Biomimetic Mineralization of CaCO3 on a Phospholipid Monolayer

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Biomimetic Mineralization of CaCO3 on a Phospholipid Monolayer: From an Amorphous Calcium Carbonate Precursor to Calcite via Vaterite Junwu Xiao, Zhining Wang, Yecang Tang, and Shihe Yang* Department of Chemistry, William Mong Institute of Nano Science and Technology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong Received September 26, 2009. Revised Manuscript Received October 27, 2009 A phospholipid monolayer, approximately half the bilayer structure of a biological membrane, can be regarded as an ideal model for investigating biomineralization on biological membranes. In this work on the biomimetic mineralization of CaCO3 under a phospholipid monolayer, we show the initial heterogeneous nucleation of amorphous calcium carbonate precursor (ACC) nanoparticles at the air-water interface, their subsequent transformation into the metastable vaterite phase instead of the most thermodynamically stable calcite phase, and the ultimate phase transformation to calcite. Furthermore, the spontaneity of the transformation from vaterite to calcite was found to be closely related to the surface tension; high surface pressure could inhibit the process, highlighting the determinant of surface energy. To understand better the mechanisms for ACC formation and the transformation from ACC to vaterite and to calcite, in situ Brewster angle microscopy (BAM), ex situ scanning electron microscopy, transmission electron microscopy, Raman spectroscopy, and X-ray diffraction analysis were employed. This work has clarified the crystallization process of calcium carbonate under phospholipid monolayers and therefore may further our understanding of the biomineralization processes induced by cellular membranes.

Introduction Living organisms, by controlling the size, shape, and crystallographic orientation of inorganic minerals, acquire a multiplicity of excellent properties with respect to mechanical support, navigation, protection, and defense.1,2 Inspired by the natural biomineralization process, researchers in the fields of chemistry and materials science are attempting to reproduce such control in the laboratory, where ordered arrays of organic molecules are used to direct crystal growth. However, no synthetic materials have until now shown properties that can rival those of their natural counterparts. Therefore, many of the secrets of biomineralization remain to be studied and understood. Lipids, proteins, and polysaccharides play vital roles in the crystallization of biominerals.1 For instance, lipids can guide the formation of a large variety of materials with controlled microand nanostructures through their ability to self-assemble, compartmentalize, and form templates1 and thus have attracted increasing attention with respect to understanding biomineralization and synthesizing new biomaterials.3-8 In particular, phospholipids as the key membrane constituents of biological vesicles are commonly involved in delineating reaction compartments for *Corresponding author. E-mail: [email protected]. (1) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: New York, 1989. (2) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: New York, 2001. (3) Tong, H.; Wan, P.; Ma, W. T.; Zhong, G. R.; Cao, L. X.; Hu, J. M. J. Struct. Biol. 2008, 163, 1–9. (4) Faunce, C. A.; Reichelt, H.; Paradies, H. H. J. Phys. Chem. B 2008, 112, 8859–8862. (5) Buijnsters, P.; Donners, J.; Hill, S. J.; Heywood, B. R.; Nolte, R. J. M.; Zwanenburg, B.; Sommerdijk, N. Langmuir 2001, 17, 3623–3628. (6) Liu, X. H.; Zhang, L. X.; Wang, Y. L.; Guo, C. L.; Wang, E. K. Cryst. Growth Des. 2008, 8, 759–762. (7) Mann, S.; Hannington, J. P.; Williams, R. J. P. Nature 1986, 324, 565–567. (8) Ozin, G. A. Acc. Chem. Res. 1997, 30, 17–27. (9) Young, J. R.; Didymus, J. M.; Bown, P. R.; Prins, B.; Mann, S. Nature 1992, 356, 516–518.

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the crystallization of biominerals, such as marine alga coccolith Emiliania huxleyi,9 the mineralizing tissues of vertebrates,10 and magnetosomes in magnetotactic bacteria.11-13 In pursuing in vitro models, Mann et al.7 used phospholipid unilamellar vesicles to study the membrane-mediated growth of iron oxide crystals. It was found that lipid vesicles not only acted as passive hosts to enclose mineralization reactions but also strongly influenced the growing inorganic phase through the molecular recognition of chemical, electrostatic, and chiral complementarity. However, with the mineral formation inside vesicles, it is still very difficult to demonstrate in a compelling manner that nucleation actually occurs on the membrane itself. A Langmuir monolayer is a useful model because of its simplicity and versatility. In particular, the molecular density of a monolayer is easily controlled at the air-water interface with a Langmuir film balance. This type of experimental design can be used to elucidate the basic mechanisms of crystal nucleation on membranes, including those relevant to biomineralization. Indeed, several essential features of organic-inorganic interfaces have been studied by using a monolayer. Mann and co-workers14 used a compressed Langmuir monolayer to control the crystal orientation of calcium carbonate from a supersaturated subphase. They proposed that the lattice match between the polar headgroups of the fatty acid and the crystal planes of CaCO3 is a critical factor in controlling the crystal orientation.14 It was also found that the degree of compression of a stearic acid monolayer sensitively influences the homogeneity of vaterite nucleation, (10) Anderson, H. C.; Hsu, H. H. T.; Raval, P.; Hunt, T. R.; Schwappach, J. R.; Morris, D. C.; Schneider, D. J. The Mechanism of Bone Induction and Bone Healing by Human Osteosarcoma Cell-Extracts; Springer: Heidelberg, Germany, 1995; pp 129-134. (11) Mann, S.; Frankel, R. B.; Blakemore, R. P. Nature 1984, 310, 405–407. (12) Mann, S.; Sparks, N. H. C.; Frankel, R. B.; Bazylinski, D. A.; Jannasch, H. W. Nature 1990, 343, 258–261. (13) Bazylinski, D. A.; Heywood, B. R.; Mann, S.; Frankel, R. B. Nature 1993, 366, 218–218. (14) Heywood, B. R.; Mann, S. Chem. Mater. 1994, 6, 311–318.

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perhaps because of the stereochemical and electrostatic matching.15 Zhang et al.16 recognized the importance of both the lattice match and electrostatic attraction between phospholipid monolayers and calcium phosphate in determining the final phase, size, and morphology of calcium phosphate minerals. With macrocyclic monolayers, Volkmer et al.17,18 found that a polymorph selection of calcium carbonate largely depended on surface charge densities and that the crystallization of calcium carbonate could switch from calcite to aragonite or vaterite above a critical charge density. Furthermore, the recent literature reported that the dominant factor in polymorph selectivity is the CO2 escape rate instead of stereochemical and epitaxial mechanisms.19,20 Therefore, it remains a challenge to tease out the primary factors that control the orientation, phase, and morphology of the organic templated crystals. Most recently, we studied the crystallization process of CaCO3 on a negatively charged stearic acid monolayer at the air-water interface. The final calcite crystals were shown to be directly transformed from an amorphous calcium carbonate (ACC) particle precursor with sizes smaller than 100 nm.21 Surprisingly, the nucleation and growth processes of calcium carbonate under phospholipid monolayers, to our knowledge, have not been studied in detail, particularly in connection to the influence of surface pressure on the mineral phase. In this article, we report a comprehensive study on the biomimetic mineralization of CaCO3 under zwitterionic phospholipid monolayers (1,2-dipalmitoyl-snglycero-3-phosphatidylcholine, DPPC) and the interactions between phospholipid molecules and calcium carbonate at various degrees of compression. Instead of straight expitaxial crystallization on the monolayer from the supersaturated solution, we found that the ACC precursor particles were initially heterogeneously nucleated at the air-water interface in a kinetically controlled regime and then were transformed into the most stable calcite phase via the metastable vaterite crystals. The degree of transformation from vaterite to calcite was closely related to the degree of compression, which revealed that the surface energy is the dominant factor in the stabilization of the metastable vaterite crystals. Our observation of a series of intermediate phases of calcium carbonate by simply tuning the surface pressure evinces the value of the zwitterionic phospholipid monolayer as a model system in studies of biomineralization and the development of functional biomaterials.

Experimental Section Preparation of the Ca(HCO3)2 Solution. All reagents were obtained from commercial sources and used without further purification. Solutions of 20 mM CaCl2 (Sigma) and 40 mM NaHCO3 (BDH) were prepared separately and mixed in a ratio of 1:1 v/v to form 10 mM Ca(HCO3)2 solutions. The Ca(HCO3)2 solutions were flushed with gaseous CO2 from a compressed-gas tank for 2 h before use for the BAM experiments. BAM Experiments. A homemade BAM device was employed, which consists mainly of a laser (PPM80 (65890B, Power (15) Mann, S.; Heywood, B. R.; Rajam, S.; Birchall, J. D. Nature 1988, 334, 692– 695. (16) Zhang, L. J.; Liu, H. G.; Feng, X. S.; Zhang, R. J.; Zhang, L.; Mu, Y. D.; Hao, J. C.; Qian, D. J.; Lou, Y. F. Langmuir 2004, 20, 2243–2249. (17) Fricke, M.; Volkmer, D.; Krill, C. E.; Kellermann, M.; Hirsch, A. Cryst. Growth Des. 2006, 6, 1120–1123. (18) Fricke, M.; Volkmer, D. Top. Curr. Chem. 2007, 270, 1–41. (19) DiMasi, E.; Olszta, M. J.; Patel, V. M.; Gower, L. B. CrystEngComm 2003, 5, 346–350. (20) Loste, E.; Diaz-Marti, E.; Zarbakhsh, A.; Meldrum, F. C. Langmuir 2003, 19, 2830–2837. (21) Chen, Y. J.; Xiao, J. W.; Wang, Z. N.; Yang, S. H. Langmuir 2009, 25, 1054– 1059.

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Figure 1. Surface pressure-area isotherms of DPPC monolayers spread at the interfaces of air-pure water (-) and air-10 mM Ca(HCO3)2 aqueous solution ( 3 3 3 ). The isotherms were recorded under linear compression at a barrier speed of 10 mm/min (* at an area per molecule of 99.4 A˚2 denotes the starting point of barrier compression after evaporation of the solvent for 15 min).

Technology), a CCD camera (Sony XC-ST50 monochrome camera, Optem), a zoom lens (Zoom 70XL w/Iris system, Wyldar Group), and a Langmuir trough. The Langmuir trough was filled with 300 mL of Ca(HCO3)2 solution prepared as described above with a given concentration. Afterward, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, Sigma) was dissolved in chloroform (Labscan) to a concentration of 1 mg/mL, and 30 μL of the solution was spread on the surface of the subphase (Ca(HCO3)2 solution at pH ∼6.5) with a Hamilton syringe. After solvent evaporation (15 min), the DPPC monolayer was compressed at a rate of 10 mm/min. We set the surface pressure to zero when the barriers were not compressed and set the reaction time to zero when the surface pressure reached a designated value for in situ BAM imaging to monitor the time course of CaCO3 crystallization at the water-monolayer interface. Characterization. Besides in situ BAM imaging, other ex situ microscopic techniques of higher spatial resolution were also used to assist in the mapping of the CaCO3 mineralization paths under a DPPC monolayer. The size, morphology, and crystal structure were studied by scanning electron microscopy (SEM, JEOL6700) and transmission electron microscopy (TEM, JEOL2010F). SEM samples were prepared by gently moving a silicon wafer to touch the air-monolayer interface and then withdrawing it, followed by washing with ethanol (>99.9%, Merck) and quick drying. The samples were then sputter coated with a thin layer of gold for SEM observations. The preparation of TEM samples was similar; namely, a copper grid with holey carbon was directed to approach the air-monolayer interface until they touched and then was withdrawn, washed with ethanol, and dried quickly. The XRD analyses were performed on Philips PW-1830 X-ray diffractometer with Cu KR radiation (λ = 1.5406 A˚) over the 2θ range of 20-60°. Raman spectroscopy was carried out on a microRaman system (RM3000, Renishaw) with an Arþ laser (20 mW, 514 nm) and a Ge detector.

Results Figure 1 shows the surface pressure-area (π-A) isotherms of DPPC molecules spread on pure water and a 10 mM Ca(HCO3)2 aqueous solution. The mean areas of DPPC per molecule obtained by extrapolating the slopes of the isotherms to zero pressure are found to be 56 A˚2 on pure water and 58 A˚2 on 10 mM Ca(HCO3)2 solution, indicating the existence of interactions between the monolayer and calcium and bicarbonate ions as suggested by Mann and Zhang et al.15,16 It can be seen that the Langmuir 2010, 26(7), 4977–4983

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Article Table 1. Quantitative Calculation of the Relative Contents (wt %) of Vaterite and Calcite in the Mineralized Samples Collected under a DPPC Monolayer at Various Surface Pressures after 9 h of Reaction Based on the Standard Reference Intensity Ratio (RIR) Method and Reference Data from Standard X-ray Cards π (mN/m) polymorph

0

2

5

7

25

40

calcitea vateriteb

100

76.4 23.6

72.1 27.9

67.1 32.9

35.8 64.2

27.7 72.3

a The RIR of calcite is considered to be 2.0. b The RIR of vaterite is estimated to be 1.0.

Figure 2. XRD patterns of the mineralized samples obtained under a DPPC monolayer at various surface pressures after 9 h of reaction: (A) 0, (B) 2, (C) 5, (D) 7, (E) 25, and (F) 40 mN/m [(9) calcite and (0) vaterite].

liquid-expanded (LE)-liquid -condensed (LC) coexistence region is slightly narrowed because of the presence of 10 mM Ca(HCO3)2 in the subphase, again signifying interactions between the lipid headgroups and the calcium and carbonate ions. Such interactions may be important to the nucleation and growth of calcium carbonate. In this work, we monitored the biomimetic mineralization of CaCO3 under a DPPC monolayer at surface pressures of 0, 2, 5, 7, 25, and 40 mN/m as a function of reaction time. To understand the effect of monolayer density on the mineralization of calcium carbonate, a series of surface pressures denoted by the short horizontal lines in Figure 1 were studied. Figure 2 shows the corresponding XRD patterns of the mineralized samples under a DPPC monolayer obtained at various surface pressures after 9 h of reaction. Foremost among the features is an interesting trend, which is immediately clear. Only the calcite phase (JCPDS 05-0586) could be found at a surface pressure of 0 mN/m (Figure 2A). However, as soon as the surface pressure was increased to 2 mN/m, vaterite (JCPDS 33-0268) and calcite were found to coexist (Figure 2B). The subsequent further increase in surface pressure resulted in a continuous expansion of the vaterite phase at the expense of the calcite phase, as can be seen in Figure 2C-F and can be more quantitatively gleaned from Table 1, which quantifies the XRD results on the basis of the standard reference intensity ratio (RIR) method. Representative SEM images of the mineralized samples collected under a DPPC monolayer at various surface pressures after 9 h of reaction are shown in Figure 3. Only calcite crystals were identified in a conventional rhombohedral form at a surface pressure of 0 mN/m (Figure 3A). When the surface pressure was increased to 2 mN/m, a floral morphology could also be spotted in addition to the rhombohedral morphology of calcite (Figure 3B). The Raman spectrum of the floret-like crystals shows two sharp peaks at 1074 and 1090 cm-1 (inset of Figure 3B), which can be assigned to the symmetric stretching of carbonate anions in the vaterite structure.22 Further increases in the surface pressure to 25 and 40 mN/m yielded more and more petal-like crystals, which are in the vaterite phase and are detached from the floret-shaped crystals because of sonication during the SEM (22) Gabrielli, C.; Jaouhari, R.; Joiret, S.; Maurin, G. J. Raman Spectrosc. 2000, 31, 497–501.

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Figure 3. SEM images of the CaCO3 particles collected under a DPPC monolayer at different surface pressures after 9 h of reaction: (A) 0, (B) 2 (the inset displays a Raman spectrum of the floretlike crystals), (C) 25, and (D) 40 mN/m. The irregularly shaped crystals in C and D are the same as the floret-like crystals in B except that the petal-like crystals have been detached via sonication. The very small debris arising from the detachment process can be clearly seen in C.

sample-preparation process (Figure 3C,D). As a whole, the SEM results revealed that with the increase in surface pressure the number of vaterite crystals rises compared to the number of the calcite crystals, directly corroborating the conclusion drawn from the XRD data. To chart the time course of the biomimetic mineralization of CaCO3 under a compressed DPPC monolayer in situ, a series of BAM images were recorded as shown in Figure 4. After 15 min of solvent evaporation, some small white dots had already appeared on the surface of a 10 mM Ca(HCO3)2 aqueous solution, as shown in Figure 4A. Conversely, the field of view was smooth on the surface of pure water and for a 10 mM CaCl2 aqueous solution after solvent evaporation for the same period of time (Figure SI-1). Therefore, it can be concluded that CaCO3 minerals had already begun to nucleate during the solventevaporation period. When the monolayer was compressed to the target surface pressure of 40 mN/m for BAM imaging, (i.e., at zero reaction time), more small white dots appeared under the DPPC monolayer (Figure 4B) because of the increasing formation of CaCO3 particles. After 30 min of reaction, the dots became brighter and larger (Figure 4C). After another hour, relatively large, bright dots came into view, which are presumably large calcium carbonate crystals under the DPPC monolayer as evidenced by their strong light scattering and reflection (Figure 4D). With further prolonging of the reaction time, the DOI: 10.1021/la903641k

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Figure 4. BAM images showing the mineralization of CaCO3 under a DPPC monolayer at the surface pressure of 40 mN/m as a function of reaction time: (A) After solvent evaporation for 15 min (0 mN/m, see the asterisk symbol at an area per molecule value of 99.4 A˚2 in Figure 1); (B) after 0 min of reaction (40.0 mN/m), (C) after 30 min of reaction (37.1 mN/m); (D) after 1 h of reaction (36.7 mN/m); (E) after 2 h of reaction (36.4 mN/m); and (F) after 6 h of reaction (35.6 mN/m).

appearance of much larger, even brighter, and new features connoted the continual growth of the calcium carbonate crystals accompanied by the formation of new crystals under the DPPC monolayer (Figure 4E, F). To be brief, the BAM images clearly revealed the formation process of calcium carbonate crystals under the DPPC monolayer: although the mineralization already started before and during the monolayer compression, the latter growth could be sustained and controlled only when the DPPC monolayer surface pressure was defined. Although in situ BAM images have portrayed a nice sequence of snapshots of the mineralization process of calcium carbonate under a DPPC monolayer, precise information about the sizes and morphologies of the structures could not be obtained at the air-water interface because of the limited lateral resolution of our BAM system (∼2 μm). This problem will be resolved below by using electron microscope techniques. The SEM images in Figure 5 show the evolution of the calcium carbonate particles with reaction time under a DPPC monolayer at a fixed surface pressure of 40 mN/m. As described above, the zero reaction time was set at the point when the designated surface pressure was reached for studying the time course of CaCO3 crystallization at the interface. At zero reaction time, we observed not only rhombohedral calcite crystals (Figure 5A) but also acicular-like crystals (Figure 5A) and tightly packed ellipsoidal particles (Figure 5B). The Raman spectrum of the acicular-like crystals shows peaks at 1460 and 1570 cm-1, which are ascribable to the asymmetric stretching of carbonate anions in the aragonite phase.22 In fact, even at a surface pressure of 0 mN/m (see the point in Figure 1 denoted by * with an area per molecule value of 99.4 A˚2), the acicular-like crystals also appeared at zero reaction time (Figure SI-2). How4980 DOI: 10.1021/la903641k

Figure 5. SEM images of the CaCO3 particles obtained under a DPPC monolayer (π = 40 mN/m) after reaction for various times: (A, B) 0, (C, D) 0.5, (E, F) 1, and (G, H) 2 h. (Inset of A) Raman spectrum of the acicular-like crystals.

ever, the tightly packed ellipsoidal particles and rhombohedral calcite crystals, different from the samples collected at zero reaction time (Figure SI-2), were formed at a surface pressure of 0 mN/m only after 1 h of solvent evaporation as shown in Figure SI-3, when the chloroform had been completely evaporated. Furthermore, from BAM images obtained after 15 min of solvent evaporation, particles had already begun to nucleate at the surface of the subphase during the solvent-evaporation period (Figure 4A). Therefore, it can be concluded that the aragonite crystals in acicular-like morphology were formed at the chloroform-water interface during the period of solvent evaporation whereas the tightly packed ellipsoidal particles were formed at the air-water interface. This seems to explain why both the acicular morphology and the tightly packed ellipsoidal particles were observed at a surface pressure of 40 mN/m at zero reaction time. Conceivably, during the solvent-evaporation period, both the chloroform-water interface and the air-water interface could exist such that the two types of particles could be formed. After the designated surface pressure of 40 mN/m was reached, the DPPC-mediated mineralization processes were more easily monitored. After 0.5 h of reaction, loosely packed hollow ellipsoidal particles composed of nanoparticles with a diameter of 70 to 100 nm appeared in the samples (Figure 5C,D). One hour later, the tightly packed ellipsoidal particles appeared again in the products (Figure 5E) but might be formed in quite different ways (e.g., transformed from the loosely Langmuir 2010, 26(7), 4977–4983

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packed hollow ellipsoidal particles). The tightly packed ellipsoidal particles are also composed of nanoparticles with a diameter of 70 to 100 nm (Figure 5F). After another 2 h of reaction, the products contained calcium carbonate particles in a typical floral morphology (Figure 5G) consisting of nanoparticles with a diameter of 70 to 100 nm (Figure 5H). The floral morphology confirms that these particles are the same as those in Figure 3B, which have already been proven to be in the vaterite phase by the Raman spectrum in the inset of Figure 3B. In fact, as will be shown below, the two types of ellipsoidal particles observed above are also in the vaterite phase. The mineralized samples collected at a surface pressure of 40 mN/m after various reaction times were subjected to TEM investigations to gain more detailed information about the nucleation and growth of the calcium carbonate particles under a DPPC monolayer. Figure 6 puts the result on view. First, spherical amorphous particles with a mean diameter of 70100 nm were found in the products at a reaction time of zero (Figure 6A). The corresponding EDX spectrum indicates that the particles are indeed made of calcium carbonate (Figure SI-4). The amorphous nature of the spherical particles is further supported by the Raman spectrum, which exhibits only a major but rather broad peak at ∼1085 cm-1 (inset of Figure 6A), in contrast to the series of relatively sharp peaks normally associated with crystalline forms of calcium carbonate.23 Additional verification of the ACC particles is from the SAED pattern in Figure 6B, which is essentially featureless. Second, crystalline particles that are similar in size to the ACC nanoparticles were also obtained at zero reaction time; these crystalline particles are in the vaterite phase according to the TEM and HRTEM images in Figure 6C,D, respectively. Third, the loosely packed hollow ellipsoidal particles collected after 30 min of reaction were also found to be in the vaterite phase but with a polycrystalline structure (Figure 6E,F). As for the tightly packed ellipsoidal particles collected after 1 h of reaction (see the top-view image in Figure 6G), we find unambiguously the corresponding SAED pattern (inset in Figure 6G) to be the same as that of a single vaterite crystal taken along the [001] direction. However, the SAED pattern taken from the side view (Figure 6H) of the ellipsoidal particles (inset of Figure 6H) conforms to the single vaterite crystal diffraction pattern along the [110] zone axis. Taken together from the TEM and SAED results, the tightly packed ellipsoidal vaterite particles created by the oriented attachment of nanoparticles with a mean diameter of 70-100 nm have such a mesocrystal structure (see also the corresponding HRTEM image in Figure SI-5), in which the constituent nanoparticles orient their crystallographic c axes uniformly in the oblate axial direction-the direction of the smallest dimension-of the ellipsoidal vaterite particles.

Discussion Among the polymorphs of calcium carbonate, calcite is thermodynamically the most stable structure, whereas amorphous calcium carbonate (ACC) is the least stable form. Our direct observation of the first nucleation of ACC precursor particles and their subsequent transformation into the most stable calcite phase via the intermediate vaterite phase under a DPPC monolayer and the final phase influenced by the degree of compression may have implications in biological mineralization processes of cellular membranes. In living organisms, the initial nucleation of the ACC precursor followed by phase transformations to final phases via a series of intermediate phases is widespread, and the (23) Addadi, L.; Raz, S.; Weiner, S. Adv. Mater. 2003, 15, 959–970.

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Figure 6. Calcium carbonate particles formed after reaction for various times under a DPPC monolayer at a surface pressure of 40 mN/m: (A) TEM image and Raman spectrum (inset) of amorphous calcium carbonate particles formed at 0 h; (B) SAED pattern of the amorphous calcium carbonate particles in A; (C) TEM image of the vaterite particles formed at 0 h; (D) HRTEM image of the vaterite particles in C; (E) TEM image of the loosely packed ellipsoidal particles formed at 0.5 h; (F) HRTEM image of the loosely packed ellipsoidal particles in E; (G) Top-view TEM image and SAED pattern (inset) of the tightly packed ellipsoidal particles formed at 1 h; (H) side-view TEM image and SAED pattern (inset) of the tightly packed ellipsoidal particles formed at 1 h. In taking the SAED patterns in the insets of Figure 6B,G,H, the electron beam was focused to diameters of 110 nm and 1 and 1 μm, respectively, and directed onto the whole particle under examination as marked by white circles in Figure 6A,G,H.

biological systems seem to be able to stop the mineralization at a particular structure.23,24 Biomimetic Mineralization Pathways of CaCO3 under a DPPC Monolayer. Although the existence of ACC as a transient precursor during the biomimetic mineralization was demonstrated previously,21,25,26 this is the first time that it is observed under a phospholipid monolayer. Here the mineralization reaction proceeds by the outgassing of CO2 from a supersaturated Ca(HCO3)2 solution under the template of a DPPC monolayer. A kinetically controlled precipitation process is (24) Aizenberg, J. Adv. Mater. 2004, 16, 1295–1302. (25) Pichon, B. P.; Bomans, P. H. H.; Frederik, P. M.; Sommerdijk, N. A. J. M. J. Am. Chem. Soc. 2008, 130, 4034–4040. (26) Pouget, E. M.; Bomans, P. H. H.; Goos, J.; Frederik, P. M.; de With, G.; Sommerdijk, N. Science 2009, 323, 1555–1458.

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believed to account for the early nucleation of the 70-100 nm ACC nanoparticles at the air-water interface. The relative supersaturation at the air-water interface is enhanced compared to that of the bulk subphase for several reasons. The first is the easier escape of CO2 from the Ca(HCO3)2 solution surface. Second, although DPPC has no net charge, its phosphate and trimethyl ammonium groups still could attract calcium and carbonate ions, respectively, through electrostatic interaction, effectively increasing the relative supersaturation at the DPPC-water interface as revealed by the π-A isotherms in Figure 1. Furthermore, DPPC molecules can significantly reduce the interfacial free-energy density of calcium carbonate nuclei, thus facilitating their formation. Understandably, at the air-water interface holding the DPPC monolayer, the facilitated escape of CO2, increased ion concentration, and enhanced calcium carbonate nuclei stabilization are more favorable to the formation of the large ACC nanoparticles than in the bulk solution. The 70-100 nm ACC nanoparticles that we observed are akin to those observed by Pouget et al. (70-120 nm) temporarily stabilized by the monolayer.26 Although this group also reported ACC nanoparticles with a diameter of 30 nm or so generated through the aggregation of prenucleation clusters in the bulk of a fresh Ca(HCO3)2 solution, the size of the nanoparticles is much smaller than those heterogeneously nucleated at the interface. The formed ACC precursor particles were unstable and thus transformed into the final mineral phases via a series of the intermediate phases assisted by DPPC, conforming to the empirical Ostwald-Lussac law. Here the intermediate phases include aragonite during solvent evaporation and vaterite after solvent evaporation. The acicular-like aragonite crystals were found in the mineralized samples at surface pressures of 0 and 40 mN/m at zero reaction time (Figures SI-2 and 5A), which were apparently formed during solvent evaporation. After the complete evaporation of solvent, however, the tightly packed ellipsoidal vaterite, rather than the acicular-like aragonite, crystals were formed from the nascent ACC particles even when the surface pressure was 0 mN/m (Figure SI-3). The transformation from ACC to aragonite instead of vaterite and calcite may be caused by the low degree of organization of DPPC molecules in the process of solvent evaporation. Similar observations were made by Kuther and co-workers,27 who related aragonite formation to the disorder in self-assembled monolayers on gold colloids, and by Sommerdijk et al.,25 who proposed the formation of aragonite crystals under a leucine derivative self-organized monolayer because of the more bulky side group of leucine that can slow the self-organization of the surfactants. Finally, with time, the aragonite crystals were transformed into calcite as revealed in Figure 7A, presumably via a solvent-mediated mechanism.28-30 After zero reaction time, the ACC precursor nanoparticles formed under a DPPC monolayer were first transformed into metastable vaterite crystals of about the same size as ACC. This transformation was likely brought about by the local highly supersaturated microenvironment via the escape of CO2 from the supersaturated Ca(HCO3)2 solution because the increase in supersaturation has a much more positive effect on the tendency to form metastable phases than to form more stable phases.2,31 (27) Kuther, J.; Seshadri, R.; Knoll, W.; Tremel, W. J. Mater. Chem. 1998, 8, 641–650. (28) Bottcher, M. E. Mar. Chem. 1997, 57, 97–106. (29) Wei, H.; Shen, Q.; Zhao, Y.; Wang, D. J.; Xu, D. F. J. Cryst. Growth 2003, 250, 516–524. (30) Shen, Q.; Wei, H.; Zhou, Y.; Huang, Y. P.; Yang, H. R.; Wang, D. J.; Xu, D. F. J. Phys. Chem. B 2006, 110, 2994–3000. (31) Tong, H.; Ma, W. T.; Wang, L. L.; Wan, P.; Hu, J. M.; Cao, L. X. Biomaterials 2004, 25, 3923–3929.

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Figure 7. SEM images showing (A) the phase transformation from aragonite to calcite (black arrow) at zero reaction time and (B) the phase transformation from vaterite to calcite (black arrow) after 1 h of reaction under a surface pressure of 40 mN/m.

This is consonant with the recent work by DiMasi et al. and by Meldrum and co-workers,19,20 who revealed a preferential precipitation of metastable vaterite crystals under a short-chain fatty acid monolayer and from a fast-outgassing Langmuir trough, respectively, highlighting the dominant role of the CO2 gas escape rate in dictating the transformation from ACC to vaterite or to calcite. Actually, vaterite crystals were also observed at the air-water interface after 2 h of reaction even in the absence of the DPPC monolayer (Figure SI-6), further buttressing this viewpoint. The numerous vaterite nanocrystals (70-100 nm in diameter) tended to aggregate into loosely packed hollow ellipsoidal particles with a polycrystalline structure to decrease the surface energy and further rearranged into the tightly packed ellipsoidal particles, which behaved like a single-crystal structure with an increase in reaction time. Most likely, such a transformation from an open structure to a dense structure arose from the orientational rearrangement of the constituent particles to maximize their surface contacts and thus lower the system free energy.31-33 For the as-formed tightly packed ellipsoidal vaterite crystals, the (001) crystal faces were found to become dominant (SAED pattern in the inset of Figure 6G). However, no (001) face was exposed in the absence of the DPPC monolayer (Figure SI-6). This is consistent with reports in the literature27,34 because vaterite does not generally expose the (001) faces owing to their high surface energy in the absence of any growth modifier. Under a DPPC monolayer, the anionic phosphate groups could act synergistically with the -N(CH3)3þ headgroups to induce the crystallization of vaterite on those faces on which the erected carbonate anions are perpendicular to the calcium ion plane, namely, the (001) faces. A similar explanation was applied to the preferential nucleation of (32) Wang, T. X.; Colfen, H.; Antonietti, M. J. Am. Chem. Soc. 2005, 127, 3246– 3247. (33) Zhu, Y. C.; Liu, Y. Y.; Ruan, Q. C.; Zeng, Y.; Xiao, J. W.; Liu, Z. W.; Chen, L. F.; Xu, F. F.; Zhang, L. L. J. Phys. Chem. C 2009, 113, 6584–6588. (34) de Leeuw, N. H.; Parker, S. C. J. Phys. Chem. B 1998, 102, 2914–2922.

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Figure 8. Schematic representation of the mineralization pathways of calcium carbonate under a DPPC monolayer. During the evaporation of chloroform, ACC particles were generated and transformed into (A) the acicular-like aragonite crystals at the chloroform-water interface and (B) the tightly packed ellipsoidal vaterite particles at the air-water interface and (C) eventually transformed into rhombohedral calcite crystals. After solvent evaporation, the newly formed ACC particles were transformed into (D) vaterite crystals, (E) self-aggregated into loosely packed ellipsoidal, polycrystalline particles and (F) evolved into tightly packed ellipsoidal, single crystalline particles and transformed into (G) rhombohedral calcite crystals or (H) floret-like vaterite crystals by radial outgrowth depending on surface pressure. (ACC amorphous calcium carbonate; A - aragonite; V - vaterite; and C - calcite).

vaterite under an octadecylamine monolayer.35 This suggests that the crystallization processes are influenced not only by the escape rate of CO2 gas but also by the template effect of the monolayer. Ultimately, the tightly packed ellipsoidal vaterite particles were transformed to either the floral-shaped vaterite crystals or the most stable rhombic-shaped calcite crystals (Figure 7B). As a summary, Figure 8 schematically sorts the mineralization pathways of calcium carbonate under a DPPC monolayer. During the evaporation of chloroform, the ACC particles were first and heterogeneously nucleated at the air-water interface. Then, the ACC precursor nanoparticles were directly crystallized into the aragonite crystals in an acicular-like shape at the chloroform-water interface (step A in Figure 8) and subsequently transformed into the most stable calcite crystals (step B in Figure 8). Another fate of the ACC nanoparticles was the crystallization into the tightly packed ellipsoidal vaterite particles (step C in Figure 8), which appeared to occur at the air-water interface instead of at the chloroform-water interface. After the solvent was completely evaporated (in 15 min) and the designated surface pressure had been reached through constant-rate DPPC monolayer compression, numerous fresh ACC precursor particles were continuously nucleated at the air-water interface and quickly transformed into vaterite nanocrystals (step D in Figure 8). Driven by the trend to decrease the surface energy, the vaterite crystal nuclei self-aggregated into the loosely packed hollow ellipsoidal particles, resulting in a polycrystalline structure (step E in Figure 8) and then gradually evolved into single-crystallike, tightly packed ellipsoidal particles by orientational rearrangement and consolidation, with the (001) crystal faces being stabilized by the DPPC molecules (step F in Figure 8). Notice the two different ways that the tightly packed ellipsoidal particles were produced: the compressed DPPC monolayer directed the mineralization to follow the (D f E f F) path in a controlled fashion, whereas the fast and direct formation of the tightly packed ellipsoidal particles (C) occurred when the DPPC monolayer was not compressed. As the reaction time was further increased, the tightly packed ellipsoidal vaterite crystals became unstable and thus transformed into the most thermodynamically (35) Heywood, B. R.; Rajam, S.; Mann, S. J. Chem. Soc., Faraday Trans. 1991, 87, 735–743.

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Article

stable calcite crystals through a solvent-dissolution mechanism (step G in Figure 8) or developed into the floret-shaped crystals with the same crystalline phase by radial outgrowth into bulk solution (step H in Figure 8), depending on the surface pressure. Surface-Energy-Dependent Polymorph Selection of CaCO3. Under a DPPC monolayer, the selection of polymorphs of calcium carbonate was found to be closely related to the degree of compression. Ultimately, the tightly packed ellipsoidal vaterite crystals tended to be transformed into the most thermodynamically stable calcite crystals via the solvent-mediated mechanism with the reaction time increasing, as revealed by the disappearance of the tightly packed ellipsoidal vaterite crystals concomitantly with the formation of small calcite crystals (Figure 7B).25,29,30 More and more tightly packed ellipsoidal vaterite crystals gradually grew into vaterite crystals in a typical floral morphology by radial outgrowth into the bulk solution with an increasing monolayer density of DPPC molecules.15 Plainly, the higher the monolayer density of DPPC, the more difficult it is for CO2 to escape and thus the lower the supersaturation of CaCO3. This would predispose CaCO3 to crystallize into the thermodynamically stable calcite phase as revealed by Meldrum et al. and by DiMasi et al.19,20 This is contrary to our observation. Therefore, we suggest that under higher surface pressure, more DPPC molecules could be more readily adsorbed onto the (001) crystal faces of vaterite crystals to decrease the surface energy and create a kinetic barrier for the transformation from vatertie to calcite. Volkmer et al.17,18 proposed that the polymorph selection of calcium carbonate is largely controlled by the surface charge density of a macrocyclic monolayer. One can expect that the surface energy can be altered as well with the modified surface charge density of the macrocyclic monolayer. Therefore, the ultimate determinant in polymorph selection appears to be the surface energy.

Conclusions We have mapped the mineralization processes of calcium carbonate under a phospholipid monolayer. Our study revealed that the ACC precursors, initially nucleated from the highly supersaturated solution, were first transformed into the intermediate vaterite phase, a process mainly determined by the escape rate of CO2 from the supersaturated solution of Ca(HCO3)2 and the stabilization effect of DPPC molecules. The vaterite phase was eventually transformed into the most thermodynamically stable calcite crystals. The sequence of phospholipid-directed mineralization processes that we identified is in accordance with the empirical Ostwald-Lussac law, which may reflect the biomineralization processes of cellular membranes. Furthermore, the extent of conversion from vaterite to calcite was found to depend sensitively on the degree of monolayer compression instead of the escape rate of the CO2, implying that surface energy is a dominant factor in the transformation. This has important implications in biomineralization because living organisms, to achieve optimized functions, are able to stop the mineralization processes at a particular stage with a desired polymorph with the assistance of appropriate surface-active biomolecules. Acknowledgment. This work was supported by the Hong Kong Research Grants Council General Research Funds, GRF No. HKUST 604107. Supporting Information Available: BAM images of a DPPC monolayer. SEM images of the products and a sample collected under a DPPC monolayer. EDX spectrum of the spherical nanoparticles shown in Figure 6A. This material is available free of charge via the Internet at http://pubs. acs.org. DOI: 10.1021/la903641k

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