The Journal of
Physical Chemistry
0 Copyright 1995 by the American Chemical Society
VOLUME 99, NUMBER 32, AUGUST 10,1995
LETTERS Influence of Supramolecular Template Organization on Mineralization Arkadi L. Litvin>’ Lynne A. Samuelson: Deborah H. Charych,B Wayne Spevak? and David L. Kaplan*J Biotechnology Division, U.S.A m y Natick Research, Development & Engineering Center, Natick, Massachusetts 01760; Geo-Centers, Inc., Natick, Massachusetts 01 760; and Lawrence Berkeley Laboratory, Berkeley, California 94720 Received: March 31, 1995; In Final Fom: May 23, 1 9 9 9
The influence of supramolecular diacetylenic template structures on spacial location and morphology of CaC03 crystals grown under the template has been demonstrated. The periodic modulations in supramolecular template textures (global geometry; millimeter scale) influence crystal location, whereas the local density of the template (local geometry; nanometer scale) influences the polymorph Selectivity. For various anionic functional groups under specific experimental conditions, it was found that the binding of Ca ions to the organic template results in an increase of -4.5-5 A2/molecule to the molecular area based on pressure-area isotherms.
Introduction In biological mineralization processes and in attempts to mimic these processes syntheti~ally,~-~ an organic matrix, usually anionic in character,’Oserves as a template for nucleation and growth of minerals. We have chosen amino acid-modified diacetylene Langmuir films as the representative templates for tunable biomineralization. Diacetylenes were selected because they have been systematically studied, they can be easily polymerized, and they have been used for modeling biomembranes including tubules and In recent studiesi3-I5 crystals have been grown by means of a liquid-crystal “templating” mechanism, in which the silicate material forms inorganic walls between ordered surfactant micelles. In the present work crystal growth occurs under the liquid crystalline template and density of nucleation sites and morphology of crystals is controlled by manipulation of the template. We report the possibility of fine tuning spatial control over nucleation and growth of minerals within certain experimental
’ U.S.Army Natick Research, Development & Engineering Center. 5 @
Geo-Centers, Inc. Lawrence Berkeley Laboratory. Abstract published in Advance ACS Abstracts, July 15, 1995.
0022-3654/95/2099-12065$09.00/0
conditions. Perry et al.I6 reported that spatial localization constitutes a requirement for controlled biological mineralization. We have demonstrated the direct influence of supramolecular organic template structures on crystal location and polymorph selectivity. An example of such highly controlled interactions is the formation of equally spaced crystals under the pseudofocal-conic template’ with the spacing between crystals exactly matching the characteristic spatial dimension of the template. The observed spatial correlation appears on a millimeter scale. Namely, the periodic modulations in supramolecular template texture (global geometry) are responsible for crystal location on a millimeter scale. Spatial correlations on a millimeter scale have been also observed in natural biominerals, such as the aragonite phase in nacre.” We also explore the role of Ca2+ binding to the monolayer functional groups as a mechanism of altering the spatial organization of the diacetylenic template and, consequently, of influencing the polymorph selectivity. We find the same increase in area per molecule upon interaction with Ca2+, as compared to a water control, for all monolayer functional groups studied. 0 1995 American Chemical Society
12066 J. Phys. Chem., Vol. 99, No. 32, 1995
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Experimental Section Our experimental setup for preparation of Langmuir monolayers and their direct observation by Brewster angle microscopy (BAM) has been described.' All BAM images presented in this paper are at the scale 1 x 1 mm. Calcium carbonate crystals were grown from a calcium bicarbonate subphase18(pH = 5.86.0, T = 21 OC, [Ca2+]= 9-9.5 mM) in the presence of stearic acid, diacetylene modified by glycine (Gly-DA), and hydroxylethylamine (EA-DA) in the surface pressure ranges 0, 10, 20 mN/m. Transmission Electron Microscopy (TEM). A single GlyDA monolayer was deposited on carbon supported grids at a surface pressure of -10 mN/m with a deposition speed of 3 mm min-'. A Philips EM400 STEM equipped with a Noran EDXS detector at 120 keV was employed to investigate crystalline features of the monolayer. Scanning Electron Microscopy (SEM). CaC03 crystals were deposited on aluminum SEM stubs at the monolayersubphase interface. The stubs were coated with a thin layer of gold (about 100 A) under vacuum on a Denton Vacuum DESK I1 to minimize charge on the samples and characterized using an Amray 1820 system.
Results and Discussion Previously, we reported the direct visualization of texture changes under pressure' in glycine-modified diacetylene monolayers Gly-(CH2)8C~C-c~c(CH2) 1 lCH3 (Gly-DA) using Brewster angle microscopy.l9 We have observed a striped (smectic) texture (Figure 1, top), which appears directly after spreading the monolayer at the airwater interface. The crystals grown under the film in the uncompressed state (-0-5 mN/m) were mostly calcite (Figure 1, bottom), with only a low percentage of vaterite. Mann et aL20 have suggested, in the case of stearic acid, that for this pressure range a certain degree of domain formation in the organic template could promote vaterite growth. The presence of some vaterite under the uncompressed modified diacetylene template could be explained by previously observed localized organization in the diacetylene film in which the striped (smectic) structure is formed even at low surface pressure.' After the pressure has been applied, a well defined pseudofocal-conic texture appears (Figure 2, top) in the pressure range 30-35 mN/m for a water subphase' and in the range 20-25 mN/m for a calcium bicarbonate saturated subphase. The appearance of a pseudo-focal-conic texture at lower pressures for a CaCO3 subphase is consistent with our observation that the presence of Ca2+resulted in a significant reduction in the surface pressure compared to a water subphase. A similar surface pressure reduction from 32 to 20 mN/m, due to the introduction of Ca2+,was observed by Losche et al. for L - a dimyristoyl phosphatidic acid.21 The influence of supramolecular texture on polymorph selectivity was also assessed. The crystals grown under the film at a surface pressure range of 20 mN/m were exclusively vaterite, which indicates that a higher degree of organic monolayer surface compression could influence and promote vaterite growth. This is consistent with the work of Mann et al. for stearate monolayers.22 The most striking finding in this study, in terms of the influence of supramoleculartexture on the growth of biominerals under the organic template, was that the distance between the foci of the pseudo-focal-conic structure (-0.32 mm) almost exactly matched the average distance between vaterite crystals [-0.33 f 0.01 mm, N = 731 (Figure 2, bottom). These crystals grow along lines connecting the foci of the supramolecular
Figure 1. Crystals grown under Gly-DA organic template at surface pressure -0-5 mN/m. Brewster angle micrograph of Gly-DA monolayer (top), illustrating striped (smectic) domain structure. Scanning electron micrograph (SEM) of CaC03 crystals grown under this type of supramolecular structure (bottom), with predominance of calcite. Calcite, rhombohedral, R-3C; vaterite, hexagonal, P63/mmc.
structure. This is one example of a direct spatial correlation on a millimeter scale between a liquid crystalline template and the corresponding biomineral structures. The same millimeter scale correlations have been presented in the description of a natural biomineral (the aragonite phase in nacre17). In this work we do not consider stereochemical relationships between various diacetylene monolayers and different CaCO3 polymorphs since a very detailed discussion on this subject has been reported for stearic acid monolayers and these polym o r p h ~ .The ~ packing parameters of stearate molecules are in the same range as that of diacetylenes (interheadgroup spacing of stearate -5 A, diacetylene monomers -4.7-5.2 A, diacetylene polymers -4.93 A).23 We note that the stereochemical relationships discussed by other author^^-^-^^ indicate correlations on a nanometer scale, whereas we have observed the spatial correlations between the organic supramolecular structure and CaC03 crystals on a millimeter scale. To further support and substantiate the explanation that localized organization of the film exists in the uncompressed state (Figure 1, top), we have undertaken a TEM study of the Gly-DA single monolayer. Figure 3 shows distinct domain structures with varying packing densities in the different domains, two examples of which are delineated by arrows.
J. Phys. Chem., Vol. 99, No. 32, 1995 12067
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Figure 2; Crystals grown under Gly-DA organic template at surface pressure -20-25 mN/m. Brewster angle micrograph of Gly-DA monolayer (top), illustrating pseudo-focal-conic texture. SEM image of vaterite crystals grown under this type of supramolecular structure (bottom). The distance between the foci of closest-neighbors matches the average distance between vaterite crystals (-0.32 mm) in the bottom micrograph.
Presumably, vaterite is growing under the more densely packed domains since a higher template packing density promotes vaterite growth. Thus, there is a correlation between the supramolecular structure of the organic template and polymorph selectivity of the biomineral grown under the template (Figures 1-3).
To explore the reasons for the observed correlation, we discuss the role of Ca2+binding to the monolayer anionic groups as a mechanism of altering spatial organization of diacetylenic template and consequently of influencing the polymorph selectivity. We have used organic templates which contain anionic functional groups, since these groups are characteristic for some natural organic matriceslo involved in mineralization. In this case Ca2+ binding serves as a necessary and important intermediate step between the supramolecular matrix structure and nucleation and growth of crystals. Pressure-area isotherms were obtained for various monolayer-subphase combinations with the water subphase serving ,as a control. The results shown in Table 1 demonstrate the increase, with respect to water control, in the molecular area, denoted [AAo(Ca2+-H20)], at pH = 5.8-6.0. This increase, due to the introduction of Ca2+, could be caused by calcium
ions binding to carboxyl, hydroxyl, or phosphate anions and disturbing the intermolecular packing of head groups. The increased area per molecule in the presence of Ca2+ and other cations is characteristic of strong cation binding and has also been observed by We find the same increase in area per molecule (-4.5-5 A2) for all monolayer functional groups studied. The increase is observed for the calcium bicarbonate subphase (pH = 5.86.0, T = 21 OC, [Ca2+ ] = 9-9.5 mM) as compared to the water control, suggesting an equal contribution for carboxyl, hydroxyl, and phosphate groups in binding Ca2+. The finding that AAo(Ca2+-H20) is in the range -4.5-5 A2/moleculefor all functional groups studied, serves as an indication that in all cases the Ca-0 bond forms with bond length in the range -2.12-2.24 A, within the 'acceptable range for this kind of These bond lengths were calculated by taking the square root of the differences in molecular area from pressurearea isotherms. Hence, pressure-area isotherms for all the compounds listed in Table 1, at the chosen values of subphase parameters, serve as an indirect estimate of the change in Ca-0 bond length due to Ca2+ binding to various anionic organic matrices. The Ca2+ influence on molecular area is valid only for the pH range given above. As shown for compound ~ C I ~ P E ~ ~ (Table l), at pH 2, no effects of Ca2+ were observed for pressure-area isotherms, and at pH 11, a decrease in the molecular area was observed. The former (pH 2) could be due to the repulsion between Ca2+ and cationic head groups, the latter (pH 11) could be due to the decrease of anionic repulsion by binding Ca2+ to phosphate head groups. It is intriguing to speculate that the modulation of supramolecular organization within liquid-crystalline phases of cell membranes, coupled with localized pH gradients, could act as modulators of crystal growth in biological systems involved in biomineralization. Spatial localization, either as membranebound organelles or localized regions in cell walls, have been described as a requirement for controlled biological mineralization.16
Conclusion The overall periodic modulations in supramolecular template textures (global geometry; millimeter scale) are responsible for crystal location, whereas the local density of the template (local geometry; nanometer scale) influences the polymorph selectivity.2 The contribution of Ca2+ to the molecular area of the organic template studied (compared to the water control) appears to be constant and equals -4.5-5 A2/molecule. We note that the influence of Ca2+binding on the monolayer molecular area becomes an important factor in determining the packing density of the monolayer molecules. The monolayer packing density, in turn, influences the polymorph selectivity.
Acknowledgment. We are very grateful to Dr. Sung, Center for Advanced Materials, University of Massachussets, Lowell, for TEM support. References and Notes (1) Litvin, A. L.; Samuelson, L. A.; Charych, D. A.; Spevak, W.; Kaplan, D. L. J. Phys. Chem. 1995, 99, 492. (2) Heywood, B. R.; Mann, S. Adv. Muter. 1994, 6, 9. (3) Mann, S. Nature 1988, 332, 119. (4) Addadi, L.; Weiner, S. Angew. Chem., Int. Ed. Engl. 1992, 31, 153. (5) Landau, E. M.; Popovitz-Biro; R., Levanon, M.; Leiserowitz, L.; Lahav, M. Mol. Cryst. Liq. Cryst. 1986, 134, 323. (6) Calvert, P. Muter. Sci. Eng. 1994, CI,69.
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12068 J. Phys. Chem., Vol. 99, No. 32, 1995 TABLE 1 no.
1 2 3 4
comDound
subphasea
COOHCH2NHCO-DA (Gly-DA) HOCH2CH2NHCO-DA (EA-DA) CH3(CHz)&OOH (stearic acid) CH3(CH2)150 ]O-[-Ol,NH3
AAo(Ca2+-H20), A2/molecule
CaCO3 CaC03 CaC03 CaC12
(1,3-dihexadecylglycer0-2phosphoethanolamine(2C16PE)) +
CH3(CH2)150
Subphase parameters were kept at pH = 5.8-6.0, T = 21 OC, [Ca2+] = 9-9.5 mM. This study.
Figure 3. TEM micrograph of single Gly-DA monolayer at low surface pressure (-10 mN/m). The darker lines with similar orientation designate different domains with various packing densities within the single monolayer. Two-dimensional lattices with distinct domain structures are observed and represent different packing densities. Two examples of these differences are noted by the white arrows on the micrograph, although the range of dimensions varies. (7) Fendler, J. H. Membrane-Mimetic Approach to Advanced Materials; Springer-Verlag: New York, 1994. (8) Zhao, X. K.; Yang, J.; McCormick, L. D.; Fendler, J. H. J. Phys. Chem. 1992, 96, 9933. (9) Mann, S.; Webb, J.; Williams, R. J. P. Biomineralization: Chemical and Biochemical Perspectives; VCH Publishers: Weinheim, 1989. (10) Sikes, C. S.; Wheeler, A. P. Sulface reactive peptides and polymers; ACS Books: Washington, 1991; Chapter 5.
(11) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Znt. Ed. Engl. 1988, 27, 113. (12) Ahlers, M.; Muller, W.; Reichert, A.; Ringsdorf, H.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1990, 29, 1269. (13) Firouzi, A.; Kumar, D.; Bull, L. M.; et al. Science 1995,267, 1138. (14) Monnier, A.; Schuth, F.; Huo, 0.; et al. Science 1993, 261, 1299. (15) Kresge, C. T.; Leonowicz, M. E.; Roth, W. J.; Vartuli, J. C.; Beck, J. S . Nature 1992, 359, 710. (16) Sikes, C. S.; Wheeler, A. P. Surface reactive peptides and polymers; ACS Books: Washington, 1991; Chapter 23. (17) Case, S . T. Structure, Cellular Synthesis and Assembly of Biopolymers; Springer-Verlag: Berlin, 1992; Chapter 1. (18) Kitano, Y. Bull. Chem. SOC.Jpn. 1962, 12, 1973. (19) Honig, D.; Mobius, D. J. Phys. Chem. 1991, 95, 4590. (20) Mann, S.; Heywood, B. R.; Rajam, S.; Birchall, J. D. Proc. R. SOC. London 1989, A423,457. (21) Losche, M.; Mohwald, H. J. Colloid Zntelface Sci. 1989, 131, 56. (22) Mann, S.; Heywood, B. R.; Rajam, S.; Birchall, J. D. Nature 1988, 334, 692. (23) Enkelman, V. Adv. Polym. Sci. 1984, 63, 91. (24) Ebara, Y.; Ebato, H.; Ariga, K.; Okahata, Y. Langmuir 1994, 10, 2267. (25) Nag, K.; Rich, N. H.; Keough, K. M. Thin Solid Films 1994,244, 841. (26) Deamer, D. W.; Meek, D. W.; Comwell, D. G. J. Lipid Res. 1967, 8, 255. (27) Gordziel, M. J.; Flanagan, D. R.; Swarbrick, J. J. Colloid. Interface Sci. 1982, 86, 178. (28) Carrell, C. J.; Carrell, H. L.; Erlebacher, J.; Glusker, P. J. Am. Chem. SOC.1988, 110, 8651. (29) Nayal, M.; Di Cera, E. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 817.
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