Langmuir 2006, 22, 3073-3080
3073
Probing Cooperative Interactions of Tailor-Made Nucleation Surfaces and Macromolecules: A Bioinspired Route to Hollow Micrometer-Sized Calcium Carbonate Particles Niklas Loges,† Karlheinz Graf,§ Lutz Nasdala,‡ and Wolfgang Tremel*,† Institute of Inorganic and Analytical Chemistry, Duesbergweg 10-14, and Institute of Geosciences, Becherweg 21, Johannes Gutenberg-UniVersita¨t Mainz, D-55099 Mainz, Germany, and Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany ReceiVed October 24, 2005. In Final Form: January 2, 2006 It is well known that the formation of biominerals by living organisms is governed by the cooperation of soluble and insoluble macromolecules with peculiar interfacial properties. To date, most of the studies on mineralization processes involve model systems that account only for the existence of one organic matrix and thus disregard the interaction between the soluble and insoluble organic components that is crucial for a better understanding of the processes taking place at the inorganic-organic interface. We have set up a model system composed of a matrix surface, which is composed of a self-assembled monolayer (SAM) and a soluble component, poly(aspartic acid). It could be demonstrated that the phase selection of calcium carbonate and the morphology of the resulting particles are determined by the stabilization of amorphous precursor particles by the polymer and the interaction between polymer and SAM. The morphology of the hollow vaterite microspheres are reminiscent to a 3D analogue of the so-called “coffee-stain effect”, where the transformation from a voluminous hydrated, amorphous material to a more dense crystalline material leads to the formation of hollow spheres from massive spherical microparticles.
Introduction Nature produces fascinating architectures of inorganic materials on a wide range of macro- and microscales and makes use of inorganic components such as calcium carbonate as a constituent of exoskeletons or as protective shells.1-3 Examples abound in nature, ranging from the egg shells of birds and snail shells to sea shells and nacreous pearls.4 From the known polymorphs of calcium carbonate, calcite and aragonite are the most prominent ones in living systems. How the complex morphologies of CaCO3 architectures are actually formed by biomineralization processes is still a matter of debate. It is generally assumed that biological macromolecules play a key role in the mineralization process and are intimately associated with the growing mineral. The nucleation process is believed to be initiated at the organicinorganic interface, the organic component providing a nucleating matrix for the inorganic mineral. However, the interface between the inorganic mineral and the organic matrix represents only one aspect of the biomineralization process. In addition to insoluble matrix proteins involved in the nucleation, there are soluble proteins exerting a major impact on the nucleation and propagation steps. Mineral-binding proteins are responsible for the modulation of the mineralization process,1,2,4 either by preventing nucleation through the complexation of ions in solution or by stabilizing precursor particles and preventing particle agglomeration by surface binding. This has been demonstrated for a prominent example, the formation of the nacreous layer of the abalone shell, Haliotis rufescens.4,5 † Institute of Inorganic and Analytical Chemistry, Johannes GutenbergUniversita¨t Mainz. ‡ Institute of Geosciences, Johannes Gutenberg-Universita ¨ t Mainz. § Max Planck Institute for Polymer Research.
(1) Weiner, S.; Addadi, L. J. Mater. Chem. 1997, 7, 689-702. (2) Mann, S. Biomineralization - Principles and Concepts in Biomaterials Chemistry; Oxford University Press: Oxford, U.K., 2001. (3) Meldrum, F. C. Int. Mater. ReV. 2003, 48, 187-224. (4) (a) Lowenstam, H. A., Weiner, S., Eds. On Biomineralisation; Oxford University Press: New York, 1989. (b) Ba¨uerlein, E. Biomineralization; WileyVCH: Weinheim, Germany, 2000.
Presently, an increasing body of experimental evidence points toward amorphous calcium carbonate (ACC) as a reactive intermediate or precursor during the formation of crystalline polymorphs in biomineralization processes.6 The formation of amorphous calcium carbonate as a transitory precursor stabilized by surface-bound macromolecules may explain the formation of rather complex structures that are seemingly unrelated to the symmetry of the crystal structure of the final polymorphs. From investigations on marine algae (Pleurochrysis and Emiliana), it is known that acidic low-molecular-weight polyanions are involved in the nucleation of CaCO3 and the growth of CaCO3 coccoliths.7,8 In these algae, calcite nucleation is believed to occur in contact with the so-called coccolith ribbon, a narrow band of organic material. Subsequently, small crystallites appear within the particle clusters. The crystallization is terminated by the dissociation of the so-called coccolithosome particles and the formation of a polyanion coating on the mineral surface.9 Because these complex interactions within the calcifying machinery of natural systems are difficult to analyze, many studies have concentrated on the nucleating matrix so far. One obvious strategy is to demineralize the skeletal matrix and to rebuild the mineralized tissue in a “retrosynthetic” step. A pitfall of this route is that soluble molecules may be extracted from the matrix as well. A related approach is to extract the soluble matrix proteins that are involved in the nucleating process from biological material and to use them afterward in in-vitro experiments.10 An alternative approach is to design simplified synthetic models of the matrix (5) (a) Weiss, I. M.; Renner, C.; Strigl, M. G.; Fritz, M. Chem. Mater. 2002, 14, 3252-3259. (b) Su, X.; Belcher, A. M.; Zaremba, C. M.; Morse, D. E.; Stucky, G. D.; Heuer, A. H. Chem. Mater. 2002, 14, 3106-3117. (6) Raz, S.; Hamilton, P. C.; Wolt, F. H.; Weiner, S.; Addadi, L. AdV. Funct. Mater. 2003, 13, 480-486 (7) Marsh, M. E. In Biomineralization: Progress in Biology, Molecular Biology and Application; Ba¨uerlein, E., Ed.; Wiley-VCH: Weinheim, Germany, 2004; pp 197-216. (8) Marsh, M. E.; Chang, D. K.; King, G. C. J. Biol. Chem. 1992, 267, 2050720512. (9) Marsh, M. E. Protoplasma 1994, 177, 108-122.
10.1021/la0528596 CCC: $33.50 © 2006 American Chemical Society Published on Web 03/07/2006
3074 Langmuir, Vol. 22, No. 7, 2006
proteins. In this context, Langmuir monolayers,11 protein-covered substrates,1,10 polymer dispersions,12 liquid-crystalline systems,13 self-assembled monolayers (SAM)14 or colloids,15 and dendrimers16 have been studied as model interfaces for the crystallization of inorganic compounds. This reaction sequence can be mimicked by a simplified model system consisting of a self-assembled thiol monolayer tethered to a Au(111) surface and acidic macromolecules in solution. The advantage of this rather simple system is that the crystallization of calcium carbonate (or other inorganic compounds) on organic monolayers11,15,16 and the interaction of the polyelectrolyte with the bivalent cations are quite well understood and can easily be monitored. In a recent study, it could be demonstrated that hierarchically ordered mineral structures were formed by the cooperative interaction, mediated by the Ca2+ cations, between a polar SAM and anionic macromolecules in solution.17 These supramolecular electrostatic interactions should be strongly dependent on (i) the surface functionality of the SAM and that of the dissolved macromolecule, (ii) the protonation equilibria associated with the dissolved macromolecules and the headgroups of the thiols (e.g., R,ω-carboxylate thiols) constituting the SAM (i.e., the pH value), and (iii) cation species involved in complexation equilibria.18 Moreover, for weakly interacting molecules and SAMs temperature effects might play a significant role when the interactions are on the order of the thermal energy. We have pursued this hypothesis by carrying out experiments on a model system, where through the interaction of a dissolved polyelectrolyte [poly(aspartic acid), Na salt] with a self-assembled organic monolayer hollow vaterite spheres were formed from transient amorphous CaCO3 nanoparticles.
Loges et al. Mineralization Procedure. Glass slides were cleaned and hydrophilized using a modified RCA method20 in which they were placed in a mixture of ultrapure water (Barnstead Easypure UV, F > 18.3 MΩ cm-1), ammonia solution (28-30%), and hydrogen peroxide (5:1:1 by volume). The mixture was heated to 80 °C and held at this temperature for 10 min. After cooling to room temperature, the slides were rinsed with ultrapure water and blown dry with nitrogen (99.999%). After transfer to the vacuum chamber of a coating unit (Edwards-FL400), the slides were coated with 1.5 nm of Cr, followed by 55 nm of Au. The slides were then exposed to a 1 mM solution of the thiol in toluene (p.a., Riedel) or ethanol (p.a., Roth) for about 24 h. After extensive rinsing with toluene or ethanol to remove unbound thiols, the surface was blown dry with nitrogen. These slides were placed on a plastic slide holder face-down in a crystallization flask, as described in ref 14b, containing 250 mL of a 10 mM CaCl2 solution, and 500 µL of a poly(aspartic acid) sodium salt solution (average molar mass: 2500 g/mol, Baypure DS 100, 40%) was added to reach a concentration of 1.0 mg/mL of poly(aspartic acid). The pH value was adjusted with 0.1 mol/L hydrochloric acid to 7.0, and the flask was placed in a desiccator. Subsequently, the crystallization process was started by placing a Petri dish with 7.0 g of (NH4)2CO3 at the bottom of the desiccator. The crystallization was carried out at room temperature (25 °C) and stopped after 2 days by elicitation of the slides, washing them with Millipore water to remove weakly adhered crystals and finally blown dry with pure nitrogen. Calculation of the Degree of Hydration of ACC. The degree of hydration of CaCO3 can be estimated in a straightforward manner on the basis of the following assumptions: (i) The final volume of the vaterite spheres is equal to the original volume of the ACC droplet. (ii) There is no growth of the vaterite spheres to the outside liquid. (iii) The amount of poly(aspartic acid) in the ACC is negligible. Therefore, the number of moles of CaCO3 in a sphere is given by
Experimental Section Reagents. Undecanethiol was bought from Sigma-Aldrich (98%), and ω-mercapto-undecanol and ω-mercaptoundecanoic acid were prepared from the corresponding ω-bromo compounds by the Bunte salt method.19 (10) (a) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science, 1996, 271, 67-69. (b) Levi, Y.; Albeck, S.; Brack, A.; Weiner, S.; Addadi, L. Chem.sEur. J. 1998, 4, 389-396. (11) (a) Jaquemain, D.; Wolf, S. G.; Leveiller , F.; Deutsch, M.; Kjaer, K.; Als-Nielsen, J.; Lahav, M.; Leiserowitz, L. Angew. Chem., Int. Ed. Engl. 1992, 31, 130-152. (b) Mann, S.; Archbald, D. D.; Didymus, J. M.; Doughlus, T.; Heywood, B. R.; Meldrum, F. C.; Reeves, M. J. Science 1993, 261, 1286-1292. (c) Donners, J. J. J. M.; Nolte, R. J. M.; Sommerdijk, N. A. J. M. J. Am. Chem. Soc. 2002, 124, 9700-9701. (12) (a) Marentette, J. M.; Norwig, J.; Sto¨ckelmann, E.; Meyer, W. H.; Wegner, G. AdV. Mater. 1997, 9, 647-650. (b) Templin, M.; Frank, A.; DuChesne, A.; Leist, H.; Zhang, Y. M.; Ulrich, R.; Schadler, V.; Wiesner, U. Science 1997, 278, 1795-1798. (c) Cha, J. N.; Stucky, G. D.; Morse, D. E.; Deming, T. J. Nature 2000, 403, 289. (d) Co¨lfen, H.; Qi, L. Chem.sEur. J. 2001, 7, 106-116. (13) Kresge, C. T.; Leonowicz, M. E.; Roth, W. J.; Vartuli, J. C.; Beck, J. S. Nature 1992, 359, 710-713. (14) (a) Ku¨ther, J.; Tremel, W. J. Chem. Soc., Chem. Commun. 1997, 20292030. (b) Ku¨ther, J.; Seshadri, R.; Knoll, W.; Tremel, W. J. Mater. Chem. 1998, 8, 641-650. (c) Ku¨ther, J.; Nelles, G.; Seshadri, R.; Schaub, M.; Butt, H.-J.; Tremel, W. Chem.sEur. J. 1998, 4, 1834-1841. (d) Ku¨ther, J.; Seshadri, R.; Nelles, G.; Butt, H.-J.; Knoll, W.; Tremel, W. AdV. Mater. 1998, 10, 401-404. (e) Aizenberg, J.; Black, A. J.; Whitesides, G. M. J. Am. Chem. Soc. 1999, 121, 4500-4509. (15) (a) Ku¨ther, J.; Seshadri, R.; Tremel, W. Angew. Chem., Int. Ed. 1998, 37, 3044-3047. (b) Ku¨ther, J.; Seshadri, R.; Nelles, G.; Assenmacher, W.; Butt, H.-J.; Mader, W.; Tremel, W. Chem. Mater. 1999, 11, 1317-1325. (c) Bartz, M.; Ku¨ther, J.; Nelles, G.; Weber, N.; Seshadri, R.; Tremel, R. J. Mater. Chem. 1999, 9, 1121-1126. (16) Donners, J. J. M.; Heywood, B. R.; Meijer, E. W.; Nolte, R. J. M.; Sommerdijk, N. A. J. M. Chem.sEur. J. 2002, 8, 2561-2567. (17) (a) Balz, M.; Therese, H. A.; Li, J.; Gutmann, J. S.; Kappl, M.; Nasdala, L.; Hofmeister, W. Butt, H.-J.; Tremel, W. AdV. Funct. Mater. 2005, 15, 683688. (b) Balz, M.; Therese, H. A.; Kappl, M.; Nasdala, L.; Hofmeister, W.; Butt, H.-J.; Tremel, W. Langmuir 2005, 21, 3981-3986. (18) Buescher, K.; Graf, K.; Ahrens, H.; Helm, C. A. Langmuir 2002, 18, 3585-3591. (19) Wolf, H. Ph.D. Dissertation, Universita¨t Mainz, Mainz, Germany, 1995.
4 NCaCO3 ) π 3
FCaCO3 [r3 - (r - d)3] M volume of the shell CaCO3
where d is the thickness of the vaterite shell, FCaCO3 is the density of calcium carbonate (vaterite, Fvaterite ) 2.65 g/cm3),21 and MCaCO3 is the molar weight of calcium carbonate (100.087 g/mol). The number of moles of water in a sphere is given by 3 4 π(r - d) FH2O NH2O ) 3 MH2O
where r is the external radius of the sphere. The degree of hydration can now be calculated according to NH2O NCaCO3
)
π(r - d)3FH2O MH2O
MCaCO3 1 ) 3 F π[r - (r - d) ] CaCO3 3
(r - d)3FH2OMCaCO3 MH2O[r3 - (r - d)3]FCaCO3 using an average value of r ) 1.75 µm and a thickness d ) 100 nm of the calcium carbonate shell. Because we cannot determine the thickness of the hollow sphere accurately, we use a constant value of 100 nm for all particles. Therefore, we can give only a rough estimate of the degree of hydration. The above equation leads to a formula of CaCO3 × 6.8H2O (variance ( 10%,) in harmony with the well-known hexahydrate of calcium carbonate. Scanning Electron Microscopy (SEM). The imaging of the resulting CaCO3 particles on the clean slides was performed on a Zeiss DSM 962 or a Zeiss DSM 940. The acceleration voltage was (20) Graf, K.; Riegler, H. Colloids Surf., A 1998, 131, 215-224. (21) Bolze, J.; Peng, B.; Dingenouts, N.; Panine, P.; Narayanan, T.; Ballauf, M. Langmuir 2002, 18, 8364-8369
Nucleation Surfaces and Macromolecules fixed to 5 kV, and the working distance was 5 to 7 mm. Small cuts from the glass slides were fastened with conductive carbon tabs on aluminum sample holders. For better conductivity, the samples were sputtered with 10 nm of gold using a Baltec MED020 coating system. Quartz Crystal Microbalance Measurements. The measurements were performed on a Q-sense D300 system (LOT, Darmstadt). AT-cut quartz crystals coated with gold films were cleaned and covered with SAMs using the procedure described above for the glass slides. The temperature was controlled with a Peltier element optimized for the temperature range between 18 and 40 °C. The sample chamber was filled with a 10 mmol/L solution of CaCl2 until constant frequency was achieved. Subsequently, the liquid was exchanged with a tempered solution of 10 mmol/L CaCl2 containing 1.0 mg/mL poly(aspartic acid). In both solutions, the pH was adjusted to 7.0 at the start of the experiments. Raman Microspectroscopy. The Raman spectrum measurement was carried out on a LabRAM HR800 (Jobin Yvon, Horiba). This confocal Raman system is based on a dispersive spectrometer with a notch filter and a focus length of 800 µm. It is equipped with an optical microscope (Olympus BX41) and a Peltier-cooled CCD detector (charge-coupled device). The spectra were excited with the 638.17 Å emission of a He-Ne laser. The lateral resolution was better than 1.5 µm, and the volume resolution was approximately 5 µm3. The wavenumber precision was 0.5 cm-1, and the spectral resolution was about 0.7 cm-1. Time-Resolved IR Measurements. Time-resolved IR measurements were carried out by interrupting the mineralization for one of several parallel reactions carried out in 20 mL flasks after time intervals of 1 and 24 h per flask. The CaCO3 content of the corresponding flask was collected by centrifugation, washed with ultrapure water, and finally dried at room temperature. FTIR spectra were recorded on solid samples in a KBr matrix. The samples were ground with dry KBr into a fine powder and pressed into transparent pellets. The spectra were recorded in the far-IR region (700-200 cm-1, ∼5 cm-1 resolution) with an FTIR spectrometer (2030 Galaxy FTIR, Mattson Instruments) equipped with a TGS/PE detector and a silicon beam splitter. pH Measurement. A crystallization flask was placed in a desiccator with a side tube through which the cable of the pH electrode was conducted outside to the processing unit. We used a WTW SenTix 81 pH electrode with automatic temperature compensation and a WTW pH 340 processing unit to monitor the pH value in intervals of 10 min for 2000 minutes.
Langmuir, Vol. 22, No. 7, 2006 3075
Figure 1. SEM micrographs of CaCO3 crystals on a thiol-modified surface with (a) C10COOH, (b) C11OH, and (c) C11H at pH 7 and at 22 ( 3 °C (scale bar ) 200 µm).
Results and Discussion
Figure 2. SEM micrographs of varite hollow spheres obtained after 2 days of crystallization on OH-terminated SAMs in the presence of poly(aspartate)- (a, scale bar ) 5 µm; b, scale bar ) 200 nm) and calcite-covered spheres obtained after 3 days of crystallization (c, scale bar ) 5 µm; d, scale bar ) 10 µm).
SAM Surface without Polymer Additive. In previous studies, it could be demonstrated that the selectivity regarding the preferential formation of a certain crystal phase is determined by the chain length and headgroups of the ω-functional thiols,14b,c,e the surface roughness,14d and the temperature.14a,b The mineralization of CaCO3 is started by the thermal decomposition of ammonium carbonate and subsequent diffusion of the evolving CO2 and NH3 into the solution whose pH was adjusted to 7.0 using a 0.1 m HCl solution. Figure 1 shows SEM micrographs of CaCO3 crystals formed on conventional SAMs, terminated by headgroups of different polarity [COOH (C10COOH ) ω-mercaptoundecanoic acid), OH (C11OH ) ω-mercaptoundecanol), CH3 (C11H ) 1-mercaptoundecane)]. On the COOH-terminated surface (Figure 1a), mainly aragonite, but without any preferred orientation, was obtained. The precipitate on the OH-terminated surface is exclusively calcite, with preferred nucleation of the crystals on the flat (104) side (Figure 1b). On the apolar CH3terminated surface, equal amounts of calcite and aragonite were formed, although with a lower nucleation density and without preferred orientation (Figure 1c). SAM Surface with Polymer Additive. Performing the above mineralization processes in the presence of poly(aspartic acid)
led to the formation of very different crystallization products. Microstructured hollow spheres of vaterite were formed on OHterminated SAMs (as well as on mica and glass) after 2 days. Parts a and b of Figure 2 show an overview SEM image of the mineralization products and a high-resolution scanning electron microscopy image (HR-SEM) of a single hollow sphere which in turn seems to be composed of smaller spherical particles, as can be seen most clearly at the edges of the mouth of the open spheres. Energy-dispersive analysis of X-rays (EDX) from the products shown in Figure 2 yielded strong Ca, C, and O signals (22.4 atom % C, 60.2 atom % O, and 17.5 atom % Ca). An interesting feature of the spherical structures is their uniformity in shape and size (particle diameters ranging between 2 and 5 µm). Most of the microspheres are closed but a smaller fraction is open, possibly because the spheres adhered to the SAM or glass support were unintentionally moved and the outer was broken. After 3 days of crystallization, the hollow spheres were either overgrown with an outer layer of calcite, the most stable polymorph, or coarse crystal aggregates of calcite were formed on the surface by secondary nucleation as illustrated in Figures 2c and d. The growth of the outer calcite shell of facetted crystals
3076 Langmuir, Vol. 22, No. 7, 2006
Loges et al.
Figure 4. pH profile of CaCO3 crystallization. (A) pH equilibrium is dominated by NH3 uptake. (B) pH equilibrium is controlled by CO2 uptake. (C) CO2 uptake and depletion by CaCO3 precipitation have similar orders of magnitude. (D) pH value is dominated by the release of aspartate.
Figure 3. (a) Raman spectra of vaterite spherules shown in Figure 2a and b. (b) Raman spectra of vaterite spherules overgrown with a calcite layer as shown in Figure 2c and d.
takes place in the later stages of the crystallization only when the level of supersaturation is low and the crystals grow more slowly. With the aid of micro-Raman spectroscopy (or IR spectroscopy),22a,b it is possible to distinguish and positionally resolve micrometer-sized aggregates of CaCO3 minerals calcite (trigonal), aragonite (rhombic), vaterite, or even amorphous CaCO3 phases.22 A typical Raman spectrum of the hollow spheres and the overgrowth layer is shown in Figure 3 with reference spectra of calcite and vaterite for comparison. Within the Raman spectrum, the most intense vibration of calcite is the symmetric stretching vibration of the carbonate group at ∼1080 cm-1. The Raman spectrum of the spherical particles also shows the characteristics of the vaterite structure. The spectrum of vaterite contains four main sharp bands at 1085, (1011), 714, and 278 cm-1 (Figure 3a). In particular, the splitting of the principal band (reflecting two distinct site symmetries for the CO32- groups in the crystal lattice) with a very weak subband at 1011 cm-1 is a prototypical feature of a vaterite polymorph, which is not observed in calcite structures.22 Interestingly, a definitive crystal structure determination of vaterite is not yet available because none of the three proposed structures for vaterite shows such a feature.23,24 At lower frequency (below 400 cm-1), the spectrum of the (22) (a) Nasdala, L.; Banerjee, A.; Ha¨ger, T.; Hofmeister, W. Microsc. Anal., Eur. Ed. 2001, 70, 7. (b) Weiss, I. M.; Tuross, N.; Addadi, L.; Weinser, S. J. Exp. Zool. 2002, 293, 478-491. (c) Becker, A.; Bismayer, U.; Epple, M.; Fabritius, H.; Hasse, B. Dalton Trans. 2003, 551-555. (23) (a) D’Souza, S. M.; Alexander, C.; Carr, S. W.; Waller, A. M.; Whitcombe, M. J.; Vulfson, E. N. Nature 1999, 398, 312-315. (24) (a) Farmer, V. C. The Infrared Spectra of Minerals; Mineralogical Society: London, 1974. (b) Nakamoto, K. Infrared Spectra of Inorganic and Coordination Compounds; Wiley: Weinheim, Germany, 1978. (c) Decius J. C.; Hexter, M. Molecular Vibrations in Crystals; McGraw-Hill: New York, 1987.
carbonate anion has Raman bands associated with the vibrations of the carbonate group relative to calcium ions. The presence of vaterite and calcite for the inner and outer shells was confirmed independently by X-ray microdiffraction. Thus, the phase identity of the hollow spheres could be identified unambiguously as vaterite (hexagonal CaCO3), whereas the outer shell in Figure 2c and d was proven to contain typical calcite rhombs, with calcite being the most thermodynamically stable calcium carbonate at ambient temperatures and pressures. The hollow spheres show slightly roughened outer and inner surfaces (Figure 2b) indicating the polycrystalline nature of the walls. From Figure 2b, the thickness can be estimated to be about 50-150 nm. From the HR-SEM image, it is difficult to discern whether the primary vaterite crystallites are oriented in any manner. The most prominent structural characteristic of the products in Figure 2 is the hollow spherical morphology. The crystal aggregates shown in Figure 2 exhibit structural organization at three levels: (i) crystallinity at the atomic level, (ii) nanometer-sized particles lacking well-defined morphology in the mesoscopic regime, which aggregate to form the open calcium carbonate spheres, and (iii) hollow spherical morphology in the microscopic range. Vaterite (the kinetically stabilized polymorph) is known to form in the presence of aspartate and glutamate additives,25 which is consistent with the selective formation of vaterite in the present case. Our interpretation is backed by the results of DSC measurements that reveal an organic portion of approximately 5 wt %. The hollow sphere formations are more difficult to explain because they preclude a templating effect from self-organized polymer structures. The strong pH dependence of the mineralization process can be deduced from the results of control experiments at pH 4 and 10. The yield of hollow vaterite spheres increases in weakly acidic solutions (pH 4), where the carboxylic groups of the polyaspartate are protonated. Coarse crystal aggregates analogous to those depicted in Figure 7b and c (vide infra) were formed at pH 10, where the deprotonation of the polymer is complete. A possible explanation for the formation of hollow spheres made up of vaterite nanocrystals may be given on the basis of the development of the solution pH during the course of the crystallization. The corresponding pH profile (Figure 4) showed a characteristic multistep process. After the start of the reaction, (25) Manoli, F.; Dalas, E. J. Cryst. Growth 2001, 222, 293-297.
Nucleation Surfaces and Macromolecules
Langmuir, Vol. 22, No. 7, 2006 3077
Figure 5. IR spectra monitoring the evolution of the crystallization process (a) after 1 h and (b) after 24 h. (c) TEM micrograph of a sample taken after 3 h of crystallization time. (d) TEM micrograph showing the agglomeration of ACC particles to larger droplets.
Figure 6. QCM measurement monitoring the binding of polyaspartate to the SAM: progression of the frequency before and after the addition of polyaspartate (Mn ) 2500 g/mol).
the pH of the solution rises within an induction period of ∼5 h from 7 to 9.7 because of the dissolution of ammonia that is formed during the decomposition of (NH4)2CO3. The better solubility of NH3 compared to that of CO2 leads to the observed sudden pH change. This equilibrium adjusts within the next 9 h until the NH3 vapor pressure in the gas phase matches the NH3 partial pressure of the solution. Now CO2 is absorbed by the solution over a period of about 6 h, during which the pH value remains constant. This is the limit of an equilibrium between uptake and CO2 in the solution and CO2 depletion of the solution due to the precipitation of calcium carbonate. According to the Ostwald rule of stages, amorphous calcium carbonate (ACC) is formed in the presence of polyaspartate by a liquid-liquid phase separation of droplets of the mineral precursor and the solution.26 The formation of amorphous calcium carbonate could be verified by time-resolved IR measurements (Figure 5a and b). In samples taken within the time slot between 1 and 5 h, we detect vibrational bands at 864 and 1082 cm-1 that can be assigned to ACC,27 whereas these absorption bands have disappeared in samples taken after 24 h. (26) Gower, L. B.; Odom, D. J. J. Cryst. Growth 2000, 210, 719-734.
Figure 7. Crystallization products on a CH3-terminated SAM in the presence of polyaspartate (Mn ) 2500 g/mol). (a) Overview (scale bar ) 200 µm), (b) magnified view of a single sphere (scale bar ) 5 µm), and (c) magnified view of the roughened surface of a single sphere composed of calcite rhombs (scale bar ) 2 µm). (d) Spheres at higher magnification (scale bar ) 20 µm). These massive particles are inadvertently overturned so that the flat surface on which they were nucleated is visible. The dark shadows (arrows) at the left and at the bottom show a part of the SAM where the spheres most probably were situated originally.
Additionally, we were able to demonstrate the presence of ACC particles in solution by TEM measurements (Figure 5c). The samples were taken 3 h after the start of the mineralization process by allowing carbon dioxide to diffuse into the solution. The sample contained small noncrystalline CaCO3 particles with diameters of ∼20 nm that had already agglomerated in solution (27) Gehrke, N.; Co¨lfen, H.; Pinna, N.; Antonietti, M.; Nassif, N. Cryst. Growth Des. 2005, 5, 1317-1319.
3078 Langmuir, Vol. 22, No. 7, 2006
to submicrometer-sized droplets (Figure 5d) and transformed (after binding to the surface) into crystalline vaterite particles after ∼12 h. The ACC is deposited on the polar OH-terminated surface in the form of droplets. The final size of these droplets after completion of the reaction was estimated by DiMasi et al.28 from light-scattering experiments to be in the range of 1-4 µm, which is in good agreement with the size of the spherules in Figure 2a and b after the mineralization is complete. The second increase in the pH value in Figure 4 is most probably caused by the incipient crystallization of the ACC droplets. According to the Ostwald rule, the transition from the amorphous phase to the thermodynamically most stable phase proceeds via the intermediate metastable phases, namely, vaterite and aragonite. This transformation from ACC to vaterite occurs at the ACCsolution interface, where the surface reactivity is highest (i.e., a vaterite shell is formed around the ACC droplet core). Because the density of the crystalline phase is larger than that of the amorphous phase, there is a transfer of Ca2+ cations to the ACC solution boundary while the droplet is depleted, the driving force for the mass transfer being the concentration gradient of CaCO3 due to the compacting. After the formation of a 100 nm shell, the Ca2+ concentration in the interior of the spherule is so small that the supersaturation limit cannot be exceeded any more. As a result, the spherule remains empty. From a comparison of the volumes of a (filled) ACC spherule and that of a vaterite shell with an outer diameter of ∼100 nm, we can estimate the degree of hydration of ACC as CaCO3 × 6.8H2O, which is in agreement with the reported degree of hydration in the literature.21 What is responsible for the formation of hollow CaCO3 spheres? We exclude the possibility of templating by micelle formation in solution because (i) the cavity size within the spheres (several micrometers) is much larger than a typical micelle diameter and (ii) the amount of polymer would be much too small to fill the cavities of the spheres. Our explanation of the hollow sphere formation bears a resemblance to that for the so-called coffeestain effect,29 where ringlike deposits are obtained on a 2D support upon the evaporation of a solvent drop. The established explanation for this effect assumes that an outward flow in a drying drop of liquid is produced when the contact line is pinned so that liquid that is removed by evaporation from the edge of the drop must be replenished by a flow of liquid from the interior. This flow is capable of transferring 100% of the solute to the contact line and thus accounts for the strong perimeter concentration of many stains. Thus, the hollow sphere formation is reminiscent of a 3D analogue of the coffee-stain effect. Interaction between Polyaspartate and OH-Terminated SAMs. A better understanding of the above results can be obtained when an interaction between the polyaspartate and the template surface is considered. In solutions with pH values between 9 and 10, we can assume that the carboxylate groups of the polymer are mostly deprotonated. It is known that the polymer adopts a β-sheet structure provided that the Ca2+ concentration is high enough.30 If the polarity of the SAM headgroups is sufficiently large to ensure the attachment and unfolding of the polymer strands by Ca2+ complexation, the surface and/or the polymer may serve as a template for the mineralization process. This condition is safely satisfied for a carboxylate-terminated monolayer,17 and it also might be fulfilled for a moderately polar (28) DiMasi, E.; Liu, T.; Olszta, M. J.; Gower, L. B. J. Colloid Interface Sci. Submitted for publication. (29) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827-830. (30) Addadi, L.; Moradian, J.; Shay, E.; Maroudas, N. G.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 2732-2736
Loges et al.
OH-terminated SAM,31 although the interaction energy between polymer and SAM (mediated by the Ca2+ cations) should be smaller by about 1 order of magnitude. We can substantiate this hypothesis by the results of measurements with a quartz microbalance (QCM), which indicate a small amount of polyaspartate binding even for OH-terminated monolayers. Monitoring the surface growth from solutions containing only Ca2+ ions and polymer with QCM as a function of time revealed that a substantial amount of material (about 0.9 µg pro cm2) is bound to the surface within a few minutes. We attribute the formation of this surface layer to the attachment of polyaspartate to the polar OH-terminated template monolayer. Because the binding energy of the polyaspartate to the OH-terminated SAM is expected to be small, we have repeated the QCM measurements at 40 °C to give a rough estimate (although from only two data points) of the binding energy of polyaspartate to the monolayer (-2.5 kJ/mol). This is close to the order of the thermal energy at room temperature. Figure 6 displays the attachment of polyaspartate onto an OH-terminated SAM surface. By replacing an aqueous solution containing Ca2+ ions only at ambient temperature after about 10 min with the same solution additionally containing polyaspartate, a pronounced frequency shift of about 25 Hz is visible because of the binding of the polyaspartate to the surface. Corresponding measurements at 40 °C resulted in almost no frequency shift, indicating no electrostatic binding of polyaspartate to the SAM.18 These findings indicate that different results may be obtained for the crystallization of calcium carbonate in the presence of polyaspartate at temperatures other than room temperature. We point out that spherical particles of CaCO3 are not new. Calcite spherules have been grown on colloidal Au templates functionalized with thiol SAMs,15 and amorphous calcium carbonate spherules have been made in solutions containing a high Mg2+ content32 or by the hydrolysis of dialkyl carbonate,33 whereas porous or hollow microparticles were obtained using double hydrophilic block copolymers as crystallization inhibitors34 or from emulsions35 and agarose gels.36 Whereas the formation of spherules may be related to templated crystal growth and the formation of hollow spherules may be due to constrained environments33 or may remained unexplained,30,32 the formation mechanism of the hollow spherules reported here seems to be fundamentally different because it is mediated by the encapsulation of small droplets of ACC and by surface adhesion through weak cooperative interactions between a surface layer and a macromolecule in solution. SAM Surface with Polymer Additive: Effect of the SAM Headgroup. It is an intriguing result that hollow vaterite spherules are formed on OH-terminated SAMs. Performing a set of experiments analogous to those described above on CH3terminated nonpolar surfaces leads, under all conditions (temperature, pH, and time) employed by us, to the formation of calcite spherules with a massive core, although with a low nucleation density, as shown in Figure 7 where a typical surface section is shown. The spherules have diameters of ∼15 µm. Some of them are seen in higher magnification (Figure 7b and c) to have well-defined surfaces displaying complex aggregates (31) Hosoda, N.; Sugawara, A.; Kato, T. Macromolecules 2003, 36, 64496452. (32) Ajikumar, P. K.; Wong, L. G.; Subramanyam, G.; Lakshminarayanan, R.; Villayaveettil, S, Cryst. Growth Des. 2005, 5, 1129-1134. (33) Faatz, M.; Gro¨hn, F.; Wegner, G. AdV. Mater. 2004, 14, 996-998. (34) (a) Co¨lfen, H.; Antonietti, M. Langmuir 1998, 14, 582-589. (b) Yu, S. H.; Co¨lfen, H.; Hartmann, J., Antonietti, M. AdV. Funct. Mater. 2002, 12, 541546. (c) Yu, J.; Yu, J. C.; Znang, L.; Wang, X.; Wu, Ling, Chem. Commun. 2004, 2414-2415. (35) Li, M.; Mann, S. Langmuir 2000, 16, 7088-7094. (36) Yang, D.; Qi, L.; Ma, J. Chem Commun. 2003, 1180-1181.
Nucleation Surfaces and Macromolecules
Figure 8. Crystallization products on a COOH-terminated SAM in the presence of polyaspartate (Mn ) 2500 g/mol). (a) Overview (scale bar ) 50 µm) and (b) magnified view of a single sphere (scale bar ) 10 µm).
Langmuir, Vol. 22, No. 7, 2006 3079
Figure 10. Crystals obtained from on OH-terminated surfaces in the presence of polyaspartate at 4 °C (a) after 2 days (scale bar ) 10 µm) and (b) after 3 days (scale bar ) 5 µm) of crystallization time.
Figure 9. Crystals obtained from a control experiment performed under the same conditions as in Figure 2 involving the homogeneous crystallization of calcium carbonate in the presence of polyaspartate but in the absence of a SAM (scale bar ) 5 µm).
Figure 11. Ring-type deposits obtained from OH-terminated surfaces in the presence of polyaspartate at 45 °C. Scale bars are (a) 200 and (b) 20 µm.
of facetted calcite crystals with slightly damaged edges (Figure 7c). The crystals seem to organize radially, with the direction of growth being the body diagonal of the rhombohedral crystals or crystallographic [001]. Figure 7d shows a number of crystallite assemblies at higher magnification. The bundle is turned over and presents the flat surface on which it was nucleated. Certainly, the number of such spheroidal crystallite assemblies that we obtain on an apolar surface is much smaller than that on polar surfaces, as shown in Figure 8. This finding reveals that surface polarity has a significant effect on crystallization. We further note that on the polar SAM not all of the spherules are formed from facetted crystals but rather have a rough surface layer consisting of crystallites with an irregular morphology and a seemingly statistical orientation. The spheroidal nature of the crystallite assemblies observed on polar surfaces might suggest that nucleation as well as the major part of crystal growth takes place in solution. Some of the growth may take place after the assemblies have settled, as indicated by the flat part of the spherule shown in Figure 8b. Assuming that the spherules start to precipitate exclusively after the Ca2+ depletion in the solution, the growth of facetted crystals might take place on the particle surface only during the later stages of crystallization, when the level of supersaturation is low and the crystals grow more slowly. Having considered the nucleation on the templated SAM, it is informative to carry out a control experiment in order to test whether concurrent homogeneous crystallization in solution also leads to the formation of spherulitic aggregates (Figure 9). MicroRaman spectroscopy indicates that mixtures of calcite and vaterite crystals are formed exclusively by homogeneous nucleation in solution. This result supports the idea that the spheroidal particles shown in Figure 8 were formed by the cooperative interaction between the SAM and the polymer on the SAM surface. SAM Surface with Polymer Additive: Effect of Temperature. The temperature-dependent QCM measurements have shown that the interaction energies between the surface and
polyaspartate are low. Therefore, temperature might have a significant influence on the outcome of the mineralization and the morphology of the products. Crystallization at 4 °C. When the crystallization experiments were performed at 4 °C on C11OH, calcium carbonate films were formed independent of the solution pH. On the basis of the data presented above, it is reasonable to explain these findings by the following model. At 4 °C, there is a substantial interaction between the SAM and the dissolved polyaspartate. In the presence of the polymer, small isotropic droplets of ACC are formed in solution. At low temperature, the crystallization velocity of the ACC droplets is so small that a substantial amount of ACC is deposited on the SAM, where it aggregates to a thin film because of the interaction between the SAM and the polar side groups of the polymer. After drying, these films start rupturing and forming scrolls of calcium carbonate polymer films because of the tension resulting from the loss of hydrate molecules at the upper surface of the film (Figure 10a). If the duration of the crystallization experiment is extended from 2 to 3 days under identical conditions, a stable ACC film is formed (Figure 10b). Small crystallites are apparent in troughs of the ACC film (arrows in Figure 10b), which are much smaller than the associated troughs. Similar observations have been described by Gower and Odom.25 Crystallization at 45 °C. When the crystallization was performed at 45 °C, distinct ringlike deposits were formed on polar OH- and COOH-terminated surfaces (Figure 11). A possible explanation for the formation of the ring-type pattern may be related to the formation of liquid ACC droplets in solution through the encapsulation of calcium carbonate precursor particles by the polyaspartate polymer. However, at 45 °C the interaction between the polymer-encapsulated ACC droplets and the surface is on the order of the thermal energy. Therefore, the wetting of the surface by ACC droplets is less pronounced, no film formation occurs, and only some spherical deposits of ACC are formed. When the transformation from ACC to the more dense vaterite
3080 Langmuir, Vol. 22, No. 7, 2006
occurs at the ACC-solution interface, a characteristic ring pattern is generated by the flow in which the pinning of the contact line of the crystallizing ACC drop ensures that liquid crystallizing from the reactive edge (with higher surface energy) is replenished by material from the interior. The resulting outward flow can carry virtually all of the dispersed material to the edge. Therefore, the formation of these ringlike vaterite patterns may be related to the coffee-stain effect originating from the phase transformation of loosely surface-bound ACC droplets (less dense) to vaterite aggregates (more dense). On the basis of our present data, we cannot distinguish whether CaCO3 nucleates directly on the SAM substrate with attached polymer or by the aggregation of polyaspartate and CaCO3 particles formed in solution. However, it seems likely that nanometer-sized ACC particles, preformed and polymerstabilized in solution, are being attached to the SAM.
Conclusions and Outlook We combined the ideas of template-induced crystallization on SAMs and polymer additives into a strategy that includes the interaction (i) of a matrix participating in the nucleation process and (ii) a polymer with anionic groups as well as (iii) the corresponding ions in solution. By the combined interaction of these components, ordered characteristic composite structures were aggregated from preformed calcium carbonate precursor particles. These experiments demonstrate that the transformation of an amorphous polymer-stabilized precursor can provide an
Loges et al.
effective synthetic route to manipulating the morphology of calcium carbonate particles. The spherical shape of the amorphous precursor is dictated by interfacial energy. The subsequent transformation from a voluminous hydrated to a more dense crystalline material leads to the formation of hollow spheres reminiscent of a 3D analogue of the so-called coffee-stain effect, where ringlike deposits are obtained on a 2D support upon evaporation of a solvent drop. An outward flow of material in a crystallizing spherical particle is produced when the outer particle boundary is pinned so that material compacting at the edge of the sphere must be replenished by mass flow from the interior. This flow is capable of transferring 100% of the ACC precursor particles to the sphere boundary and thus accounts for the formation of a hollow sphere. The associated model of cooperative formation of the hollow vaterite microspheres represents an alternative to models of structure formation, where two-phase systems (e.g., microemulsions or foams) act as structure-directing interfaces. Acknowledgment. We are grateful to the Deutsche Forschungsgemeinschaft (DFG) for support within the priority program “Prinzipien der Biomineralisation.” We are indebted to Dr. M. Huth (MPI fu¨r Chemie/Mainz) for help with the HRSEM, and we acknowledge a generous donation of gold from Degussa AG, Hanau. LA0528596
