Nanostructure of Biogenic Calcite Crystals: A View by Small-Angle X

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Nanostructure of Biogenic Calcite Crystals: A View by Small-Angle X-Ray Scattering Christoph Gilow,† Emil Zolotoyabko,†,‡ Oskar Paris,§ Peter Fratzl,† and Barbara Aichmayer*,† †

Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany Department of Materials Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel § Institute of Physics, Montanuniversitaet Leoben, 8700 Leoben, Austria ‡

bS Supporting Information ABSTRACT: One of the most fascinating topics currently being discussed in the field of biomineralization is the occlusion of organic macromolecules within mineral crystals. It is already known that intracrystalline organic inclusions in biogenic calcite improve the fracture behavior and anisotropically distort the calcite lattice. However, the detailed structure of the crystals and the underlying processes leading to the incorporation of the organic molecules are poorly understood. In this work, we investigate calcite prisms extracted from the shell of Pinna nobilis by means of three-dimensional synchrotron small- and wide-angle X-ray scattering (SAXS/WAXS). Organic
inorganic interfaces within the single crystals give rise to a strong, anisotropic SAXS signal. The results are shown as a stereographic projection of the integrated SAXS intensity (gray scale) together with the wide-angle spots (colored) of different calcite lattice planes. A comparison of native (left) and annealed (right) prisms, where the contrast for the latter is enhanced due to the removal of organics, shows a preferential orientation along the highly charged (001) lattice planes, which strongly interact with negatively charged aspartate groups of intracrystalline proteins. Our findings on the nanostructure help to understand how biogenic calcite crystals achieve their remarkable properties and thereby open up ways for the development of bioinspired hybrid materials.

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iogenic crystals produced by organisms have long been the focus of researchers due to their superior properties, which originate from the interplay between organic macromolecules and an intricate microstructure.1,2 Examples include outstanding mechanical,3 magnetic,4 and optical5 characteristics. The organisms exert a fascinating degree of control on the morphology,6 orientation,7 and polymorphism8
10 of biogenic crystals. Through the assistance of organic macromolecules guiding the biomineralization process, organisms can even precipitate both crystalline and amorphous forms of chemically similar minerals.11 The structure of biominerals and the processes by which these structures are created are of special interest for biomimetics, which is attempting to fabricate advanced materials by using routes and structural principles inspired by nature.12
14 For the design of novel materials, a deep understanding of the interaction between the mineral and organic substances on a nanometer scale and below is of fundamental importance. The interaction of organic molecules with specific mineral lattice planes has previously been studied using model systems, e.g., calcite nucleated on selfassembled monolayers (SAMs)15 or calcite with intracrystalline hydrogel inclusions,16 highlighting the importance of hydrophobicity/hydrophilicity as well as of stereochemical and geometrical effects. In this work, we investigate the nanostructure of biogenic calcite crystals from Pinna nobilis by means of 3-dimensional r 2011 American Chemical Society

small- and wide-angle X-ray scattering (SAXS/WAXS). Observing the orientation and structure of intracrystalline organic
mineral interfaces and their changes after a heat treatment allows us to shed new light on the interplay between organic inclusions and calcite. Calcite, a rhombohedral polymorph of calcium carbonate, which is thermodynamically stable at room temperature and normal pressure, is one of the most abundant biogenic minerals. Biogenic calcite is known to comprise intracrystalline organic macromolecules within individual crystallites, as it was first proposed for sea urchin skeleton parts.17 It was suggested that these organic macromolecules adhere to specific crystal planes,17 thus controlling the biomineralization process. Since then, other systems have also been found to contain intracrystalline macromolecules,18 such as calcitic prisms from the outer prismatic layer of the mollusk shells Pinna nobilis and Atrina rigida, both belonging to the Pinnidae family. The prisms are single crystals up to hundreds of micrometers in width and millimeters in length. The organic matrix (about 0.3 wt %) within the prisms of Atrina rigida was found to comprise a network of β-chitin fibers with a diameter Received: January 27, 2011 Revised: May 1, 2011 Published: May 04, 2011 2054

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Figure 1. Axonometric projection of the calcite lattice perpendicular to the c
a plane (hexagonal notation). The straight lines indicate traces of the (001), (104), and (
102) planes. The (001) and (
102) planes contain pure sublayers of positively charged Ca-ions, whereas the (104) plane is electrically neutral.

of 15
20 nm and highly acidic proteins, with the latter being assumed to strongly interact with the mineral and to partially bind to β-chitin.19 Some of the proteins extracted from calcitic prisms, such as Asprich in Atrina rigida19 and Caspartin in Pinna nobilis,20 have more than 50% aspartic acid residues in their sequences. As will be discussed later on, this highly acidic nature of the proteins is of major relevance for the specific interactions between the growing mineral and proteins during biomineralization. Despite containing a significant amount of intracrystalline organics, sea-urchin skeletal parts as well as calcitic prisms extracted from mollusk shells scatter X-rays as nearly perfect single crystals, providing well-defined diffraction spots17,21,22 with a low mosaicity (about 0.03° for the prisms extracted from Atrina serrata).21 The measured widths (fwhm) of the synchrotron powder diffraction peaks of the biogenic calcite are about 0.01°,21,23 which corresponds to crystallite sizes up to 700 nm.23 High-resolution synchrotron X-ray diffraction measurements with powdered specimens of calcitic prisms extracted from Pinna nobilis and Atrina rigida revealed anisotropic lattice distortions as compared to geological or synthetic calcite.24 The maximum distortion (up to 0.24% in Pinna nobilis25) was found along the c-axis (hexagonal notation). Besides that, significant changes in the C
O bond length (up to 0.7% in Pinna nobilis) were detected by high-resolution neutron powder diffraction.25 It was assumed that the observed structural changes are the result of interaction between organic macromolecules and the growing mineral.26 In fact, the lattice distortions are effectively relieved after mild annealing for 0.5 h at temperatures above 200 °C, during which intracrystalline organics are defragmented and partially removed from the calcitic prisms.24 In addition to the decomposition of macromolecules, other processes may occur in this temperature range, such as water elimination and possibly near surface crystal reconstruction. This relaxation process is accompanied by a drastic (two-three times) broadening of diffraction peaks and a respective decrease in the sizes of crystal blocks which coherently scatter X-rays.23 The maximum broadening effect was found for

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the (006) and (012) diffraction peaks. Note that the (001)- and (012)-types of crystallographic planes (including the structurally equivalent (
102)- and (1
12)-planes) are the only planes in the calcite structure which contain pure sublayers of positively charged Ca-ions (see Figure 1). Therefore, the (001)- and (012)planes in calcite are believed to be natural places for the attachment of acidic proteins (comprising negatively charged residues) to the mineral (or vice versa). These findings and related considerations led to a model of interconnected, coherently scattering calcite unit cells deposited along complex trajectories, which are guided by chitin fibers coated by acidic proteins.26 However, the nanostructure of native calcitic prisms and, in particular, the orientation of the internal organic/inorganic interfaces with respect to the calcite lattice remains unaddressed. In this work, we investigate calcitic prisms extracted from the shells of Pinna nobilis. As our main experimental technique, we use a combination of small-angle X-ray scattering (SAXS), which is sensitive to variations in the electron density at the organic
inorganic interfaces,27,28 and X-ray diffraction (WAXS) within the same experimental setup at the BESSY II storage ring to study individual calcitic prisms. Orientation-averaged information on the nanostructure of internal interfaces was first obtained from laboratory SAXS measurements (for details, see the Supporting Information) with powdered specimens annealed for 1 h at different temperatures ranging from 50 to 300 °C (Figure 2a). No change was observed with respect to native prisms for any heat treatment up to 50 °C. All curves show a typical behavior with a linear intensity drop (on a log
log scale) at large Q-values, with Q being the modulus of the scattering vector. The slope of the intensity decay at higher Q-values gives information on the structure of the organic
inorganic interfaces. For systems comprising homogeneous phases and well-defined sharp interfaces (i.e., when the jumps of the electron density across the interfaces can be described by stepfunctions), the SAXS intensity exhibits the so-called Debye
Porod law in the large Q limit, where the intensity decays as Q
4.28
30 Positive deviations (i.e., slopes
4 < h <
3) indicate density fluctuations within the phases31 or the presence of rough surfaces with a fractal dimension of D = 6
h.32,33 In native calcitic prisms, the SAXS intensity at large Q-values decays as Q
3.4, corresponding to a fractal dimension of 2.6 (see Figure 2b). Annealing at temperatures of 250 °C and higher results in the emergence of a Porod regime with a slope equal to h =
4 (see Figure 2b), indicating predominantly sharp interfaces. The crystallographic orientation of the organic
inorganic interfaces with respect to the single crystalline calcite lattice was studied by synchrotron SAXS/WAXS measurements (for details, see the Supporting Information) at the μ-Spot beam line of the BESSY II storage ring (Helmholtz
Zentrum Berlin).34 Simultaneous detection of the SAXS signal and diffraction peaks as a function of specimen rotation allowed us to find the orientation of the SAXS intensity distribution with respect to certain crystallographic directions of the calcite lattice. Typical 2D SAXS signals before and after annealing at 300 °C are shown in Figure 3a and b. The SAXS signal is strongly anisotropic, and this anisotropy is drastically changed after annealing. Native prisms reveal a SAXS streak toward the (104)-node of the reciprocal space and an additional, less pronounced streak in the [001]-direction (see Figure 3a). Quantitative data on the intensity levels are given below the corresponding 2D scattering patterns in Figure 3c and R d, showing the integral SAXS intensity ( I(Q)Q2 dQ) integrated over the Q-range 0.3
3.3 nm
1, as a function of the azimuthal 2055

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Figure 2. (a) Spherically averaged SAXS signal from Pinna nobilis prisms annealed at temperatures between 50 and 300 °C, plotted against Q using logarithmic scales. The corresponding annealing temperature is indicated below each curve. (b) The slope h of the linear region at high Q values for a native sample (room temperature, without annealing) and 2
3 samples after annealing at different temperatures is plotted against the temperature. Before annealing, the signal exhibits a slope of h =
3.4, indicating the absence of a Porod regime. Annealing up to 300 °C leads to a transition of the slope to h =
4, which corresponds to a Porod regime indicating the emergence of smooth interfaces.

angle χ. After annealing, the strongest SAXS streak is toward the [001]-direction, while a weak additional intensity remains along the directions distributed between the (104)- and (202)-nodes of the reciprocal space (see Figure 3b). For a better visualization of the orientation distribution of the Q-integrated SAXS intensity, we have produced pole figures, where the [001]-direction of calcite is chosen as the stereoprojection axis (see Figure 3e and f). In order to be able to mark specific nodes of the reciprocal lattice, we combined the stereoprojection of the integral SAXS intensity distribution with the rescaled projections of relevant diffraction peaks. The green dot in the center indicates the calculated position of the (006)-diffraction peak, which cannot be measured in the particular experimental geometry. Native prisms produce a SAXS signal which reflects the 3-fold symmetry of the calcite lattice (about the c-axis) with pronounced streaks aligned toward the (104)-, (0
14)-, and (
114) nodes of the reciprocal lattice (see Figure 3e). A broad and much weaker maximum in the SAXS intensity also appears in the center of the stereoprojection close to the (001)-position. After annealing at 300 °C, the SAXS intensity was drastically redistributed, while still preserving the 3-fold symmetry (see Figure 3f). Now the predominant feature is a strong and sharp intensity maximum in the center of the stereoprojection, which corresponds to the SAXS streak in the [001]-direction. Due to the experimental geometry, the (006) reflection was not excited; the green dot at the pole indicates the calculated position of the c-axis. The artifact visible as a slightly tilted line in the stereographic projection is caused by slight deviations in the normalization over the course of the experiment and is not relevant for the discussion of the results. The untreated prisms exhibit a SAXS signal with streaks toward the (104)-type nodes of the reciprocal lattice (Figure 3e). Upon annealing, the signal changes and a strong streak can clearly be attributed to the (001) direction (Figure 3f). A weaker anisotropic signal is smeared within three zones, which include the (001)-, (104)-, and (101)-planes, corresponding to the [010] zone axis, as well as symmetry-equivalent crystallographic directions (Figure 3f). In addition to these changes in the distribution of the SAXS intensity, we found an annealing-induced broadening of the (104)and (202)-diffraction spots normal to the corresponding radial directions in the collected 2D scattering images. This indicates an

increased mosaicity (up to 5°) of individual crystallites within the calcitic prisms as a result of the heat treatment. However, as can easily be seen when comparing Figure 3e and f, these changes in the crystallite orientation (WAXS) are much less pronounced than the changes in the distribution of the SAXS signal, with the latter reflecting the modified structure of internal interfaces within the prisms. The key experimental finding, that is, an appearance of a much stronger SAXS streak in the [001]-direction as a result of annealing, is explained as follows. It was already mentioned that the (001)planes (basal planes) in calcite contain pure sublayers of positively charged calcium ions (see Figure 1). Hence, it is very reasonable to assume that these ions act as preferred binding sites for the negatively charged residues in the acidic proteins extracted from prisms of Pinna nobilis.20 After annealing at elevated temperatures, the intracrystalline organics are partially removed from the [001]-oriented interfaces. This is clearly seen as a substantial intensity increase of the (001)-streak in the SAXS signal (see Figure 3b and f) after annealing due to an increased scattering contrast. In fact, the intensity contrast in SAXS is proportional to the square of the difference between the electron densities of the system’s components.30 By using structural data for calcite (e.g., ref 25) and crystalline β-chitin,35 we find the electron densities (in electron units, e) to be 0.815 and 0.511 e/Å3, respectively. The typical electron density of proteins is around 0.42 e/Å3 (ref 36) and, hence, similar to the one of chitin. Removing intracrystalline organics and, consequently, forming calcite
air interfaces will drastically increase the SAXS contrast by a factor between 4 and 7, depending on which fraction of intracrystalline organics (chitin or proteins) is preferably removed. The removal of the organic phase also leads to a smoothening of the internal interfaces, as demonstrated by the SAXS studies with powdered samples (see Figure 2). The corresponding transition temperature coincides exactly with that one found before for the relaxation of lattice parameters (see ref 23). Since the latter process is driven by partial removal of intracrystalline organics from calcitic prisms, it seems likely that the removal of organics also causes the interface smoothening after annealing at elevated temperatures. Besides that, the annealing also leads to an increase in the angular misorientation (up to 5°) between crystalline calcite blocks, as revealed by the broadening of the WAXS spots 2056

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Figure 3. (a and b) Typical SAXS signals for calcitic prisms of Pinna nobilis before (a) and after (b) annealing at 300 °C. The colored arrows point into the direction of the positions of the indicated WAXS peaks. Prior to annealing, the anisotropy of the signal is mainly related to the (104) peaks. This is greatly changed after annealing, where a clear correlation between the SAXS signal and the (001) orientation can be found. BS indicates the shadow of a capillary used to mount the beamstop. (c and d) show the corresponding integral SAXS intensity, integrated between Q = 0.3 and 3.3 nm
1 as a function of the azimuthal angle χ. Vertical lines indicate the angular positions of WAXS peaks, and the yellow rectangle indicates the region affected by the beam stop. (e and f) Stereographic projection of the integral SAXS intensity (Q = 0.3
3.3 nm
1, gray scale) along the (001) direction of an isolated calcitic prism before (e) and after (f) annealing at 300 °C. The corresponding stereographic projections for the Bragg reflections are overlaid in red for the (104) reflection (20.6
20.9 nm
1) and in yellow for the (202) reflection (29.8
30.3 nm
1).

(Figure 3f). Only in the native state do the the calcite prisms scatter like single crystals (mosaicity less than 1°). The concentration of SAXS intensity toward the {104}-type nodes (see Figure 3e) before annealing and the redistribution of SAXS intensity between planes entering the [010]-zone axis (as well as the symmetry-equivalent directions) are less clear. In fact, the (104) plane of calcite (see Figure 1) is composed of successive positively charged calcium ions and negatively charged carbonate groups, and thus, it is electrically neutral. Due to this fact, it has the lowest surface energy and serves as a prominent cleavage plane in geological calcite. Therefore, it is difficult to imagine that the (104)-planes can provide binding sites for proteins containing negatively charged residues. For this reason, we consider possible arrangements of differently oriented facets which would produce a SAXS signal similar to the one of a planar (104)-oriented interface, as long as the

facets are smaller than the coherence length of the used (synchrotron) X-rays. Hence, also taking into account previous findings based on high resolution X-ray diffraction23 that highlighted the importance of both the (001)- and (012)-type planes, we suggest the following plausible scenario: A part of the organic
inorganic interfaces follows the appropriate (001)- and (012)types of planes, which contain pure sublayers of positively charged calcium ions, but on average the resultant interface “trajectories” coincide with the traces of the (104)-planes. In other words, we suggest that part of the organic/inorganic interfaces in calcitic prisms have complex zigzag-type trajectories, comprising highly charged facets covered by organic matter. As a result, the average orientation of the SAXS signal in native prisms is aligned with the (104)-directions in reciprocal space. However, when the intracrystalline organics are partially removed, mainly from the (001)oriented interfaces, the remaining zigzag trajectories, still filled by 2057

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Crystal Growth & Design organics, change their effective orientation, while staying within the [010]-zone. The (001)-type interfaces, empty of organics (after annealing), produce a strong SAXS streak in the respective direction, as was mentioned above. In conclusion, the incorporation of organic macromolecules into single crystal calcite prisms occurs on well-defined (highly charged) crystallographic planes, mostly on basal planes of the (001)-type. These planes are well revealed in SAXS measurements only after annealing when the organic phase is partially destroyed, thus increasing the X-ray scattering contrast. The original coverage of these crystalline planes by intracrystalline organics also makes them fractally rough, as shown by SAXS measurements with powdered samples. This proves that the intracrystalline organics adhere primarily to the charged basal planes of the growing calcite lattice, in that way, most probably, governing the prismatic growth of individual crystallites.

’ ASSOCIATED CONTENT

bS

Supporting Information. Details of sample preparation, experimental setups (laboratory SAXS and synchrotron 3D SAXS/WAXS), data treatment, and reciprocal space reconstruction. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: þ49-331-567 9463. Fax: þ49-331-567 9402.

’ ACKNOWLEDGMENT We acknowledge Frederic Marin (Universite de Bourgogne, Dijon, France) for supplying calcitic prisms for this research. We also thank Anna Schenk, Stephan Siegel, Chenghao Li, and Ivo Zizak for their support and help at the μ-Spot beamline (BESSY II, Helmholtz-Zentrum Berlin) as well as Ingrid Zenke for support with operating the Nanostar instrument. Furthermore, we are grateful to Till Hartmut Metzger for helpful discussions.

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