubation in the aqueous solution with or without dopamine (2 mg/mL), and their morphologies and sizes 14732 DOI: 10.1021/la1027509

were characterized by SEM and TEM analysis (Figures 1b-h). CaCO3 that mineralized without dopamine resulted in the formation of large rhombohedral calcite crystals with an average size over 25 μm (Figure 1b). In contrast, CaCO3 precipitates that mineralized in the presence of dopamine exhibited predominantly spherical morphologies with diameters ranging from 3 to 10 μm as shown in Figure 1c. Magnified SEM images (Figure 1d,f) and the TEM image (Figure 1e) reveal that the individual microsphere consists of much smaller nanoparticles that are less than 100 nm in size. The cross-sectional image exhibits a spherulitic growth of microspheres by the assembly of CaCO3 nanoparticles, resulting in the microspheres’ high porosity (Figure 1g,h). The crystalline phases of CaCO3 precipitates were verified using XRD analysis (Figure 2); Figure 2a shows the XRD pattern of CaCO3 precipitates that were formed in the absence of dopamine, revealing strong reflection peaks for calcite crystals. Conversely, the XRD pattern for CaCO3 mineralized in the presence of dopamine shows noticeably strong vaterite peaks (Figure 2b) indicating that the major phase is crystalline vaterite. These results indicate that the presence of dopamine has a stabilizing effect on the unstable vaterite phase, maintaining the spherical structure and blocking its transition to the calcite phase. The vaterite microspheres that were formed in the presence of dopamine were highly stable and dominantly observed after further incubation for over two weeks (as shown in Supporting Information, Figure S1), and even after two months. We performed a UV-vis spectroscopic analysis on dopamine solution to verify that the polymerization of dopamine was ongoing in the solution during the mineralization of CaCO3. According to our observation (Figure 3a), the absorbance intensity of the solution increased as time passes from 0 min to 6 h showing a saturation profile, which is consistent with the observation by Bernsmann et al.,23 a continuous increase in UV absorbance until it reaches dark brown color according to time passage indicating (23) Bernsmann, F.; Richert, L.; Senger, B.; Lavalle, P.; Voegel, J. -C.; Schaaf, P.; Ball, V. Soft Matter 2008, 4, 1621–1624.

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Figure 4. FT-IR spectra of CaCO3 precipitates formed in the presence of dopamine (2 mg/mL). The absorption peak for vaterite at 745 cm-1 (n2) emerged after two minutes of incubation and remained throughout the mineralization time, whereas a peak for calcite at 712 cm-1 (n1) did not appear. The splitting bands at around 1398 and 1475 cm-1 (n3) were observed throughout the entire incubation period (4 days). Each SEM image shows corresponding CaCO3 precipitates formed after 2 min, 1 day, and 4 days of incubation, respectively. These results indicate that the presence of dopamine in the incubation solution induced a rapid emergence and prolonged preservation of the spherical vaterite phase.

Figure 3. (a) UV absorbance profile change of a fresh dopamine solution (2 mg/mL) at 280 nm for the time passage of 6 h. Inset is the comparison between the UV spectra attained at time passages of 0 min and 6 h with wavelength range from 260 to 300 nm. (b) DispersiveRaman spectra of CaCO3 crystals formed after 4 days in the presence of dopamine (2 mg/mL). The polymerization of dopamine monomers to polydopamine is confirmed by peaks at Raman shifts of 1575.38 and 1341.19 cm-1, which indicate aromatic ring chains and bonds between phenyl groups in polydopamine, respectively. A weak peak at 3149.12 cm-1 indicates hydroxyl groups of polydopamine. The inset structural formula for polydopamine was suggested by H. Lee et al.21

the polymerization of dopamine. On the basis of the observation, we infer that the polymerization of dopamine was ongoing during CaCO3 mineralization, especially in the early phase of the mineralization. We analyzed the chemical properties of thus-formed CaCO3 precipitates using dispersive-Raman spectroscopy. The solid line in Figure 3b indicate the spectrum for the CaCO3 precipitates formed with dopamine. The spectrum shows peaks with Raman shifts of 1341.19 and 1575.38 cm-1. The two peaks respectively designate the stretching and deformations of aromatic ring chains, which correspond to peaks from the Raman spectra of polydopamine.24 This indicates the formation of polydopamine in the CaCO3 microspheres. According to literature,21 the oxidative polymerization of dopamine monomers results in the formation of polydopamine with cross-linked (24) Fei, B.; Qian, B.; Yang, Z.; Wanga, R.; Liu, W. C.; Mak, C. L.; Xin, J. H. Carbon 2008, 46, 1792–1828.

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Figure 5. FT-IR spectra of CaCO3 precipitates formed in the absence

of dopamine. The calcite peak at 712 cm-1 (n1) emerged after 1 h of incubation. After 4 days, only the calcite peak at 712 cm-1 (n1) remained while the vaterite peak at 745 cm-1 (n2) disappeared, and the split bands at 1398 and 1487 cm-1 (n3) merged into one peak at 1400 cm-1. Accordingly, SEM images show a complete phase transformation from heterogeneous spherical phase to rhombohedral calcite in 4 days. DOI: 10.1021/la1027509

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Figure 6. SEM images of CaCO3 precipitates mineralized for 24 h in the presence of various dopamine concentrations: (a) 2 mg/mL, (b) 0.2 mg/mL, and (c) 0.02 mg/mL. (d) Corresponding XRD patterns for the precipitates mineralized with different concentrations of dopamine in the reaction mixture.

5,6-dihydroxyindoles through a multistep reaction mechanism; the peaks mentioned earlier and another weak peak at 3149.12 cm-1 for -OH in catechol moieties are in accordance with the previous report. These results suggest that the formation and stabilization of spherical vaterite structure was induced by the polymerization of dopamine monomers into polydopamine during the CaCO3 mineralization. Our thermogravimetraic analysis (TGA) results further support the existence of polydopamine in the resulting CaCO3 precipitates. Unlike the pure CaCO3, precipitates mineralized in the presence of dopamine showed dual weight losses of 3.03 wt % at around 350-470 °C and 40.70 wt % at around 620-700 °C due to the decomposition of polydopamine and CaCO3, respectively (Supporting Information, Figure S2). To further investigate the effects of dopamine on the mineralization process of CaCO3, we analyzed the time-dependent phase transitions of CaCO3 precipitates using FT-IR spectroscopy. According to our results (shown in Figure 4), CaCO3 precipitates that were formed in the presence of dopamine exhibits a clearly different time profile of FT-IR spectra compared to precipitates that were formed in the absence of dopamine (Figure 5). FT-IR peaks at 712 (n1) and 745 cm-1 (n2) are known to be unique to the crystalline calcite and vaterite phases, respectively.25-27 Although a slight peak at 730 cm-1 might be considered as a shifted characteristic peak for minor calcite phase existing in the precipitates, no exact characteristic peak for calcite was observable at (25) Addadi, L.; Raz, S.; Weiner, S. Adv. Mater. 2003, 15, 959–970. (26) Li, C. M.; Botsaris, G. D.; Kaplan, D. L. Cryst. Growth Des. 2002, 2, 387– 393. (27) Vagenas, N. V.; Gatsouli, A.; Kontoyannis, C. G. Talanta 2003, 59, 831– 836.

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712 cm-1 (n1) in the presence of dopamine (Figure 4); whereas it appeared within an hour of incubation without dopamine (Figure 5). The peak at 745 cm-1 (n2) and a split in band at around 1398 and 1475 cm-1 (n3) remained constant throughout the incubation period for the precipitates mineralized with dopamine (Figure 4). In contrast, the peak for vaterite (n2) disappeared after 4 days of incubation for the precipitates without dopamine (Figure 5) and sharp peak for calcite at 712 cm-1 (n1) remains, corresponding to a FT-IR result for CaCO3 mineralized without additives shown in a previous report.28 Note that the two split carbonate absorptions (n3) appear in ACC or the vaterite phase by an asymmetric stretch of carbonate ions, according to a previous report.25 These results suggest that the addition of dopamine preserves the vaterite phase for a prolonged period of time. Accordingly, the vaterite’s spherical morphology did not change throughout the time periods as shown in SEM images for precipitates mineralized for 2 min, 1 day, and 4 days, although the sizes of microspheres did increase slightly and heterogeneously after 4 days of mineralization (Figure 4), in contrast to the SEM images in Figure 5 that show clear morphologic change from heterogeneous spheres to rhombohedral caclite for the precipitates formed without dopamine. We performed SEM and XRD analyses of CaCO3 precipitates that were formed in the presence of different dopamine concentrations (2, 0.2, and 0.02 mg/mL) for 24 h to verify the stabilizing effect of dopamine on vaterite microspheres. The SEM images (Figure 6a-c) and the XRD pattern (Figure 6d) clearly illustrate the dependency of morphologies and phase transformations of CaCO3 on dopamine concentration. According to our results, (28) Wang, X.; Kong, R.; Pan, X.; Xu, H.; Xia, D.; Shan, H.; Lu, J. R. J. Phys. Chem. B 2009, 113, 8975–8982.

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spherical vaterite was a major phase when CaCO3 was mineralized in the presence of 2 mg/mL dopamine (Figure 6a). In contrast, the SEM image of the precipitates mineralized under 0.2 mg/mL dopamine shows significantly increased rhombohedral calcites with 10 to 20 μm in size coexisting with remaining vaterite microspheres (Figure 6b). In the case of a lesser amount of dopamine (0.02 mg/mL), it shows dominant calcite phase with apparently larger crystal sizes of mostly over 25 μm, which is similar to the size of calcite crystals that were mineralized in the absence of dopamine (Figure 1b) with enlarged agglomerates of vaterite microspheres (Figure 6c). In both cases of reduced dopamine conditions (i.e. 0.2 mg/mL and 0.02 mg/mL), vaterite transforming into calcite were found as shown in Supporting Information, Figure S3. The calcite crystals grows from dissolving surface of vaterite microsphere (Supporting Information, Figure S3a) and the dissolution of vaterite leaves the round traces on the calcite surface (indicated by white arrows, Supporting Information, Figure S3b, S3c). The vaterite microspheres and their agglomerates exhibited destructed surfaces and internal structures along with debris (Supporting Information, Figure S3c, S3d), implying that the vaterite microspheres were undergoing a dissolution process before they recrystallized into rhombohedral calcite. Note that our observations on vaterite dissolution and the growth of calcite are in a good agreement with the previous reports showing similar visual evidence suggesting the dissolution of vaterite and recrystallization to calcite.28,29 Thereby, we attribute the size difference between the calcite crystals in Figure 6b,c to the effect of polymerization of dopamine, which delayed the dissolutionrecrystallization process, resulting in a retarded growth of calcite crystals in Figure 6b (with 0.2 mg/mL dopamine) compared to those in Figure 6c (with 0.02 mg/mL dopamine). The corresponding XRD patterns for the precipitates observed in Figure 6b,c show typical, dominant peaks for calcite crystals (Figure 6d), suggesting that the stabilizing effect is dependent on dopamine concentration, and that a sufficient amount of dopamine is needed to fully stabilize vaterite microspheres. Many efforts had been made so far to stabilize vaterite phase by using additives such as amino acids,30 polycarboxylates,31,32 polypeptides,33,34 surfactants,35 double-hydrophilic block copolymers,36-38 dendrimers,39,40 and carbon nanotubes,41 although explanations about stabilization mechanisms vary depending on their specific assays.7 Regarding the enhancement of vaterite stability by dopamine in our study, we analogize that the strong adhesive property induced by the catechol group in dopamine20-22 may keep the vaterite nanoparticles together and maintain the spherical structure. Such hypothesis is based on the previously reported affinitive interaction between calcium ions and the (29) Rieger, J.; Thieme, J.; Schmidt, C. Langmuir 2000, 16, 8300–8305. (30) Tong, H.; Ma, W.; Wang, L.; Wan, P.; Hu, J.; Cao, L. Biomaterials 2004, 25, 3923–3929. (31) Xie, A. -J.; Yuan, Z. -W.; Shen, Y. -H. J. Cryst. Growth 2005, 276, 265–274. (32) Meng, Q.; Chen, D.; Yue, L.; Fang, J.; Zhao, H.; Wang, L. Macromol. Chem. Phys. 2007, 208, 474–484. (33) Zhang, Z. P.; Gao, D. M.; Zhao, H.; Xie, C. G.; Guan, G. J.; Wang, D. P.; Yu, S. H. J. Phys. Chem. B 2006, 110, 8613–8618. (34) Sugawara, T.; Suwa, Y.; Ohkawa, K.; Yamamoto, H. Macromol. Rapid Commun. 2003, 24, 847–851. (35) Dupont, L.; Portemer, F.; Figlarz, M. J. Mater. Chem. 1997, 7, 797–800. (36) Sedlak, M.; Antonietti, M.; C€olfen, H. Macromol. Chem. Phys. 1998, 199, 247–254. (37) C€olfen, H.; Antonietti, M. Langmuir 1998, 14, 582–589. (38) Yu, S.-H.; C€olfen, H.; Hartmann, J.; Antonietti, M. Adv. Funct. Mat 2002, 12, 541–545. (39) Naka, K.; Tanaka, Y.; Chujo, Y.; Ito, Y. Chem. Commun. 1999, 1931–1932. (40) Naka, K.; Tanaka, Y.; Chujo, Y. Langmuir 2002, 18, 3655–3658. (41) Li, W.; Gao, C. Langmuir 2007, 23, 4575–4582. (42) Holten-Anderson, N.; Mates, T. E.; Toprak, M. S.; Stucky, G. D.; Zok, F. W.; Waite, J. H. Langmuir 2009, 25, 3323–3326.

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Figure 7. SEM images for CaCO3 mineralized for 24 h with or without dopamine in the reacting mixture (a,c), and corresponding precipitates after being resuspended in a dopamine-free system (a filtered solution saturated with respect to calcite) for 36 h (b,d). (a) CaCO3 mineralized for 24 h in the absence of dopamine in the solution showing calcite crystals with minor remaining vaterite. (b) Rhombohedral calcite crystal showing an evident crystal growth after suspending the precipitates in (a) in the dopamine-free system for 36 h. (c) CaCO3 mineralized for 24 h in the presence of 2 mg/mL dopamine in the solution mainly showing vaterite microspheres. (d) After suspending the precipitates in (c) in dopamine-free system for 36 h showing mixture of remaining vaterite microspheres and calcite crystals whose sizes are similar to the surrounding vaterite microspheres. (e) Representative morphology of a vaterite microsphere in (c). (f) Morphology of a vaterite microsphere in (d), which has undergone dissolution process of surface showing urchinlike structure. (g) A vaterite microsphere in (d) showing a partial growth of calcite crystal on its surface. (h) A calcite crystal in (d) showing the size similar to those of surrounding vaterite microspheres indicating that the growth of calcite is hindered.

catechols,42 which would further prevent the dissolution of vaterite and recrystallization of calcite. Figure 7 demonstrates that the presence of dopamine hinders both dissolution of vaterite and growth of calcite. When precipitates were resuspended in a dopamine-free system for 36 h, the vaterite microspheres that were mineralized in the presence of 2 mg/mL dopamine showed a strong resistivity against the complete dissolution, and a significantly reduced crystal growth of calcite (Figure 7c-h), whereas the precipitates that were mineralized without dopamine showed a substantial crystal growth of calcite crystals (Figure 7a,b) with hardly found remaining vaterite crystals. The inhibitory effect of dopamine on the transformation of vaterite to calcite phase is further supported by the fact that the vaterite crystals, which were observed in the presence of 2 mg/mL dopamine after incubation for 4 days (Figure 1c), maintained their original, spherical morphology compared to the crystals shown in Figure 7d,f-h that exhibits partly dissolved surfaces and slight growth of calcite phase. Hence, the presence of dopamine in the solution should have a significant effect in hindering the calcite recrystallization as well as in preventing the dissolution of vaterite. To observe the effect of dopamine polymerization on CaCO3 mineralization, we mineralized CaCO3 in the presence of alreadypolymerized dopamine (i.e., polydopamine) that was prepared DOI: 10.1021/la1027509

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by incubating a fresh dopamine solution for 48 h. As shown in Supporting Information, Figure S4, the formation of vaterite microspheres were significantly reduced in the case but a massive formation of rhombohedral calcite phase was observed. We attribute this to the aggregation of polydopamine itself resulting in reduced catechol moieties exposed outward that could interact with calcium in the vaterite to reduce dissolution of vaterite and growth of calcite when participated in the mineralization process. Supporting Information, Figures S4b-e show polydopamine agglomerates adhered on the surface of CaCO3 precipitates. It is further supported by Supporting Information, Figures S4f-h showing the comparison between Si wafers dip-coated with 2 mg/mL dopamine solution which had been polymerized for 2 min and 48 h beforehand, which clearly demonstrate that the polydopamine agglomerates are formed when dopamine molecules are excessively polymerized (i.e., for 48 h) before coating. The mechanism of transformation from ACC to crystalline vaterite remains controversial yet,43 but it is often referred to as a solid-state transformation, which implies that a short-range local order already exists in ACC and subsequently leads to the transition of ACC to a certain crystalline polymorph.44 As the mechanism of a size-increase of spherulitic crystalline particles is an unresolved issue that is often disputed in terms of nanoaggregation and crystal growth,45,46 the exact mechanism of stabilization effect of dopamine polymerization upon growth of vaterite spherulites would also remain as another topic to be further elucidated. Future investigation will examine the interaction between the polymerization of dopamine and the recently discovered prenucleation clusters that are found at the primitive stage (i.e., the stage before ACC phase) of CaCO3 mineralization8,47 to cover the full mechanism underlying the biomineralization process. We expect that porous CaCO3 microspheres hybridized with adhesive polydopamine may become effective templates for layer-by-layer assemblies, microcapsules for encapsulating bioactive compounds, (43) Meldrum, F. C.; C€olfen, H. Chem. Rev. 2008, 108, 4332–4432. (44) Levi- Kalisman, Y.; Raz, S.; Weiner, S.; Addadi, L.; Sagi, I. Adv. Funct. Mater. 2002, 12, 43–48. (45) Andreassen, J.-P. J. Cryst. Growth 2005, 274, 256–264. (46) Beck, R.; Andreassen, J.-P. Cryst. Growth Des. 2010, 10, 2934–2947. (47) Gebauer, D.; V€olkel, A.; C€olfen, H. Science 2008, 322, 1819–1822.

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Kim and Park

or precursors for other biomineralization processes to fabricate novel inorganic-organic hybrid materials.

Conclusion Dopamine, a biomimetic small molecule inspired from mussel adhesive foot proteins, has been introduced for the facile synthesis of CaCO3 vaterite microspheres. We found that the vaterite microspheres were preserved for a prolonged time when mineralized in the presence of 2 mg/mL dopamine, whereas complete transition to the calcite phase occurred within four days in the absence of dopamine. The vaterite microspheres consisted of 50 to 100 nm-sized nanoparticles, and their cross sections exhibited high porosity and spherulitic orientation. The presence of polydopamine in the microspheres was confirmed by Raman spectroscopy and TGA, indicating the oxidative polymerization of dopamine during the mineralization of CaCO3. We suggest that the calcium-binding catechol moieties of dopamine are adsorbed onto CaCO3 nanoparticles to induce a prolonged conservation of vaterite microspheres by retarding the dissolution and recrystallization into the calcite phase. Acknowledgment. This study was supported by the National Research Foundation (NRF) via National Research Laboratory (NRL) (R0A-2008-000-20041-0) and Converging Research Center (2009-0082276) Programs. This research was also partially supported by the BioGreen 21 Program (20070301034038), Republic of Korea. Supporting Information Available: SEM images and XRD pattern of CaCO3 precipitates that were mineralized for two weeks in the presence of dopamine. TGA result for the CaCO3 mineralized with or without dopamine. SEM images of vaterite microspheres undergoing dissolution and recrystallization process in 24 h in lesser dopamine concentrations (i.e., 0.2 mg/mL, 0.02 mg/mL) in the reaction mixture. SEM images for CaCO3 mineralized in a solution that contains polymerized dopamine for 48 h before the mineralization of CaCO3. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2010, 26(18), 14730–14736