Langmuir 2008, 24, 10579-10582
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Observation of an Organic-Inorganic Lattice Match during Biomimetic Growth of (001)-Oriented Calcite Crystals under Floating Sulfate Monolayers Sumit Kewalramani,† Kyungil Kim, Benjamin Stripe, Guennadi Evmenenko, Geoffrey H. B. Dommett, and Pulak Dutta* Department of Physics and Astronomy, Northwestern UniVersity, EVanston, Illinois 60208 ReceiVed July 7, 2008. ReVised Manuscript ReceiVed August 24, 2008 Macromolecular layers rich in amino acids and with some sulfated polysaccharides appear to control oriented calcite growth in living organisms. Calcite crystals nucleating under floating acid monolayers have been found to be unoriented on average. We have now observed directly, using in situ grazing incidence X-ray diffraction, that there is a 1:1 match between the monolayer unit cell and the unit cell of the (001) plane of calcite. Thus, sulfate head groups appear to act as templates for the growth of (001)-oriented calcite crystals, which is the orientation commonly found in biominerals.
Introduction Calcite is the primary component of many biominerals. Highly oriented calcite crystals are found in shells where they provide mechanical strength and in trilobite eyes where they are used as optical elements.1 It was recently noted that “This precise orientation of crystals is the big mystery of biomineralization. Organisms know how to do it; we do not yet know how they know”.2 It is presumed, but not proven, that calcite nucleation and growth is governed by the organic matrices that are found within the biomineral. These consist of macromolecules rich in amino acids, such as aspartic acid, and sulfated polysaccharides.3 To understand the biological process of calcite nucleation, bulk precipitation studies of calcite have been performed in the presence of biological macromolecules4,5 and synthetic polymers containing sulfate and carboxylate functionalities.6 An alternative method is to use self-assembled structures of carboxylate rich polymers,7 biological macromolecules,3 or monolayers of carboxylates and sulfates adsorbed onto solid substrates8-11 or floating over aqueous subphases (Langmuir films)1,12-20 as potential templates * Corresponding author. E-mail:
[email protected]. † Current address: Brookhaven National Laboratory, Upton, New York 11973. (1) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: Oxford, U.K., 2001. (2) Towe, K. M. Science 2006, 311, 1554. (3) Addadi, L.; Moradian, J.; Shay, E.; Maroudas, N. G.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 2732. (4) Arias, J. L.; Neira-Carrillo, A.; Arias, J. I.; Escobar, C.; Bodero, M.; David, M.; Fernandes, M. S. J. Mater. Chem. 2004, 14, 2154. (5) Aizenberg, J.; Hanson, J.; Koetzle, T. F.; Weiner, S.; Addadi, L. J. Am. Chem. Soc. 1997, 119, 881. (6) Pai, R. K.; Hild, S.; Ziegler, S.; Marti, O. Langmuir 2004, 20, 3123. (7) Berman, A.; Ahn, D. J.; Lio, A.; Salmeron, M.; Reichert, A.; Charych, D. Science 1995, 269, 515. (8) Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature 1999, 398, 495. (9) Aizenberg, J.; Black, A. J.; Whitesides, G. M. J. Am. Chem. Soc. 1999, 121, 4500. (10) Travaille, A. M.; Donners, J. J. J. M.; Gerritsen, J. W.; Sommerdijk, N. A.J. M. AdV. Mater. 2002, 14, 492. (11) Travaille, A. M.; Kaptijn, L.; Verwer, P.; Hulsken, B.; Elemans, J. A. A. W.; Nolte, R. J. M.; van Kempen, H. J. Am. Chem. Soc. 2003, 125, 11571. (12) Dimasi, E.; Olszta, M.; Patel, V. M.; Gower, L. B. Cryst. Eng. Commun. 2003, 5, 346. (13) Kmetko, J.; Yu, C.-J.; Evmenenko, G.; Kewalramani, S.; Dutta, P. Phys. ReV. B 2003, 68, 085415. (14) Heywood, B. R.; Mann, S. Chem. Mater. 1994, 6, 311. (15) (a) Volkmer, D.; Mayr, N.; Fricke, M. Dalton Trans. 2006, 41, 4889. (b) Fricke, M.; Volkmer, D. Top. Curr. Chem. 2007, 270, 1.
for calcite nucleation. The monolayer approaches are attractive from the standpoint of understanding and examining the organic-inorganic structural relationships that drive the nucleation process, which is a key step in the biomineralization process.1 Calcite crystals have been grown in oriented fashion on selfassembled monolayers of carboxylates and sulfates on solid substrates7-11 and also under monolayers floating on an queous subphase.1,14,16-20 The observed orientations of calcite are frequently explained in terms of a stereochemical (orientational) match between the headgroup and carbonate ion of the calcite lattice and/or an epitaxial match (lattice match). However, in most studies the organic molecules are not actually observed during the nucleation process. The monolayer matrix structure is often assumed to be the same as the structure observed under different conditions or is estimated on the basis of isotherms. However, our GID studies performed during the oriented nucleation of BaF2 under Langmuir films21 showed that neither the inorganic nor the organic lattices are rigid; they exert a mutual influence on each other. In addition to the need to determine actual in situ structures, there is also a need to determine inorganic crystal orientations using methods that average over a large number of crystals. Calcite crystals that are analyzed by in situ or ex situ microscopy techniques after being grown under carboxylate monolayers show two distinct morphologies.1,14,16-18,20 It has been argued1,16,17 that both crystal forms arise as a result of the nucleation of calcite with its {1-10} crystal face parallel to the monolayer surface. Unexpectedly, however, in situ structural studies,12,13 which average over all crystals within a macroscopic area (several square centimeters) have shown that there is growth but no average preferential alignment under carboxylate monolayers: the nucleate (16) Heywood, B. R.; Rajam, S.; Mann, S. J. Chem. Soc., Faraday Trans. 1991, 87, 735. (17) Rajam, S.; Heywood, B. R.; Walker, J. B. A.; Mann, S. J. Chem. Soc., Faraday Trans. 1991, 87, 727. (18) Ahn, D. J.; Berman, A.; Charych, D. J. Phys. Chem. 1996, 100, 12455. (19) Lahiri, J.; Xu, G.; Dabbs, D. M.; Yao, N.; AksayI. A.; Groves, J. T. J. Am. Chem. Soc. 1997, 119, 5449. (20) Loste, E.; Diaz-Marti, E.; Zarbakhsh, A.; Meldrum, F. C. Langmuir 2003, 19, 2830. (21) Kmetko, J.; Yu, C. J.; Evmenenko, G.; Kewalramani, S.; Dutta, P. Phys. ReV. Lett. 2002, 89, 186102.
10.1021/la802124v CCC: $40.75 2008 American Chemical Society Published on Web 09/06/2008
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is a 3D powder. The acid surface causes induced mineralization22 but not controlled mineralization.23 In this letter, we turn our attention to the other major organic component associated with biological calcite growthsmolecules with sulfate functionality. Previous studies have shown that calcite will nucleate under Langmuir monolayers of alkyl sulfates.1,14,15,18 These have uniform pyramidal morphology with a basal (001) crystallographic plane and three symmetric thermodynamically stable {104} planes. On the basis of the symmetry of grown crystals, it was concluded that calcite nucleates with its (001) plane parallel to the sulfate monolayer surface. (001)-oriented calcite growth in the presence of sulfated molecules has also been noted in other systems. Addadi et al.3 found that ordered polyaspartic acid adsorbed onto solid substrates leads to the nucleation of rhombohedral calcite (only {104} faces expressed) but the presence of sulfonated polystyrene structures on these substrates promoted the growth of hexagonal calcite ((001) faces expressed). Aizenberg et. al found that calcite crystals on selfassembled monolayers of sulfate on palladium-coated substrates are (001) oriented8 (on gold- and silver-coated substrates the orientations were found to be different9). The (001) orientation is the one most frequently observed in biological minerals, for example, in the prismatic layers of mollusk shells, even though it is not a stable face in vacuo. However, the unexpected discrepancy (in the case of growth under acid monolayers) between conclusions based on specific crystals and the results of X-ray studies that average over many crystals underscores the need for in situ experiments. Under conditions in which calcite is seen to grow with an average (001) orientation, is there a commensurate relationship between the monolayer and the calcite (001) interfacial lattices? To answer this question, we have studied calcite nucleation under arachidyl sulfate monolayers using grazing incidence X-ray diffraction, a surface-sensitive technique in which X-rays probe a very narrow region (∼5-10 nm thick) below the Langmuir film.24
Experimental Section Spreading solutions of arachidyl sulfate sodium salt (CH3(CH2)19OSO3Na) were prepared following the method of Hendrikx.25 Monolayers were spread over supersaturated solutions of calcium carbonate that were prepared by Kitano’s method.26 The concentration of calcium ions was measured by EDTA titration and was found to be ∼7.33 mM. The pH of the solution was ∼5.8. The concentration and the pH of the supersaturated solutions prepared by this method are similar to those reported by others.10,11,19,20,27 Some details of Kitano’s method are presented in ref 28. Secondary experiments were also performed with supersaturated aqueous subphases containing soluble peptide polyaspartic acid (concentration ∼0.2 µM, 〈Mw〉 ) 6000, Sigma-Aldrich). Apart from small differences in the degree of preferential alignment of the calcite nucleate, the results obtained in the two cases were found to be identical. All experiments were performed at 20 °C and with the monolayer pressure maintained at 15 mN/m. Synchrotron X-rays with λ ) 1.5498 or 1.3776 Å were incident upon the water surface in grazing incidence geometry. Horizontal and vertical soller slits in front of the detector defined a horizontal resolution of KXY ≈ 0.01 Å-1 (fwhm) and a vertical resolution of KZ≈ 0.05 Å-1 fwhm. Other details of the setup can be found in ref 30. (22) Frankel, R. B.; Bazylinski, D. A. ReV. Mineral. Geochem. 2003, 54, 95. (23) Bazylinski, D. A.; Frankel, R. B. ReVi. Mineral. Geochem. 2003, 54, 217. (24) Fontaine, P.; Goldmann, M.; Bordessoule, M.; Jucha, A. ReV. Sci. Instrum. 2004, 75, 3097. (25) Hendrikx, Y. J. Colloid Interface Sci. 1979, 69, 493. (26) Kitano, Y. Bull. Chem. Soc. Jpn. 1962, 35, 1980. (27) Mann, S.; Heywood, B. R.; Rajam, S.; Birchall, J. D. Nature 1988, 334, 692.
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Figure 1. Calcite structure during growth under a sulfate monolayer. (a) Contour plots derived from scattering data collected during late crystal growth stages show four strong diffraction peaks from (001)oriented calcite crystals. The unindexed, vertically extended peaks in the lower left corner are from a thick organic structure, apparently a trilayer. (b) Debye ring scans (equivalent to rocking curves) for the three strongest calcite peaks. The position of maxima on the {110} and {012} ring scans unambiguously indicate that calcite is (001) oriented on average, with a misorientation of ∼(5° fwhm.
Results and Discussion GID scans performed during crystal growth under arachidyl sulfate reveal four different calcite peaks (Figure 1a) in scan ranges of 0 e KXY e 3 Å-1 and 0 e KZ e 0.9 Å-1. The peak positions match those of bulk calcite. The peak intensities are distributed along Debye rings; rocking curves for the three strongest calcite peaks are shown in Figure 1b. The {110} peak, which is normal to the {001} peak, has a maximum close to the horizontal plane (i.e., KZ ≈ 0). The {012} peak, which makes an angle of 63° with the {001} peak, has a maximum 26° above the horizontal plane. These data establish that calcite nucleates with its {001} peak close to vertical (where it cannot be directly observed in our apparatus), thus the (001) crystal face is parallel to the surface of the water. The maximum for the ring scan of the {1-13} peak is expected to be at KZ )1.08 Å-1, which is slightly outside the scan range of our apparatus, but the partial data that we have obtained (Figure 1) are also consistent with (001) growth. The fwhm of the peaks along the ring scans indicates that the nucleated calcite has a small but nonzero range of orientations ((5°), which we ascribe to disorder introduced as the crystals grow larger and are no longer locked to the surface plane. Thus sulfate monolayers, unlike acid monolayers, nucleate calcite that is oriented when averaged over a macroscopic area. But what is the face-selection mechanism? To answer this question, we turn our attention to the structure of the monolayer. Alkyl sulfates are more hydrophilic than the corresponding alkyl
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Figure 3. Sulfate-calcite interface. Schematic diagram of a structure consistent with the evidence shown in this letter. The repeat distances of the two lattices have been experimentally established, although the position of each atom has not: (orange spheres) Ca, (black spheres) C, (blue spheres) O, and (yellow spheres) S. Figure 2. Arachidyl sulfate monolayer structure. In plane scans at different Kz values (9, 0 Å-1; 2, 0.2 Å-1; [, 0.4 Å-1; b, 0.8 Å-1) (a) on a pH 1.8 water subphase showing an in-plane (strongest at Kz ) 0) monolayer peak at KXY ) 1.455 Å-1 and (b) on a dilute aqueous solution containing 10 mM calcium ions, showing a stronger and sharper monolayer peak at the same position plus off-plane peaks from a thicker layer at KXY ≈ 1.34 and 1.36 Å-1.
carboxylates, and arachidyl sulfate does not form a stable structure on pure water subphases even at the relatively low pH of 5.5. The molecules desorb from the air-water interface and dissolve into the subphase.31 However, arachidyl sulfate shows stable surface pressure-area isotherms either at pH < 3 31 or in the presence of calcium ions in the subphase even at pH ∼6.15,18 We have investigated the organic structures in both cases and also during calcite nucleation. At pH ∼1.8, arachidyl sulfate forms a stable monolayer structure, and there is a single in-plane diffraction peak at KXY ≈ 1.455 Å-1 within our scan range (Figure 2a). When the subphase is a dilute solution of calcium ions (0.5 -10 mM) at pH 5.5-5.8, then two distinct structures are observed (Figure 2b). The monolayer peak is still at the same position but is much more intense and sharply peaked than on a pure water subphase. In addition, peaks from a much thicker bulk structure, possibly a collapsed phase, are observed in the range of 1.31 < KXY < 1.36 Å-1. All of the peaks from the monolayers are spread along Bragg rods (vertical lines in the KXY-KZ plane) because of their small thickness; notice in Figure 2 that the KXY position of the (28) Supersaturated bicarbonate solutions were prepared by bubbling carbon dioxide for ∼5 h through a stirred aqueous suspension of calcium carbonate (0.8 g/L). The solution was then filtered through 0.2 µm porous membranes to remove the undissolved calcium carbonate particles. Carbon dioxide was rebubbled through the filtered solution for an additional hour prior to use. Calcite nucleation at the monolayer surface is based on the following chemical equilibrium: 2HCO3(aq) + Ca2+(aq)fCaCO3(s) + CO2(v) + H2O(l) Titration analysis for calcium ions allows for an estimation of maximum attainable supersaturation, namely, that generated under conditions of spontaneous outgassing of all CO2. Mann et. al27 have estimated this value to be ∼103 at the experimental pH of 5.8. The outgassing of carbon dioxide causes the crystallization to be localized at the air-water interface. This outgassing also shifts the equilibrium of aqueous carbonic acid, carbon dioxide, bicarbonate, and carbonate in such a way that the pH of the solution increases as the equilibrium shifts to the right. Microelectrode measurements near the air-water interface showed that during calcite nucleation the pH increases linearly at a rate of ∼0.003 pH units/min.29 In our experiment, the first diffraction signal from calcite crystals were obtained at nucleation times of ∼30 min after spreading the monolayer. Thus, observable crystallization occurs at an estimated pH of ∼5.9. (29) Dobson, P. S.; Bindley, L. A.; Macpherson, J. V.; Unwin, P. R. ChemPhysChem 2006, 7, 1019. (30) Barton, S. W.; Thomas, B. N.; Flom, E. B.; Rice, S. A.; Lin, B.; Peng, J. B.; Ketterson, J. B.; Dutta, P. J. Chem. Phys. 1988, 89, 2257. (31) Wustneck, R.; Siegel, S.; Ebish, Th.; Miller, R. J. Colloid Interface Sci. 1998, 203, 83.
monolayer peak at KXY ) 1.455 Å-1 does not change as KZ goes from 0 to 0.2 Å-1. Because of this characteristic, monolayer peaks can easily be distinguished from calcite peaks, which form the rings shown in Figure 1 as a result of their small misorientation. The width of the vertical (Bragg rod) scan indicates that the peak at KXY ≈ 1.455 Å-1 arises from a structure that is ∼25 Å thick (data not shown). This is consistent with the expected monolayer thickness of arachidyl sulfate (∼28 Å). Because there are no other monolayer peaks in the vicinity, we conclude that the lattice is hexagonal and the sulfate molecules are aligned along the water surface normal.32 The spacing between molecules is 4.99 Å, and the area per molecule is ∼21.6 Å2. (In contrast, the Bragg rod scans for the peaks at KXY ≈ 1.34 and 1.36 Å-1 with a width of ∆KZ ≈ 0.08 Å-1, indicating a bulk structure with a thickness of ∼80 Å. This value is close to the thickness of a trilayer of arachidyl sulfate. The thick organic structure covers only a small part of the surface33 and does not appear to play a role in calcite nucleation.34) The Ca-Ca distance of the hexagonal calcite (001) plane is exactly the same as the intermolecular distance in the hexagonal sulfate monolayer (4.99 Å), and the tridentate headgroup of untilted molecules replicates the trigonal planar geometry of carbonate ions in (001) calcite orientation. Thus, we suggest that the sulfate monolayer is the template for calcite (001) nucleation, with an exact 1:1 commensurate relationship. These results have specific implications for mineral nucleation in biological systems. It has been proposed that nucleation in a biological system is a two-step process.3 One set of molecules enhances supersaturation at the nucleation site by virtue of strong but nonspecific electrostatic interactions. A second set of ordered molecules then selects the nucleating crystal face. Sulfated molecules have a high affinity for calcium ions35 whereas carboxylates interact weakly with these ions. Thus, in mollusk shells, sulfates are thought to play the first role, and ordered polypeptide arrays play the second. Our results show that ordered sulfate molecules are capable of performing both of these functions. Sulfated macromolecules called proteoglycans are closely associated with (001)-oriented calcitic regions in mollusk, (32) Kaganer, V. M.; Mo¨hwald, M.; Dutta, P. ReV. Mod. Phys. 1999, 71, 779. (33) The surface pressure-area isotherms for arachidyl sulfate give the limiting area/molecule to be 22-23 Å2, which is very close to the value that we obtain from X-ray scattering (∼21.6 Å2). This means that the thicker structure occupies no more than 2 to 3% of the surface. (34) The monolayer interacts strongly with calcium ions, as evidenced by the enhancement of the monolayer diffraction signal by at least a factor of 3 (Fig. 2). Also, there is no obvious relationship between the calcite surface structure and the trilayer structure. (35) Worms, D.; Weiner, S. J. Exp. Zool. 1986, 237, 11.
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barnacle, and chicken egg shells.3,4,36,37 The conformations of these molecules are not known, but even in a solution phase, polysaccharide chain sections can be considered locally to be straight rods of length ∼100 Å.38,39 Furthermore, the spacing between the constituting units is known from bulk crystallography studies40 to be ∼5 Å, which is the same characteristic length as in the sulfate monolayer lattice and the calcite (001) crystal lattice. It is therefore possible for sulfated polysaccharides to be attached (36) Aria, J. L.; Fernandez, M. S. Mater. Charact. 2003, 50, 189. (37) Sharp, R. H.; Silyn-Roberts, H. Biophys. J. 1984, 46, 175. (38) Reed, W. F. In Macro-ion Characterization: From Dilute Solutions to Complex Fluids; Symposium Series 548; Schmitz, K. S., Ed.; American Chemical Society: New York, 1984. (39) Rinaudo, M. Macromol. Symp. 2006, 245-246, 549. (40) Samuels, R. J. J. Polym. Sci., Part A-2 1969, 7, 1197.
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to the nucleating calcite such that the sulfate groups match the calcium ion positions and thus energetically stabilize the (001) crystal face. Thus, sulfate groups may not only drive the calcium ions to nucleation sites but also drive the (001) face to grow along the organic surface. Acknowledgment. This work was supported by the U.S. Department of Energy under grant no. DE-FG02-84ER45125. It was performed at beamline X-14A of the National Synchrotron Light Source (NSLS) and at beamline 33 BM-B at the Advanced Photon Source (APS). We thank Dr. J. Bai for his valuable assistance at the NSLS and Dr. P. Zschack and Dr. E. Karapetrova at the APS. LA802124V
