Amino Acid Assisted Incorporation of Dye Molecules within Calcite Crystals

Abstract Biomineralisation processes invariably occur in the presence of multiple organic additives, which act in combination to give exceptional control over structures and properties. However, few synthetic studies have investigated the cooperative effects of soluble additives. This work addresses this challenge and focuses on the combined effects of amino acids and coloured dye molecules. The experiments demonstrate that strongly coloured calcite crystals only form in the presence of Brilliant Blue R (BBR) and four of the seventeen soluble amino acids, as compared with almost colourless crystals using the dye alone. The active amino acids are identified as those which themselves effectively occlude in calcite, suggesting a mechanism where they can act as chaperones for individual molecules or even aggregates of dyes molecules. These results provide new insight into crystal–additive interactions and suggest a novel strategy for generating materials with target properties.

Abstract: Biomineralisation processes invariably occur in the presence of multiple organic additives,w hicha ct in combination to give exceptional control over structures and properties. However,f ew synthetic studies have investigated the cooperative effects of soluble additives.T his work addresses this challenge and focuses on the combined effects of amino acids and coloured dye molecules.The experiments demonstrate that strongly coloured calcite crystals only form in the presence of Brilliant Blue R( BBR) and four of the seventeen soluble amino acids,ascompared with almost colourless crystals using the dye alone.T he active amino acids are identified as those which themselves effectively occlude in calcite,s uggesting am echanism where they can act as chaperones for individual molecules or even aggregates of dyes molecules.T hese results providen ew insight into crystal-additive interactions and suggest an ovel strategy for generating materials with target properties.
With their versatility and ease of application, soluble additives are widely used to control crystallisation processes. There is no better demonstration of the power of this approach than the process of biomineralisation, in which organisms achieve exceptional control over mineral formation to generate structures such as bones and seashells. [1] While this is achieved by many biogenic strategies,a ll are united by one common feature:t he use of organic mole-cules. [2] Significant efforts have therefore been made to identify biomolecules associated with specific roles such as selecting crystal polymorphs and tuning crystal textures,a nd to translate the underlying principles to synthetic systems. [3] Many small organic molecules, [4] and larger species such as polyelectrolytes [5] and block copolymers [4a,6] have to-date been identified that are active in controlling crystallisation processes.
While the focus of these studies has principally been on the activities of individual additives,c rystals in biology typically grow under the influence of as uite of additives. This has the potential for greater control, where individual additives could be deployed at different time-points in the reaction, [7] or they could act in combination to deliver an outcome that could not be achieved with individual additives. While an umber of bio-inspired studies have demonstrated the cooperative effects of soluble additives and insoluble organic matrices, [8] fewh ave addressed the effects of combinations of soluble additives, [9] possibly due to the challenge of investigating alarge reaction space.
This article describes an investigation into the crystallisation of calcium carbonate in the presence of mixtures of organic additives,where we focus on the combined effects of amino acids and coloured dye molecules.T he selection of ac oloured dye as one of our additives is crucial to our strategy,where it allows us to go beyond basic properties such as morphology and use optical microscopy to determine how the additives cooperate to drive occlusion within the crystal lattice. [10] Such occlusion is key to the superior mechanical properties of single-crystal biominerals, [11] to the generation of characteristic crystallographic textures, [3a, 12] and can be used to generate novel nanocomposites. [13] Our study shows that certain amino acids can vastly increase the occlusion of ac ommon dye-Brilliant Blue R( BBR)-within calcite, generating strongly coloured single crystals.T his demonstrates that selected combinations of additives can facilitate the formation of composite materials that would not be accessible with traditional methods and proposes an incorporation mechanism that is relevant to biological and synthetic systems.
Calcium carbonate was precipitated by mixing equal volumes of 10 mm solutions of CaCl 2 and NaHCO 3 ,a nd adding aliquots of amino acids and BBR at concentrations that yielded single crystals of calcite.Crystallisation was then allowed to proceed for two days.Inall cases the polymorphs generated were confirmed by using powder XRD and Raman microscopy ( Figures S1 and S2 (Figures 1a and e). Very weakly coloured rhombohedral calcite crystals precipitated in the presence of BBR alone, while aspartic acid (Asp) alone generated calcite crystals that were elongated along the c-axis,a si sc haracteristic of this additive. [11,14] In combination, however, these additives generated intensely blue calcite crystals whose morphologies were unchanged as compared with Asp alone.I dentical results were obtained using either d-o rl-Asp.
As mall number of vaterite particles were sometimes observed and were always strongly coloured, even in the absence of Asp ( Figure 1c). This is due to the polycrystalline structure of the vaterite,where the dye is entrapped between the crystalline units.S trong colouration of single crystals,i n contrast, only occurs if the dye becomes occluded within the crystal lattice.T hat dye was incorporated within the calcite crystals was confirmed by washing them with sodium hypochlorite to degrade any surface-bound molecules,d issolving them in acid, and then characterising the resulting solution using UV/Vis spectroscopy.C rystals were also embedded in epoxy resin and polished to expose their interiors,w here optical microscopy and energy-dispersive X-ray analysis (EDX) confirmed occlusion ( Figure S3).
Further examination of the dyed calcite crystals showed that many exhibited zoning effects,w here the dye was preferentially associated with the {hk0} [15] faces that define the equatorial zone (Figure 1d). At ransmitted light image therefore appears as an hour-glass structure.N on-uniform occlusion within calcite can be attributed to differential adsorption of additives to the acute and obtuse step edges, [16] where the amino acids that give occlusion here are expected to preferentially bind to the acute over the obtuse steps.T his effect will be less prominent at higher concentrations of amino acids when binding to both acute and obtuse steps occurs. [17] Such anisotropic partitioning of molecules within calcite has previously been observed for small ions such as Mg 2+ , [18] Sr 2+ and SO 4 2À , [19] and larger fluorescent dyes. [17,20] Theg enerality of these cooperative effects was then explored by screening all 17 soluble amino acids with BBR under the same reaction conditions.O nly 4a mino acids supported significant BBR occlusion:a spartic acid (Asp), glycine (Gly), glutamic acid (Glu), and asparagine (Asn) (Figures 1a and e) and all exhibited zoning;t hat the crystal containing Glu/ BBR appears to be uniformly coloured in Figure 1i sd ue to the intense colour. Forc omparison, an image is also shown of ac rystal grown in the presence of valine (Val) (Figure 1e). This amino acid does not occlude in calcite,a nd supports little dye incorporation. An umber of initial experiments were also performed to explore whether this "chaperone" strategy is unique to BBR. Having demonstrated that Asp effectively chaperones BBR into calcite,w ei nvestigated the effects on the crystal lattice using synchrotron high-resolution powder X-ray diffraction (HR-PXRD). Thedata were analysed using Rietveld analysis ( and in the presence of Asp or BBR alone.A ll samples comprised calcite,t ogether with 7-13 %v aterite,a nd the calcite crystals exhibited coherence lengths comparable to geological calcite (550-700 nm). Calcite crystals precipitated in the presence of Asp alone comprised 0.26 wt %A sp and exhibited ap eak shift towards lower angles due to lattice distortions of Dc/c = 3.05 10 À4 and Da/a = 5.21 10 À5 , whereas calcite containing Asp and BBR displayed smaller lattice distortions of Dc/c = 1.86 10 À4 and Da/a = 1.40 10 À5 , but broader peaks.T his anisotropic lattice distortion is consistent with the elastic anisotropy of calcite. [11] Calcite crystals precipitated in the presence of BBR alone exhibited as mall increase in peak width, which is indicative of low levels of occlusion, while those formed at higher Asp concentrations ([Ca 2+ ]:[amino acid]:[dye] = 250:250:1) exhibited greater lattice distortions,but no further peak broadening ( Figure S5). These data show that the peak shifts were exclusively due to the incorporated Asp,b ut that BBR contributes to peak broadening.S imilar effects have been observed for calcite containing ar ange of nanoparticles. [21] Further information about the cooperation between Asp and BBR was obtained by using in situ atomic force microscopy (AFM) using al iquid cell (Figure 4), where the methods are described in detail in the Supporting Information. AFM analysis was performed under slow flow of four variants of the calcium carbonate solutions: [22]  [dye] = 100:1:1. Calcite grows via as tep-growth mechanism under these conditions,a nd steps originate from screw dislocations present on the {104} faces (Figure 4a).
Addition of Asp to the reaction solution at these concentrations had minor effects on the shapes of the steps and their rates of propagation (Figures 4b). Preferential binding to the acute steps was seen at higher Asp concentrations,asisconsistent with the literature ( Figure S6). BBR, in contrast, adsorbed to all exposed faces (Figure 4c). The images are indicative of the low solubility BBR forming aggregates in solution, as occurs for many dyes.A ddition of amixture of Asp and BBR molecules to the growth solution, in contrast, caused severe roughening of the acute 44 " 1 and 44 " 8 steps,w hile most of the obtuse steps preserved their well-defined shapes (Figure 4d). This pattern of behaviour is also characteristic of Asp alone at higher Asp concentrations, where binding to the acute over the obtuse steps occurs due to an enhanced stereo-chemical fit. [16] Asp and BBR therefore together have agreater effect on calcite growth than identical concentrations of the individual additives.
These data demonstrate that certain amino acids can drive the occlusion of BBR in calcite,w here minimal occlusion occurs in their absence.But what is the mechanism by which this takes place?Ofthe 17 amino acids tested, Asp,Glu, Gly and Asn were the most active.N otably,t hese activities correlate closely with their occlusion efficiencies in calcite. [4c,11, 23] This suggests three potential scenarios,where (1) the active amino acids and BBR may complex in solution, such that the amino acid chaperones BBR into the crystal lattice, (2) the amino acids retard crystal growth, where the longer  residence time of strongly binding additives at step edges/kink sites gives greater occlusion and (3) these amino acids facilitate direct binding of BBR to the crystal surface, facilitating occlusion.
Thef irst scenario was investigated using Molecular Dynamics (MD) simulations.T hese were performed at 300 Ka nd utilised the AMBER force field to model the dye and amino acid molecules. [24] Asp was assigned anet charge of À1, where this is consistent with the experimental pH of % 8.5. Thec alculations showed that there was no association between Asp and BBR, where any transient complexes rapidly separated. Multiple other variables including changes in the pH (where this was achieved by assigning ad ifferent charge to the amino acid) and the addition of aCa 2+ ion to the Asp molecules were also explored, but no association between BBR and Asp was ever observed. Given that Asp and BBR do not associate in bulk solution-when they have full conformational freedom-we find it extremely unlikely that BBR complexes to Asp molecules bound to the calcite surface.N MR spectroscopy was also explored, but the solubility of BBR in water was too low to provide any information on Asp-BBR interactions.
We are also able to rule out the second scenario,where no visible increase in BBR occlusion was obtained on reduction of the calcium concentration (and thus supersaturation). In addition, analysis of the quantities of Asp and BBR occluded showed that the amount of BBR occluded correlates with the amount of Asp occluded, rather than the amount of Asp in solution ( Figure 5), where the latter would be expected if dye occlusion occurred due to ar etardation of the growth rate.
Considering then the third scenario,the binding of amino acids to calcite has previously been studied using simulations and experiments.Acomplex picture emerges which shows that occlusion efficiency cannot be predicted based on binding strength to the crystal surface.Under the experimental conditions employed here,the amino acids are zwitterionic and preferentially bind through the carbonyl oxygen and the amide hydrogen of the peptide functionality. [16,25] Alanine, Asp,Glu, Gly,leucine and tyrosine all bind to calcite in water at pH 8t oas ignificant degree, [26] and Asp and Gly exhibit almost identical adsorption free energies. [25b] However,t here are enormous differences in their incorporation efficiencies and Asp is occluded far more efficiently than Gly at identical concentrations. [23] Simulations provide an explanation for these effects.Asp binds directly to the calcite surface,stabilised by interactions of the side-chain carboxy group with local water molecules. [25c] Figure 4. a-d) AFM images of calcite crystals grown in the presence of additives recorded using aliquid cell, and associated schematic images of the additive-binding that occurs under each condition.a)Screw dislocation observed for acrystal grown in the absence of additives displayed well defined step boundaries (white) and c-glide (black) + and Àsigns indicate obtuse and acute steps, respectively.b)Little change occurs in the step shape followingintroductiono fl ow concentrations of Asp to the growth solution,w here Asp binds preferentially to acute step edges(see Figure S6). c) when BBR is the only additive, it is strongly adsorbed to the calcite {104} faces and shows no preference for either step edge. d) BBR and Asp act in combination to preferentially bind to the acute steps. a, b) were obtained in contact mode, whereas c, d) were obtained in Tapping Mode. Asp may therefore promote BBR occlusion by disrupting the first hydration layer and allowing its intimate association with the crystal surface.Gly,incontrast, is thought to substitute for water molecules in the second hydration layer rather than binding directly to the mineral surface-from where it cannot be occluded. [27] However,t hat Gly is effectively occluded at high solution concentrations indicates that aproportion of the adsorbed molecules must displace water molecules to bind directly to the calcite surface.A sB BR alone is occluded at very low levels suggests that it does not bind directly to the calcite surface.T his mechanism is illustrated schematically in Figure 4. Further modelling and experimental work is required to fully understand the complex mechanisms at play.
Finally,i ti si nteresting to consider the generality of this mechanism. That the amino acids can chaperone aggregates of BBR into the crystal lattice likely contributes to the strong colouration observed. We therefore performed additional experiments with Asp and the highly soluble fluorescent dye HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) to confirm that Asp is equally effective in driving the occlusion of individual molecules in calcite.C onfocal fluorescence microscopy of the product crystals confirmed that the dye is uniquely located in the zone occupied by Asp ( Figure 2)that is,t hat defined by binding to acute steps-which shows that this "partnership" mechanism can potentially be applied to aw ide range of additives.
This work demonstrates that greater control over the growth and properties of crystals can be achieved if organic additives are employed in combination, rather than individually.Focusing on the attractive test-system of acoloured dye (BBR) and amino acids,w es how that four amino acidsthose that are themselves effectively occluded in calcitedrive the occlusion of BBR at levels that vastly exceed those achieved with the dye alone.Importantly,our work also shows that these cooperative effects are not restricted to the BBR/ Asp partnership.T hese results therefore suggest an ovel strategy for generating materials with target properties,where combinatorial methods [9b, 28] and high-throughput screening approaches [4c] could be employed to rapidly explore the large reaction space created by multiple additives.T hey are also of particular relevance to biomineralisation processes,w here multiple additives are invariably present within the biological environment.