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Influence of Ovalbumin on CaCO3 Precipitation during in Vitro Biomineralization Xiaoqiang Wang,† Congmeng Wu,† Kai Tao,† Kang Zhao,† Jiqian Wang,† Hai Xu,*,† Daohong Xia,† Honghong Shan,† and Jian R. Lu*,‡ State Key Laboratory of HeaVy Oil Processing and Centre for Bioengineering and Biotechnology, China UniVersity of Petroleum (East China), 66 Changjiang West Road, Qingdao Economic DeVelopment Zone, Qingdao 266555, China, and Biological Physics Group, School of Physics and Astronomy, UniVersity of Manchester, Schuster Building, Manchester M13 9PL, United Kingdom ReceiVed: January 27, 2010; ReVised Manuscript ReceiVed: March 18, 2010
As a major constituent of egg white matrix, ovalbumin has long been perceived to be implicated in the formation of avian eggshells, in particular, the mammillary layer. However, very little is known about the detailed mechanism by which this protein mediates shell calcification. By the combined studies of AFM, SEM, and TEM, we have investigated the influence of ovalbumin on CaCO3 precipitation under in Vitro mineralization conditions. We observed that the influence was multifold. This protein modified the morphology of calcite crystals through a distinct anisotropic process with respect to the four crystal step edges. AFM characterization revealed that the modification was initiated at the obtuse-obtuse step corner and propagated predominantly along the obtuse steps. Furthermore, the protein favored the existence of unstable phases such as amorphous calcium carbonate and crystalline Vaterite. In contrast, lysozyme, another protein also present in the system, played a very different role in modifying calcite morphology. The mechanistic understanding gained from this study is clearly also of practical significance in developing advanced inorganic CaCO3 materials with the aid of morphological manipulation of crystalline structures via different protein mediation. 1. Introduction Minerals are ubiquitous in biological systems. Nearly all biominerals are employed by living organisms to achieve functional purposes, for instance, through mechanical, magnetic, optical, and piezoelectric actions.1-5 These minerals are usually organic-inorganic hybrids, with well-defined morphologies and structures. Although their organic contents are quite low, the unique combination with inorganic matters, together with their well-ordered structures, makes them far superior to current manmade materials in terms of function and performance. Furthermore, these biological materials are generated by organisms typically at ambient temperature and pressure, which is in stark contrast to the synthesis of many man-made hierarchical structures where harsh conditions such as higher temperature and pressure are prerequisite. Inspired by Nature, researchers from many disciplines have now made tremendous advances in understanding biomineralization, with the benefits of not only uncovering the strategies adopted by organisms but also applying them to the synthesis of advanced materials. It has been widely perceived that the formation of biominerals including the nucleation, growth, and assembly of inorganic particles is finely regulated by organic macromolecules such as proteins, glycoproteins, polysaccharides, and lipids, which are mostly secreted by specific cells for this purpose.1-8 The involvement of biomacromolecules has been shown to be manifold, not only acting as templates for inducing nucleation of crystals and controlling their orientation but also serving as modifiers for decorating the habit of crystals and, even in some cases, directing the assembly of nanoscale particles to form * Corresponding authors. Phone: 86-532-86981569 (H.X.); 44-1612003926 (J.R.L.). E-mail:
[email protected] (H.X.);
[email protected] (J.R.L.). † China University of Petroleum. ‡ University of Manchester.
hierarchical structures. Over the past decade, many researchers have used liquid atomic force microscopy (AFM) to undertake in situ monitoring of the development of steps of dislocation hillocks on the (104) face of an existing calcite crystal at the molecular level, and many biomolecules including acidic amino acids, peptides, and proteins have been shown to significantly influence the step advancement speed and morphology, presumably via step-specific interactions.9-15 So far, more than 60 different minerals have been revealed to form biologically, and calcium carbonate is among the most investigated systems because of its abundance in biology as well as importance in many industrial applications. The avian eggshells are probably the most relevant systems, consisting of ∼95% calcium carbonate as well as the organic matrix.16,17 As typical biogenic minerals, the avian eggshells have attracted considerable attention, owing to their formation rate, welldefined structure, superior mechanical properties, and other advantageous features.16-24 The mineralization of eggshells occurs quite rapidly (approximately 20 h), in an acellular uterine fluid supersaturated with calcium and carbonate ions and also containing the soluble organic precursors of the shell matrix, with their composition varying at different stages of shell formation.19,20 Although exclusively in the form of calcite, the mineral in eggshell exhibits different crystallization patterns (orientation, morphology, and structure) at different zones. In general, the calcified shell exhibits two distinct morphological portions: the inner part is composed of irregular cones, known as the mamillary knob layer, and next to this layer is the palisade layer where columnar crystals are aligned perpendicular to the shell surface. It is this sophisticated structure that provides the eggshell with various essential functions such as exchange of water and gas with the outer environment, provision of calcium for embryonic development, sheltering the egg from external aggression while allowing the hatching embryo to readily break
10.1021/jp1008237 2010 American Chemical Society Published on Web 04/06/2010
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through.20 Within the mammillary layer, for instance, calcite microcrystals possess spherulitic texture and are readily dissolved to mobilize calcium to meet the needs of the growing embryo.24,25 It is thought that organic matrix components exert exquisite control over this polymorphism and the sequential incorporation of different matrix macromolecules during eggshell formation leads to distinct localization patterns.21-23 To date, many matrix biomacromolecules have been found in eggshell and they are roughly categorized into three groups: (i) ubiquitous proteins such as osteopontin and clusterin, (ii) egg white proteins such as ovalbumin, lysozyme, and ovotransferrin, and (iii) other biomacromolecules such as glycosaminoglycans and ovocleidins that are only synthesized by tissues involved in the eggshell calcification such as distal isthmus and uterus.18,20,23,25 Their distribution varies across the calcified regions of shell, implying different roles in controlling morphological structures of crystals.19-21 For instance, as the first and major egg white protein revealed in eggshell matrix, ovalbumin, representing 54% of the egg white, is found only in the mammillary bodies of the decalcified shell and is not distributed throughout the shell matrix.19,26 Furthermore, ovalbumin is also present in the uterine fluid where the shell calcification takes place, and is particularly abundant during the initial stage of shell formation.19,26 This is well consistent with the as-indicated fact that this protein is only found in the mammillae of the calcified shell that is generated at the initial stage, indicating that ovalbumin is highly likely to be implicated in the formation of the mammillary cone layer, thereby being incorporated into this part of the biomineral. However, the exact mechanism by which ovalbumin regulates the crystalline nature and morphology of the inorganic phase, particularly at the molecular scale, is not well understood. In this work, we report a carefully designed study, with the aid of flow cell AFM, scanning electron microscopy (SEM), and transmission electron microscopy (TEM), to assess the effects of ovalbumin on the growth of calcium carbonate under in Vitro mineralization conditions. Our primary aim was to observe nanometer-level alterations in the step morphology of calcite hillocks within minutes of surface exposure to ovalbumin and micrometer-level changes in calcite habits over a longer time scale (12 h). Our results demonstrated that ovalbumin had a distinguished ability to mediate CaCO3 deposition, especially in modifying the morphology of calcite crystals. Very interestingly, AFM imaging revealed that the morphological alterations were anisotropic in the presence of ovalbumin, with more pronounced changes occurring at the obtuse steps. These changes at the elementary step scale correlated well with the modifications in the macroscopic habits of calcite crystals as revealed by SEM. Additionally, both AFM and TEM analyses implied the existence of amorphous calcium carbonate (ACC) in the presence of ovalbumin during the early stage of mineralization. These findings are highly important to the understanding of well-defined structural characteristics of the mammillary layers in the avian eggshells. 2. Experimental Section Materials. Analytic grade anhydrous calcium chloride (purity g99.0%), ammonium carbonate (purity g99.0%), and sodium hydrogen carbonate (purity g99.0%) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai). Ovalbumin and lysozyme from chicken egg white were purchased from Sigma-Aldrich. All silicon slices used as substrate were cut from silicon 〈111〉 wafers (Compact Technology Ltd., U.K.), with a native oxide layer of ca. 13 Å determined by spectroscopic
Wang et al.
Figure 1. SEM images of (a) calcite rhombohedra produced at 12 h crystallization in the absence of any protein as additive and (b) modified calcite at 12 h crystallization in the presence of 2 g/L lysozyme. The scale bars represent 10 µm.
ellipsometry. Prior to each mineralization experiment, the slices were cleaned with 5% (by weight) Decon90 solution (Decon Laboratory, U.K.), followed by copious rinsing with Milli-Q water (resistivity >18 MΩ · cm). All glasswares were cleaned by sequential washing of strong acid, Decon90, and ultrapure water before use. All chemicals and biochemicals were used as supplied without further purification. Supersaturated calcium carbonate solutions (σ ) 2.0) were prepared by mixing a certain amount of NaHCO3 solution with CaCl2 solution. Supersaturation (σ) refers to ln[a(Ca2+)a(CO32-)/ Ksp], where “a” is the actual ion activity calculated from numerical code PHREEQC and “Ksp” is the equilibrium solubility product of calcite. Protein-containing solutions were filtered through a 0.22 µm filter, and the pH of all of the solutions was adjusted to 8.5 by adding dilute NaOH solution for in situ AFM observations. Biomineralization Process. In resemblance to the system described by Addadi et al.,27 we have constructed a laboratory setup to study CaCO3 crystallization in a well controlled manner so that experiments with and without ovalbumin could be directly compared.28 Our setup, consisting of a glass beaker (containing CaCl2 solution) and four smaller glass beakers (containing (NH4)2CO3) sitting around the CaCl2 beaker, all being contained inside a desiccator, allowed the vapor of ammonium carbonate (NH4)2CO3 to diffuse into the CaCl2 solution at an ambient temperature of 20-22 °C. Although the method has been widely applied, the outcome is highly dependent on the experimental details relating to the diffusion and salts used.29 For example, our data and those of Gehrke et al. have revealed the predominant production of hexagonal vaterite instead of rhombohedral calcite crystals via the method as described above, due to the role of ammonium ions.28,29 While the experimental setup was kept the same as that used for the lysozyme study,28 the number of pinholes on the Parafilm covering the CaCl2-solution-containing beaker was reduced to 2 in this work instead of 6 (identical diameter: ca. 2 mm). The slower vapor diffusion rate was found to facilitate the formation of perfect calcite rhombohedra (Figure 1a), confirming the dominant effect of vapor diffusion in this case. Note that the initial pH of the CaCl2 solution was adjusted to around pH 6.5, and as the (NH4)2CO3 vapor diffused in, the solution pH was observed to increase but was stabilized at around pH 8.5 after 1.5 h. At the end of CaCO3 growth, the substrates (silicon wafers) covered by the precipitates were taken out, and were immediately rinsed using Milli-Q water and ethanol. After being dried under a nitrogen stream, the mineralized silicon wafers were stored in a desiccator for the subsequent characterization. Characterization. The morphological features of micrometersized CaCO3 crystals were characterized by SEM, on a JEOL JSM-840 microscope at an accelerating voltage of 15 kV. The X-ray diffraction (XRD) patterns of CaCO3 precipitates were
Influence of Ovalbumin on CaCO3 Precipitation obtained with a Philips X’Pert Pro diffractometer, using nickel filtered Cu KR radiation (λ ) 1.5418 Å). The intensities were recorded over the 2θ range from 10 to 70° with a step size of 0.0167° at the ambient temperature. For infrared (IR) spectroscopy and transmission electron microscopy (TEM) characterizations, the CaCO3 precipitates were gently scratched off from the silica wafer surface. IR spectra were recorded with a Nicolet 6700 Fourier transform IR (FTIR) spectrometer. TEM and selected area electron diffraction (SAED) were performed on a JEOL JEM-1200EX microscope. In Situ AFM Imaging. To assess the effects of ovalbumin on the calcite growth at the nanometer level, we performed fluid cell AFM experiments on a commercial Nanoscope IVa Scanning Probe Microscope (Digital Instruments, Santa Barbara, CA), equipped with a J-type scanner (maximum scan area 125 µm × 125 µm). Using established methods, a freshly cleaved calcite sample (2 × 2 × 1 mm3, optical quality Iceland spar from Ward’s Scientific, Chihuahua, Mexico) was glued onto a steel puck first, and the O-ring in the liquid cell was then placed on top of the calcite (104) face. The supersaturated calcium carbonate solution (σ ) 2.0) was continuously injected into the cell via a peristaltic pump, and a flow rate of 0.5 mL/min was chosen so that calcite surface growth was not affected by diffusion limitations. Once well-defined hillocks on the (104) face were observed, the supersaturated solution containing ovalbumin was introduced to ascertain its influence. In situ AFM images were collected in contact mode at room temperature, by using Si3N4 tips with a nominal spring constant of 0.06 N/m. The scan rates were in the range 5-20 Hz, with a resolution of 512 × 512, and the contact force was carefully minimized in order to avoid any artificial effects on crystal morphology and step motion. 3. Results and Discussion Under the present experimental conditions, the diffusion of (NH4)2CO3 vapor into the CaCl2 aqueous solution was limited by the size and numbers of pinholes opened on the top of the CaCl2 solution beaker. In contrast to six pinholes as used in ref 28, two pinholes were opened in this study using otherwise the same setup. Thus, the diffusion process was much slower, leading to a very gradual buildup of supersaturation. In such a case, calcite was predominately generated in the absence of any protein, as manifested by the SEM characterization (Figure 1a) and the XRD and FTIR analyses (date not shown). The outcome is well consistent with the “Ostwald’s step rule” that indicates that at low supersaturation the difference of the supersaturation ratio (S ) C/Ce, C is the solution concentration and Ce is the solubility) affects the crystallization and the stable form may preferentially precipitate.30 Furthermore, these calcite crystals were perfect rhombohedra with six well-expressed {104} faces, as indicated in Figure 1a. Previous studies also showed that, in the presence of lysozyme, calcite was always preferred, but macrosteps formed at the corners of the calcite crystals (Figure 1b). In addition to the common (104) face, the (110) face of calcite could also be expressed, and the morphological modification was lysozyme concentration dependent.28 However, the scenario changed upon ovalbumin addition: although the precipitates were still primarily composed of calcite crystals, their habit was significantly modified. Figure 2a-d shows representative SEM images of calcite crystals collected after 12 h of crystallization in the presence of 0.5 and 2 g/L ovalbumin, respectively. To facilitate morphological analysis, two differently orientated crystals are shown for each ovalbumin concentration. Figure 2a and b exhibits the crystals in the
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Figure 2. (a-d) SEM images of calcite crystals collected at 12 h in the presence of 0.5 g/L ovalbumin (a,b) and 2 g/L ovalbumin (c,d), respectively. The scale bars represent 10 µm. The c-axis for modified calcite crystals with different orientations is marked for guiding the eyes. (e) Schematic representation of calcite crystal expressing {110}, {012}, and {018} faces (painted with gray). Note that the {110} face includes the (11j2), (102j), and (012) faces; the {012} face includes the (110) and (21j0) faces; the {018} face includes the (11j8) and (018) faces in this case.
presence of 0.5 g/L ovalbumin, while Figure 2c and d shows those in the presence of 2 g/L ovalbumin. It can be seen that, in both cases, the growth of the crystal edges of {104} faces were modified. The most striking feature is that these effects were highly anisotropic in terms of the four edges of each {104} face. Specifically, the two neighboring crystal edges formed by the obtuse steps of each {104} face (indicated by red arrows and the definitions of obtuse and acute steps will be given below) were more significantly modified than those formed by the acute steps (indicated by blue arrows); in the presence of 0.5 g/L ovalbumin, the crystal edges arising from the obtuse steps were markedly rounded and the edges from the acute steps remained relatively straight. As the concentration was increased to 2 g/L, the rounding of the former was enhanced and that of the latter was also visualized. Note that, besides the above asymmetry with respect to the crystal edges, the most significant morphological modification occurred at the obtuse-obtuse corner. In fact, the crystal step rounding or roughening in the presence of ovalbumin was initiated at this corner, as noted below in our AFM studies. The above macroscopic differential effects of ovalbumin on the calcite habit were further demonstrated by in situ AFM assessment, where earlier events after the introduction of ovalbumin were observed at the nanometer level. As shown in Figure 3a, the crystal growth at a (104) face of existing calcite crystal was well represented by the advancement of monomolecular straight steps comprising the growth hillock in the absence of any impurities, after the introduction of the super-
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Figure 3. Growth hillocks of calcite before and after the introduction of ovalbumin (0.044 g/L): (a) before ovalbumin addition; (b) 10 s, (c) 5 min, (d) 10 min, (e) 13 min, and (f) 15 min after ovalbumin addition. All AFM images are shown in the same orientation as part a with two obtuse steps at the top of the image. The AFM image sizes are as follows: (a) 10 µm × 10 µm; (b) 5 µm × 5 µm; (c) 5 µm × 5 µm; (d) 5 µm × 5 µm; (e) 5 µm × 5 µm; (f) 2 µm × 2 µm. (g) Schematic cross section of the (104) terrace showing differences in step structure.
saturated solution. Note that such a growth hillock is often initiated at dislocations,31 and the dislocation in this case had a Burgers vector of 2. As indicated above in Figure 1a, the growth of pure calcite produced rhombohedral crystals with six crystallographically equivalent {104} faces. The advancement of the growth hillock on an existing (104) face adequately reflected this rhombohedral calcite morphology, with two steps acute to the cleavage plane and two steps obtuse to the cleavage plane (Figure 3a and g), thus forming a rhombus.9,10 The two pairs of structurally distinct steps are related to one another by C-glide plane steps, as shown in Figure 3a. All AFM images in Figure 3 were oriented in such a way that the two obtuse steps were always shown in the upper part of the images. Upon introduction of 0.0443 g/L (1 µM) ovalbumin, the hillock morphology was significantly altered (Figure 3b and c, with Figure 3a as a reference). Consistent with the above SEM characterization, the AFM characterization also revealed that the morphological alteration was initiated at the obtuse-obtuse
Wang et al. corner (Figure 3b) and this alteration was anisotropic with respect to the steps (Figure 3c-f). The step roughening (mainly “bunching”14) first occurred at the intersection of the obtuse step edges at the hillock corner, as revealed by the image (Figure 3b) collected after 10 s exposure to ovalbumin. Within the first 5 min, the propagation of step bunching primarily occurred along the obtuse step at the [4j41] direction (Figure 3c). After 13 min of exposure to ovalbumin, the obtuse steps at both the [4j41] and the [481j] directions became roughened (Figure 3e). As the exposure time further increased, there was no apparent alteration in the step morphology. Although the magnified AFM image at 13 min indicated a bit of roughening occurred at the acute steps (primarily at the [4j41] direction), they remained relatively straight. The above anisotropic effect as to the steps of calcite crystals has been demonstrated by several studies in the presence of acidic amino acids, peptides, and proteins.9-15,32 For instance, Orme and colleagues have revealed that addition of aspartic acids induced morphological changes in the acute growth steps of calcite crystals and the hillocks formed in the presence of Dand L-aspartic acids were the mirror images of one another.9,10 In the study by Elhadj et al.,11,32 a series of poly-L-aspartates (Asp1-6) was tested with respect to their effects on the morphology and growth kinetics of calcite crystals. They have demonstrated that Asp1,2 had stronger effects on the acute step edges and a crossover occurred for the longer Asp4,5,6 peptides that preferentially influenced the obtuse steps. Their molecular modeling indicated that longer Aspn (n > 2) peptides displaced more water molecules adsorbed at the acute step edges than at the obtuse step edges; as a result, more energy “penalty” was required for Asp3,4,5,6 to adsorb at the acute steps than at the obtuse steps, therefore favoring Asp3,4,5,6 binding to the obtuse steps. As an acidic protein with an isoelectric point of around pH 4.5, ovalbumin is composed of a polypeptide chain containing 386 residues and the constituent acidic amino acid residues (14 Asp and 33 Glu residues) are much more in the number than the basic residues (20 Lys and 15 Arg residues).33 Thus, it can be readily expected that this protein behaved just as longer Aspn (n > 2) peptides, i.e., preferentially adsorbing on the obtuse steps and therefore affecting the morphology and growth kinetics of the obtuse steps. The work of Elhadj et al. has shown that the overall growth kinetics is closely related to the additive concentration and its molecular size.11 Specifically, they showed that there was a critical concentration for the additive (C0) below which increase in calcite growth was induced and above which the inhibitory effect occurred. Furthermore, they demonstrated that C0 decreased with increasing molecular size. For example, C0 is around 100 and 1 µM for Asp2 and Asp6, respectively, possibly due to the increasing binding strength. In contrast to the Aspn series, ovalbumin is a much larger molecule. It contains more acidic amino acid residues and other potential functional residues. It is thus expected that this protein is a more potent step inhibitor. Note that the above effects generated by ovalbumin are closely related to its concentration. For instance, at the lower concentration of 0.1 µM, there was no obvious variation observed in the morphology of steps. At the higher concentrations around 10 µM, morphological variations occurred too fast for us to observe the dynamic processes. There are four models to depict the step modifications by additives: (1) step pining, (2) incorporation, (3) kink poisoning, and (4) surfaction.9,10,12,34,35 Thus, our finding of ovalbumin concentration dependent effects on the bunching of the obtuse steps and their growth modifica-
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Figure 4. Schematic illustration of the (a) {104}, (b) {012}, (c) {110}, and (d) {018} faces of calcite. Within the {104} face, the directions of [4j41], [481j], [010], and [421j] were indicated as dashed lines. Note the distances between the two adjacent calcium ions along the above four directions are 6.425, 6.425, 4.990, and 4.048 Å, respectively. The {012} face is only composed of a single component layer of calcium or carbonate ions, and the {110} and {018} faces are composed of equal Ca and CO3 groups.
tion are consistent with the step pining mechanism where the additive must adsorb onto the step edge, thereby hindering the step advancement and producing dead zones, causing complete blocking of growth in some step segments. Because the critical length Lc scales inversely with the supersaturation σ,9 the relatively high supersaturation (σ ) 2.0) in this case favored the advancement of macrosteps but the newly expressed macrosteps displayed the “bunching” feature. The adsorption of ovalbumin at the step edges thus caused modification of the step growth in terms of both morphology and kinetics. Macroscopically, several new faces in addition to the {104} face were developed, evident from the SEM characterization (Figure 2). They mainly correspond to the {110}, {012}, and {018} faces. As shown in Figure 4b, the {012} face is only composed of a single component layer of calcium or carbonate ions, including the (11j2), (102j), and (012) faces in this case (Figure 2e). The (102j) face intersects the {104} face at the [010] direction, as indicated in Figure 4a, and this direction is perpendicular to the C-gliding plane, with only Ca atoms or CO3 groups being exposed along this direction. As a result, the direction should be stabilized by acidic ovalbumin through electrostatic interaction. The stack of the step edges along this direction leads to the formation of the (102) face. Such a mechanistic interpretation is well supported by the AFM image collected at 10 s of exposure to ovalbumin (Figure 3b). The (11j2) and (012) faces intersect the (104) face at the [421j] direction (parallel to the C-gliding plane). The Ca-Ca distance along this direction is 4.048 Å, smaller than that (4.99 Å) along
the [010] direction. As a result, the step along this direction is likely to possess a higher step edge free energy. This energetic discrepancy could partly explain why little modification occurred at the obtuse-acute step corners relative to the obtuse-obtuse corners at the relatively low ovalbumin concentration of 0.0443 g/L (1 µM), as revealed by AFM (Figure 3). As the additive concentration was increased to 0.5 and 2 g/L, the [421j] direction should be well developed and the stack of the step edges along this direction led to the formation of the (11j2) and (012) faces. The {110} face includes the (110) and (21j0) faces, and the {018} face is composed of the (11j8) and (018) faces. These {110} and {018} faces all intersect the {104} face at the [4j41] and [481j] directions, respectively. The Ca-Ca distance along the two directions is the same as 6.4254 Å, and the C atom is intermediate between the two Ca atoms. Consequently, the step edge energy along the two directions is low and they are normally developed with the {104} face. Moreover, the two faces are composed of equal Ca and CO3 groups and are thus not dipolar like the {012} face, as evident from Figure 4c and d. As suggested by Teng et al., the mechanistic basis for the formation of this kind of faces is not apparent.36 Their formation is partly related to the stereochemical and geometric constraints,37 caused by the combination of other unstable directions or faces.36 As shown in Figure 3f, although the acute steps are relatively straight, the small jagged appearance might provide a clue for the occurrence of some unusual directions. The detailed mechanism as for the occurrence of {110} and {018} faces needs to be further explored. However, it can be expected
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Figure 5. TEM images and corresponding SAED patterns (insets) of CaCO3 precipitates collected 1.5 h after the start of the mineralization process in the presence of 10 g/L ovalbumin: (a) amorphous CaCO3 nanoparticles; (b) an irregular crystalline CaCO3 plate.
that decreasing the ovalbumin concentration and/or the supersaturation to suitable concentration range would facilitate their formation. Lysozyme is another egg white protein (representing 3.5% of the egg white) but with a high isoelectric point at around pH 11.0. Although its addition also modified the morphology of calcite crystals as shown in Figure 1b, this modification was virtually of little significance when compared with that in the presence of ovalbumin. No rounding occurred along the crystal edge, and the inhibition in the growth edge seems to be highly discontinuous. Virtually, the {110} faces as revealed in our previous study correspond to the limited inhibition of the crystal edges.28 In addition to the step modification in the presence of ovalbumin, the AFM characterization also revealed the emergence of some circular islands (diameters less than 100 nm) on terraced surfaces, as shown in Figure 3. These islands are randomly distributed on the terraces. Such a phenomenon has been observed in the presence of other acidic proteins and peptides. These islands have been suggested to be amorphous calcium carbonate (ACC)-protein/peptide aggregates.14 The emergence of ACC in the presence of ovalbumin was further demonstrated by TEM characterization. TEM results showed that CaCO3 nanoparticles with a size range of 20-50 nm primarily formed after 1.5 h of mineralization in the presence of 10 g/L ovalbumin (Figure 5a). These nanoparticles were either connected or discrete, and more importantly, they are amorphous in nature, as evident from the corresponding SAED patterns (inset in Figure 5a). In addition, a few irregular plates were also observed in this early stage. They are mainly single crystals despite bearing a low degree of crystallinity, as shown in Figure 5b. In contrast, calcite rhombohedra were mainly observed at
Wang et al. 1 h after the start of the biomineralization process from TEM and SAED (data not shown) in the absence of ovalbumin. In fact, ACC in the pure form is highly unstable and very quickly transforms into the crystalline forms (typically within minutes).38,39 However, some organisms can produce stable biogenic ACC and the above phase transformation has been demonstrated to considerably retard in the presence of some acidic biomolecules such as ovalbumin.8,38,39 Hence, it is most likely that organisms exploit this phase as the transient precursor to generate the complicated morphologies.8,38 The XRD pattern of CaCO3 precipitates collected at 12 h in the presence of 0.5 g/L ovalbumin is shown as curve A in the left panel of Figure 6, indicating that the crystal produced in this case is exclusively composed of crystalline calcite. The corresponding FTIR spectroscopy is also given in Figure 6 (right panel, curve A). Apart from the pronounced absorption peak at 712 cm-1 that is unique to the calcite crystal,40 there is no other characteristic peak of other phases of CaCO3, suggesting again that the collected precipitates consist exclusively of calcite crystals. As the concentration of ovalbumin was increased to 2 g/L, however, the XRD analysis (Figure 6, left panel, curve B) indicates that there is a minor amount of vaterite phase (less than 5%) in addition to calcite, the dominant crystalline precipitates. The corresponding FTIR spectroscopy (Figure 6, right panel, curve B) is also consistent with the XRD analysis: except for the pronounced peak at 712 cm-1, there is a weak broad absorption at 745 cm-1, which is the characteristic peak of crystalline vaterite.40 Anhydrous crystalline CaCO3 has three polymorphs: vaterite, aragonite, and calcite. Calcite is the most thermodynamically stable, while vaterite is the least stable. Being inherently unstable and highly soluble, vaterite may not be abundant in Nature. However, this phase has been reported to exist for long periods in some biological systems.41-43 It is widely perceived that the kinetic stabilization of vaterite is achieved primarily through the involvement of acidic macromolecules such as proteins and polysaccharides. In a recent study, we have demonstrated that ovalbumin from chicken egg white displayed a distinguished stabilizing effect on the vaterite phase.39 Virtually, vaterite can be readily produced by the rapid mixing of calcium and carbonate solutions under the high degree of supersaturation at the ambient temperature, but it is usually transformed into calcite within the first few hours if not leaving the liquor. In the presence of 2 g/L ovalbumin, some microspheres of vaterite were still present in the mother liquor at 24 h of crystallization. We attributed this stabilization effect to ovalbumin adsorption onto the unstable crystal surface.39 As indicated above, because
Figure 6. (left) XRD patterns and (right) FTIR spectra of CaCO3 precipitates that were produced after 12 h in the presence of (A) 0.5 g/L ovalbumin and (B) 2 g/L ovalbumin, respectively. Notes: C-calcite (JCPDS: 05-0586), V-vaterite (JCPDS: 33-0268).
Influence of Ovalbumin on CaCO3 Precipitation
Figure 7. Typical calcitic twins formed in the presence of (a) 0.5 and (b) 2.0 g/L ovalbumin. These crystals were collected after 12 h of crystallization. The scale bars are 10 µm.
the rate of (NH4)2CO3 vapor diffusion was slow and the overall supersaturation is relatively low in this case, the precipitate is exclusively composed of calcite in the absence of any additive. As an acidic protein, ovalbumin could capture Ca2+ through carboxylates at the periphery of the protein (Ca2+ has a relatively higher “complex binding constant” to carboxylate groups than other conventional metal ions such as Na+ and Mg2+ 44), thereby leading to a local enrichment of calcium ions. Upon introduction of CO32- and diffusion into the solution, their distributions would be enriched by Ca2+ ions around the protein. According to the Ostwald rule, the metastable crystalline form may tend to precipitate under the higher local supersaturation, resulting in the production of vaterite crystals around the protein molecules. Furthermore, ovalbumin will adsorb onto the surface of vaterite crystals upon their formation, thereby retarding its transformation into more stable crystalline forms. In contrast, the presence of lysozyme was found to favor the formation of calcite and the influence is also proportional to the protein concentration.27 As lysozyme is positively charged under the present experimental conditions and does not concentrate Ca2+ ions locally, it must lower the “effective” supersaturation by binding with diffused carbonate ions through electrostatic interaction. It is also very interesting to note that, in the presence of ovalbumin, some calcite particles were observed to be interconnected as twins, as shown in Figure 7. This situation is similar to the phenomenon observed in the formation of vaterite microspheres in the presence of ovalbumin.39 The twinning could be related to the bridging between the soft ovalbumin layers bound on the two crystal surfaces, but the detailed mechanism needs to be investigated further. 4. Conclusions This work, undertaken by combining AFM, SEM, and TEM measurements under in Vitro biomimetic conditions, has demonstrated that ovalbumin, a key component of eggshell protein matrix, was effective at mediating CaCO3 precipitation in terms of morphology, growth kinetics, and unstable phases. The effects of ovalbumin on calcite growth and morphology were anisotropic. The AFM analysis revealed that this protein preferentially interacted with the obtuse rather than the acute steps on the (104) calcite face, causing obvious morphological modification and relative growth inhibition in the obtuse steps. The difference could be caused by the lesser amount of displacement of adsorbed water molecules along the obtuse steps when interacting with ovalbumin. Similarly, SEM also exhibited significant morphological modification and growth inhibition along the obtuse steps. On the other hand, due to the adsorption of ovalbumin at the step edge, some new directions were developed and several unusual faces were expressed as a result of the stacking of the step edges along these new directions. ACC is usually difficult to be observed due to its high instability. In the presence of ovalbumin, however, it was readily
J. Phys. Chem. B, Vol. 114, No. 16, 2010 5307 observed in the form of nanoparticles or nanoclusters. In addition, even under low supersaturation, unstable crystalline vaterites were also produced, whose transformation into the more stable calcites was significantly retarded in the presence of ovalbumin. This inhibitory effect is attributed to the enriching of calcium ions around ovalbumin, and the stabilization of the unstable ACC phase upon its formation could well arise from electrostatic interaction. Understanding the mechanistic processes of ovalbumin modifications to calcite growth could well be transformed into technologies for controlling ACC and vaterite phases and generating active hybrid biomaterials. References and Notes (1) Simkiss, K.; Wilbur, K. M. Biomineralization: Cell Biology and Mineral Deposition; Academic Press: San Diego, CA, 1989. (2) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: Oxford, U.K., 1989. (3) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: New York, 2001. (4) Aizenberg, J.; Tkachenko, A.; Weiner, S.; Addadi, L.; Hendler, G. Nature 2001, 412, 819–822. (5) Weiner, S.; Addadi, L. J. Mater. Chem. 1997, 7, 689–702. (6) Meldrum, F. C. Int. Mater. ReV. 2003, 48, 187–224. (7) Addadi, L.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4110– 4114. (8) Xu, A. W.; Ma, Y. R.; Co¨lfen, H. J. Mater. Chem. 2007, 17, 415– 449. (9) Teng, H. H.; Dove, P. M.; Orme, C. A.; DeYoreo, J. J. Science 1998, 282, 724–727. (10) Orme, C. A.; Noy, A.; Wierzbicki, A.; McBride, M. T.; Grantham, M.; Teng, H. H.; Dove, P. M.; DeYoreo, J. J. Nature 2001, 411, 775–779. (11) Elhadj, S.; Salter, E. A.; Wierzbicki, A.; DeYoreo, J. J.; Han, N.; Dove, P. M. Cryst. Growth Des. 2006, 6, 197–201. (12) Fu, G.; Qiu, S. R.; Orme, C. A.; Morse, D. E.; DeYoreo, J. J. AdV. Mater. 2005, 17, 2678–2683. (13) Thompson, J. B.; Paloczi, G. T.; Kindt, J. H.; Michenfelder, M.; Smith, B. L.; Stucky, G.; Morse, D. E.; Hansma, P. K. Biophys. J. 2000, 79, 3307–3312. (14) Kim, I. W.; Darragh, M. R.; Orme, C.; Evans, J. S. Cryst. Growth Des. 2006, 6, 5–10. (15) Delak, K.; Giocondi, J.; Orme, C.; Evans, J. S. Cryst. Growth Des. 2008, 8, 4481–4486. (16) Nys, Y.; Hincke, M. T.; Arias, J. L.; Garcia-Ruiz, J. M.; Solomon, S. E. AVian Poult. Biol. ReV. 1999, 10, 142–166. (17) Arias, J. L.; Fink, D. J.; Xiao, S.; Heuer, A. H.; Caplan, A. I. Int. ReV. Cytol. 1993, 145, 217–250. (18) Panheleux, M.; Bain, M.; Fernandez, M. S.; Morales, I.; Gautron, J.; Arias, J. L.; Solomon, S. E.; Hincke, M.; Nys, Y. Br. Poult. Sci. 1999, 40, 240–252. (19) Gautron, J.; Hincke, M. T.; Nys, Y. Connect. Tissue Res. 1997, 36, 195–210. (20) Nys, Y.; Gautron, J.; Garcia-Ruiz, J. M.; Hincke, M. T. C. R. PaleVol 2004, 3, 549–562. (21) Hincke, M. T.; Bernard, A. M.; Lee, E. R.; Tsang, C. P. W.; Narbaitz, R. Br. Poult. Sci. 1992, 33, 505–516. (22) Hincke, M. T.; Gautron, J.; Panhe´leux, M.; Garcia-Ruiz, J. M.; McKee, M. D.; Nys, Y. Matrix Biol. 2000, 19, 443–453. (23) Arias, J. L.; Carrino, D. A.; Ferna´ndez, M. S.; Rodrı´guez, J. P.; Dennis, J. E.; Caplan, A. I. Arch. Biochem. Biophys. 1992, 298, 293–302. (24) Nys, Y.; Gautron, J.; Garcia-Ruiz, J. M.; Hincke, M. T. C. R. PaleVol 2004, 3, 549–562. (25) Rose, M. L. H.; Hincke, M. T. Cell. Mol. Life Sci. 2009, 66, 2707– 2719. (26) Hincke, M. T. Connect. Tissue Res. 1995, 31, 227–233. (27) Addadi, L.; Moradian, J.; Shay, E.; Maroudas, N. G.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 2732–2736. (28) Wang, X.; Sun, H.; Xia, Y.; Chen, C.; Xu, H.; Shan, H.; Lu, J. R. J. Colloid Interface Sci. 2009, 332, 96–103. (29) Gehrke, N.; Co¨lfen, H.; Pinna, N.; Antonietti, M.; Nassif, N. Cryst. Growth Des. 2005, 5, 1317–1319. (30) Kitamura, M. J. Cryst. Growth 2002, 237-239, 2205–2214. (31) Teng, H. H.; Dove, P. M.; DeYoreo, J. J. Geochim. Cosmoschim. Acta 2000, 64, 2255–2266. (32) Elhadj, S.; DeYoreo, J. J.; Hoyer, J. R.; Dove, P. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19237–19242. (33) Stein, P. E.; Leslie, A. G.; Finch, J. T.; Carrell, R. W. J. Mol. Biol. 1991, 221, 941–959.
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