Morphology and arrangement of the crystals in the calcitic layer

The elongated crystals constituting the calcite layer in the shell of both Pinna nobilis and Atrina rigida have a hexagonal prismatic shape typically measuring several hundreds of micrometers in length and 20–50 µm in diameter (Figure 2b–d).^{Reference Nudelman, Chen, Goldberg, Weiner and Addadi7} These prisms, whose crystallographic c-axes are oriented perpendicular to the shell surface^{Reference Berman, Hanson, Leiserowitz, Koetzle, Weiner and Addadi20} and thus coincide with the long axis of the elongated crystals, are embedded in an organic interprismatic matrix mainly composed of glycine-rich proteins.^{Reference Nudelman, Chen, Goldberg, Weiner and Addadi7} Recently, Bayerlein et al. demonstrated, using advanced synchrotron microtomography (Figure 2b), that the evolution of the prismatic layer in the bivalve Pinna nobilis can be fully described by classical grain growth models, where the organism controls the thermodynamic boundary conditions in terms of the mobility and surface energy of the calcite/matrix interface.^{Reference Bayerlein, Zaslansky, Dauphin, Rack, Fratzl and Zlotnikov21}

Each individual prism shows an x-ray diffraction pattern composed of narrow spots^{Reference Berman, Hanson, Leiserowitz, Koetzle, Weiner and Addadi20,Reference Gilow, Zolotoyabko, Paris, Fratzl and Aichmayer22,Reference Wolf, Lieberwirth, Natalio, Bardeau, Delorme, Emmerling, Barrea, Kappl and Marin23} with low mosaic spread.^{Reference Olson, Metzler, Tamura, Kunz, Killian and Gilbert19,Reference Berman, Hanson, Leiserowitz, Koetzle, Weiner and Addadi20} Astonishingly, while their scattering behavior can be associated with a nearly perfect single crystal of calcite, it has been found that the prismatic particles occlude around 0.3 wt% of intracrystalline organic material.^{Reference Nudelman, Chen, Goldberg, Weiner and Addadi7,Reference Metzger, Politi, Carbone, Bayerlein, Zlotnikov, Zolotoyabko and Fratzl24} Therefore, the incorporation of these impurities (soft occlusions) does not seem to have a detrimental effect on the continuity of the mineral lattice.

Microstructure of individual prisms

The intercalation of these organic molecules, however, significantly affects the internal structural architecture, and hence the mechanical properties of the prism.^{Reference Kunitake, Mangano, Peloquin, Baker and Estroff15} In order to understand the chemical and structural role of the “soft” occlusions, it is of great scientific interest to identify their location and distribution within the crystalline host material. On the length scale of micrometers, initial etching experiments, which are designed such that the mineral content is selectively removed, uncovered a fibrous network with alternating zones of higher and lower density of organic material in both Pinna nobilis ^{Reference Dauphin, Cuif, Doucet, Salome, Susini and Williams25} and Atrina rigida.^{Reference Nudelman, Chen, Goldberg, Weiner and Addadi7} These characteristic bands, which are aligned perpendicular to the c-axis of the prism, have been generally associated with an oscillatory growth pattern of the calcite crystals in the shell. In Pinna nobilis, Dauphin et al. could correlate this periodic topographic pattern with a chemical zonation by using wavelength dispersive spectroscopy and x-ray absorption near-edge spectroscopy to map the elemental distribution of magnesium and sulphur, where the latter is associated with sulphated organic species preferentially accumulated at the growth lines.^{Reference Dauphin, Cuif, Doucet, Salome, Susini and Williams25} Besides a water-soluble fraction of intracrystalline organic material, which is discussed in detail later, Nudelman et al. identified the polysaccharide chitin as a major component of the water-insoluble fibrous network penetrating Atrina rigida prisms (Figure 2e).^{Reference Nudelman, Chen, Goldberg, Weiner and Addadi7} Atomic force microscopy of polished sections^{Reference Dauphin, Cuif, Doucet, Salome, Susini and Willams26} and scanning electron microscopy (SEM) of partially etched prisms^{Reference Nudelman, Chen, Goldberg, Weiner and Addadi7} visualized mineral particles in the size range of ∼50 nm, which appeared to be tightly associated with the organic material (Figure 2f), thus indicating the nanocomposite character of the prismatic crystals.

Nanostructure and distribution of intracrystalline organics within single crystals

Significant research efforts have recently been directed toward the analysis of the three-dimensional volume structure of individual prisms with nanometer resolution. Nondestructive techniques are preferentially applied in order to avoid artifacts arising from sample preparation. Li et al. used annular dark field scanning transmission electron microscopy (ADF-STEM) and ADF electron tomography to visualize organic–inorganic interfaces within prismatic single crystals extracted from Atrina rigida. ^{Reference Li, Xin, Kunitake, Keene, Muller and Estroff27} Electron transparent slices cut along well-defined crystallographic directions of the prism were prepared by a focused ion beam, where the tomographic technique images the bulk structure of the composite material. The authors demonstrated that the occluded biomolecules were present in the form of isolated nanopatches (20–50 nm) distributed within a host of single crystalline calcite. These organic discs, which presumably correspond to aggregated biomacromolecules, were preferentially oriented perpendicular to the c-axis of the prisms, while their distribution in the basal plane was more uniform (Figure 2g–i).

Such an alignment of platelet-shaped accumulations of intracrystalline organics with the (001)-type planes of the calcite lattice has also been observed by Gilow et al., who studied individual prisms from the species Pinna nobilis by combined small-angle x-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS) under stepwise rotation of the crystal in a microfocused x-ray beam (Figure 3a–d).^{Reference Gilow, Zolotoyabko, Paris, Fratzl and Aichmayer22} This nondestructive technique provides quantitative three-dimensional structural information on both the nanometer-sized macromolecular occlusions (SAXS signal reflecting the diffuse scattering intensity around the origin of reciprocal space) and the calcite mineral lattice (diffraction spots in the WAXS regime) averaged over a comparably large sample volume in native (Figure 3a–c) and heat-treated (Figure 3b, d) prisms. In particular, orientation correlations between certain directions of the crystal lattice and the intensity distribution of the diffuse scattering signal arising from nanostructural heterogeneities could be identified. In these experiments, it was observed that pronounced intensity streaks in the SAXS signal, which correspond to directions of strong variations in electron density, are largely aligned with only two sets of lattice planes in the calcite unit cell. One set of streaks is aligned with the (001)-type planes, which are charged (as they are constituted from either calcium ions or carbonate ions in an alternating pattern), and the other set of streaks is aligned with low-energy neutral (104)-type planes. This observation suggests preferential adhesion of organic macromolecules specifically to these crystallite facets.^{Reference Gilow, Zolotoyabko, Paris, Fratzl and Aichmayer22}

Figure 3.

Nanostructure and lattice distortions in prismatic calcite from Pinna nobilis studied by x-ray scattering techniques. (a–d) Small- and wide-angle x-ray scattering (SAXS and WAXS) plots of individual calcite prisms. (a–b) Two-dimensional (2D)-detector images of simultaneously recorded SAXS and WAXS before (a) and after (b) annealing at 300°C. The SAXS signal (center of the 2D detector) is clearly anisotropic, and intensity streaks point toward the azimuthal position of specific Bragg spots (arrows), indicating non-homogenous incorporation of organic material aligned with the corresponding families of lattice planes. After annealing (decomposition of intracrystalline organics), the orientation of nanostructural features toward the [001]-direction of the mineral lattice becomes more pronounced. (c–d) Integral SAXS intensity plotted over the azimuthal angle χ before (c) and after (d) annealing. Drop lines indicate the angular position of (006) and (104) Bragg peaks. The yellow bar marks the region affected by the beamstop (BS). (e–f) Lattice parameters (a- and c-axes) calculated from high-resolution powder diffraction extracted from high-resolution powder diffraction data as a function of the annealing temperature. Significant anisotropic lattice distortions are seen with respect to a control sample. Mild annealing at temperatures above 200°C induces relaxation of the crystal lattice. (a–d) Reproduced with permission from Reference 22. © 2011 Wiley. (e–f) Reproduced with permission from Reference 14. © 2006 Elsevier.

Metzger et al. additionally performed a detailed investigation of the distribution of the diffuse scattering intensity near selected Bragg diffraction spots. While the reflections were isotropic in the pristine prisms, intense streaks along the [001]-direction appeared after mild annealing.^{Reference Metzger, Politi, Carbone, Bayerlein, Zlotnikov, Zolotoyabko and Fratzl24} In analogy to the SAXS-studies performed by Gilow et al., these experiments indicate that the charged (001)-lattice planes are particularly sensitive to structural modifications induced via the removal of the organic component by the heat treatment. Interestingly, the authors could correlate the observed rearrangement of the internal interfaces, which become notably smoother upon annealing, with a reduced hardness of the material in nanoindentation measurements. In this way, indirect evidence was provided that the presence of the intracrystalline biomolecules and the thus generated nanoscopic organic/mineral interfaces are of key importance for the mechanical behavior of the shell prisms.

Crystallographic structure in the mineral phase and chemical composition of intracrystalline organics

Based on high-resolution synchrotron powder x-ray diffraction data, Pokroy et al. have shown that the incorporation of intracrystalline organics affects the internal structure of mineral prisms considered herein down to the atomic scale, resulting in anisotropic distortions of the mineral lattice (as compared with geological calcite).^{Reference Pokroy, Fitch, Marin, Kapon, Adir and Zolotoyabko14} The observed strains were most pronounced along the c-axis of the calcite unit cell and relaxation occurred upon annealing, which led to the conclusion that thermally unstable organic molecules induce these significant lattice distortions (Figure 3e–f). Powder neutron diffraction further revealed a significant elongation of the carbon–oxygen bond length in Pinna nobilis as compared to its geological purely inorganic counterpart.^{Reference Zolotoyabko, Caspi, Fieramosca, Von Dreele, Marin, Mor, Addadi, Weiner and Politi28} The potential contribution of Mg²⁺ ions, which are often present in substantial amounts in biogenic calcite (2.65 wt% in Pinna nobilis),^{Reference Zolotoyabko, Caspi, Fieramosca, Von Dreele, Marin, Mor, Addadi, Weiner and Politi28} to the x-ray and neutron scattering signals was carefully corrected for by construction of calibration curves. In combination, these studies suggest that the intracrystalline biomacromolecules are tightly associated with the mineral phase, and their distribution with respect to the mineral lattice is nonuniform.

Numerous studies have addressed the chemical composition of intracrystalline organics. Besides the insoluble chitin matrix, as identified by Nudelman et al.,^{Reference Nudelman, Chen, Goldberg, Weiner and Addadi7} water-soluble proteins are involved in the biomineralization of mollusk shell calcite. The sequence of these macromolecules is now known to be particularly rich in amino acids bearing a negative charge such as aspartate and glutamate.^{Reference Evans29,Reference Marin, Amons, Guichard, Stigter, Hecker, Luquet, Layrolle, Alcaraz, Riondet and Westbroek30} Specifically, Marin et al. extracted two unusually acidic proteins—caspartin and calprismin—from calcite prisms of the species Pinna nobilis. In Atrina rigida, the Asprich family of acidic proteins was identified as a constituent of the soluble organic matrix present in the prismatic calcite crystals.^{Reference Gotliv, Kessler, Sumerel, Morse, Tuross, Addadi and Weiner31} It is widely believed that due to their chemical functionalization, acidic proteins are prone to electrostatic interaction with with charged planes of the calcite lattice, and are therefore often found aligned with the (001) faces of calcite crystallites. Wolf et al. has recently suggested the potential role of caspartin and calprismin in the formation of a transient amorphous mineral phase with liquid-like characteristics.^{Reference Wolf, Lieberwirth, Natalio, Bardeau, Delorme, Emmerling, Barrea, Kappl and Marin23}

Water-soluble additives: Large and small

The most accessible and prevailing strategy for the generation of bioinspired composite structures is the precipitation of the mineral in the presence of water-soluble organic additives—a concept that has been explored extensively over the last decades.^{Reference Yu and Cölfen52,Reference Gower53} Here, inspiration is taken from the soluble organic matrix identified in biogenic calcite. Additives frequently applied include highly acidic polyelectrolytes^{Reference Gower53} and block-copolymers,^{Reference Yu and Cölfen52} where the latter usually contain at least one block bearing carboxylate or sulfonate functional groups as a common active component.^{Reference Kim, Schenk, Ihli, Kulak, Hetherington, Tang, Schmahl, Griesshaber, Hyett and Meldrum45,Reference Meldrum and Cölfen54,Reference DeOliveira and Laursen55}

However, additives are not limited to these components.^{Reference Schenk, Cantaert, Kim, Li, Read, Semsarilar, Armes and Meldrum56,Reference Cantaert, Kim, Ludwig, Nudelman, Sommerdijk and Meldrum57} A multitude of polyanionic and polycationic macromolecules with different chemistries and architectures have been identified to be excellent modulators of morphology and/or to act as growth-controlling agents of calcite crystals,^{Reference Yu and Cölfen52–Reference Chen, Qi, Tao, Zuckermann and De Yoreo58} and the versatile roles of different types of polyelectrolytes interfering with the calcium carbonate precipitation process at various stages have been classified.^{Reference Verch, Gebauer, Antonietti and Cölfen59,Reference Gebauer, Cölfen, Verch and Antonietti60} Studies addressing the nano/microstructures of synthetic calcite composites precipitated in the presence of commercial polyelectrolytes, polystyrene sulfonate (PSS),^{Reference Schenk, Zlotnikov, Pokroy, Gierlinger, Masic, Zaslansky, Fitch, Paris, Metzger, Cölfen, Fratzl and Aichmayer61} and poly(4-styrene sulfonate-co-maleic acid) (PSS-MA)^{Reference Kim, Schenk, Ihli, Kulak, Hetherington, Tang, Schmahl, Griesshaber, Hyett and Meldrum45} revealed that the distribution of polymers in mineral crystals (1–4 wt% occlusion level) is inhomogeneous, with preferential occlusion along the (001) and (104) lattice planes.

As opposed to macromolecules, which show cooperative effects of multiple functional groups, the occlusion of small molecules in calcite has been investigated only very recently. Borukhin et al. screened 20 common amino acids for their capacity to become incorporated into calcite crystals.^{Reference Borukhin, Bloch, Radlauer, Hill, Fitch and Pokroy43} They found substantial variation in the occlusion levels depending on the type of amino acid. A particularly strong interaction with the mineral phase was observed for aspartic acid, where this observation is in agreement with the composition of soluble biomacromolecules extracted from biogenic calcite (as discussed in the previous section). In general, charge, size, rigidity, and the relative pKa of the terminal groups in aqueous solution were suggested as governing factors for uptake into the crystalline host. Cysteine and glycine, however, as non-acidic amino acids, represent an unusual case in that they showed incorporation levels as high as those seen for aspartic acid.

Particles

In many cases, the soluble additives entrapped in minerals form very small clusters of organic material providing low electron density contrast and hence are practically “invisible” in electron microscopic investigations using routine equipment. This substantially complicates their localization within the mineral host. A new methodology, which uses additives in particulate form, was developed in order to generate better defined organic–inorganic interfaces and to obtain more accurate information on the quantity and distribution of the occluded phase (Figure 5a–b). A variety of particles ranging from latex spheres^{Reference Kim, Ribeiro, Maillot, Ward, Eichhorn and Meldrum51} over micelle-forming block copolymers,^{Reference Kim, Ganesan, Yang, Kulak, Borukhin, Pechook, Ribeiro, Kroger, Eichhorn, Armes, Pokroy and Meldrum46} to inorganic/metal nanoparticles^{Reference Kulak, Yang, Kim, Armes and Meldrum49,Reference Kulak, Semsarilar, Kim, Ihli, Fielding, Cespedes, Armes and Meldrum50} were successfully incorporated. The surface of the particles is functionalized with long, flexible charged polymer chains, which provide a strong binding capacity to crystal faces, thus playing a crucial role for particle incorporation. Micelle-forming copolymers in particular shared many common features with proteins, in terms of their composition, size, and shape (the folding structure of common proteins involved in calcite biomineralization is closer to a globular form than to a fully extended chain^{Reference Li, Xin, Kunitake, Keene, Muller and Estroff27}). This system has demonstrated a surprisingly high upper limit of particle occlusion (13 wt%) in the mineral single crystal (Figure 5b), exceeding the levels seen in other synthetic and biogenic systems. For example, latex particles with a diameter of ∼250 nm could be uniformly incorporated into calcite at an occlusion level of 9.3 wt% (Figure 5a), and the typical range of occlusion seen for soluble organic additives is 1∼4 wt%, with the exception of a 42-amino acid polypeptide, for which incorporation of 9.2 wt% in calcite was estimated.^{Reference Metzler, Tribello, Parrinello and Gilbert62}

Figure 5.

Scanning electron microscope images showing internal surfaces of synthetic calcite single crystals grown in the presence of polymer-based particles and gels. (a) Polystyrene latex particles (focused ion beam cross-sectioned), (b) block copolymer micelles (fractured), (c) in xyloglucan gel matrix functionalized with Fe₃O₄ nanoparticles (fractured). (a) Reproduced with permission from Reference 51. © 2010 Wiley. (b) Reproduced with permission from Reference 46. © 2011 Nature Materials. (c) Reproduced with permission from Reference 68. © 2014 Royal Society of Chemistry.

Gels

Another important synthetic strategy inspired by biomineralization is growing crystals in a gel medium.^{Reference Asenath-Smith, Li, Keene, Seh and Estroff47} As one of the oldest crystal growth methods, these processes have been well-studied using various gels, such as gelatin,^{Reference Metzler, Tribello, Parrinello and Gilbert62–Reference Nindiyasari, Fernandez-Diaz, Griesshaber, Astilleros, Sanchez-Pastor and Schmahl64} silica,^{Reference Fernandez-Diaz, Astilleros and Pina66} polyacrylamide,^{Reference Grassmann and Löbmann67} and agarose.^{Reference Li, Xin, Muller and Estroff48} Conceptually, gels were revitalized as a suitable model system to study crystal growth constrained by a viscous biological medium and to learn about interactions between the insoluble matrix and the crystal (detailed investigations on calcite crystals grown in gels are further discussed in the Hendley et al. article in this issue).

Combinations of synthetic approaches: Better together?

Combinations of different strategies have also been explored in order to achieve control over the distribution of the guest phase and for introducing diverse structures and functionalities into the single crystal. As an example of bringing particle and gel incorporation together, crystals were grown within a xyloglucan gel pre-functionalized with metal-oxide nanoparticles.^{Reference Kim, Schenk, Walsh, Kulak, Cespedes and Meldrum68} As an advantage of this approach, the nanoparticles could be nicely dispersed in the mineral phase due to their attachment to the gel network, while no special surface treatment of the particles was required. However, a relatively low level of occlusion was achieved in comparison to a system of free particles with adequate surface functionalization.^{Reference Kulak, Semsarilar, Kim, Ihli, Fielding, Cespedes, Armes and Meldrum50} This is possibly due to restricted transport of particles onto crystal growth fronts in a viscous medium^{Reference Kim, Schenk, Walsh, Kulak, Cespedes and Meldrum68} (Figure 5c). By growing calcite in agarose gel attached to self-assembled monolayers as substrates, Li et al. achieved both occlusion of an organic network and controlled orientation of macroscopic calcite crystals.^{Reference Li and Estroff69}