Haofei
Gong
a,
Yi
Yang
b,
Manuela
Pluntke
c,
Othmar
Marti
c,
Zsuzsa
Majer
d,
Norbert
Sewald
b and
Dirk
Volkmer
*a
aInstitute of Physics, Chair of Solid State and Materials Chemistry, Augsburg University, Universitätsstrasse 1, D-86159 Augsburg, Germany. E-mail: dirk.volkmer@physik.uni-augsburg.de
bDepartment of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, PO Box 100131, D-33501 Bielefeld, Germany
cInstitute of Experimental Physics, Ulm University, Albert-Einstein-Allee 11, D-89081 Ulm, Germany
dInstitute of Chemistry, Eötvös Loránd University, PO Box 32, H-1518 Budapest 112, Hungary
First published on 22nd September 2014
Four amphiphilic peptides with designed hairpin structure were synthesized and their monolayers were employed as model systems to study biologically inspired calcium carbonate crystallization. Langmuir monolayers of hairpin peptides were investigated by surface pressure area isotherms, surface potential isotherms, Brewster angle microscopy (BAM), atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy. A β-hairpin conformation was found for all peptides at the air–water interface although their packing arrangements seem to be different. Crystallization of calcium carbonate under these peptide monolayers was investigated at different surface pressures and growth times both by in situ optical microscopy, BAM and ex situ investigations such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). An amorphous calcium carbonate precursor was found at the initial crystallization stage. The crystallization process occurred in three stages. It starts from the nucleation of amorphous particles being a kinetically controlled process. Crystal nuclei subsequently aggregate to large particles and vaterite crystals start to form inside the amorphous layer, with the monolayer fluidity exerting an important role. The third process includes the re-crystallization of vaterite to calcite, which is thermodynamically controlled by monolayer structural factors including the monolayer flexibility and packing arrangement of the polar headgroups. Thus, the kinetic factors, monolayer fluidity and flexibility as well as structure factors govern the crystal morphology and polymorph distribution simultaneously and synergistically.
Interfacial properties of polypeptides in living organisms seem to play a key role in modifying calcium carbonate crystal growth since it is assumed that the initial crystal nucleation takes place at peptide-modified surfaces or interfaces. However, only few investigations employed artificial peptides as CaCO3 crystal growth modifiers, which included water-soluble peptides,18–20 and water-insoluble Langmuir monolayers.21,22 Insoluble monolayers of peptides in particular might form an organized supramolecular template matrix with a well-defined two-dimensional structure, thus providing a potential means to control the nucleation and crystallization of inorganic crystals. From an experimentalist's point of view it is straight-forward to adjust monolayer properties such as molecular conformation and/or charge density by changing the experimental conditions (i.e. monolayer surface pressure, pH and ion conc. of the aqueous subphase). The crystallization beneath peptide monolayers can be initiated at different conditions and in a controlled manner. In addition, the two-dimensional monolayer matrix structure can be characterized structurally, both by in situ methods and by ex situ characterization of the transferred Langmuir–Blodgett films, finally revealing the key factors which determine the surface-initiated crystallization process. Langmuir monolayers of peptides thus constitute reductionistic model systems for peptide-induced biomineralisation, i.e. to investigate crystal growth at self-organized organic matrices.
However, although monolayer-controlled crystal growth might seems to be a simplistic model system, diverse and partly contradictory hypotheses can be found in literature about calcium carbonate crystallization beneath monolayers of amphiphilic molecules covering a wide range of different molecular structures. In seminal studies, Mann and Heywood et al. attributed the oriented nucleation to the geometrical and/or stereochemical match between the monolayer packing arrangement and the oriented mineral surface.23–26 Later, a multistep assembly process from an amorphous to a crystalline phase was suggested by Xu et al.27 Other groups demonstrated that the CO2 evaporation rate effectively influences crystal growth beneath monolayers, which may add to or even overlay the template effect of the monolayer.28In situ synchrotron X-ray diffraction studies have demonstrated that additives such as poly(sodium acrylate) and Mg2+ ions dissolved in the aqueous subphase lead to changes in the kinetics of the crystallization process.29 Employing a vast range of structurally different, tailored amphiphiles it was concluded by Volkmer et al. that non-specific factors such as the monolayer's surface charge density or its effective dipole moment seems to be key factors that determine the morphology and polymorph of the crystals grown underneath the monolayer.30–33 In addition, monolayer flexibility and its corresponding dynamic structural changes during crystallization were shown to be crucial factors in the mineralization process.34
Continuing these model studies we here report on CaCO3 crystallization underneath monolayers of amphiphilic, structurally well-defined β-hairpin peptides which were chosen for reasons of structural analogy with natural polyacidic peptides often found in biominerals. These hairpin peptides are composed of alternating hydrophobic and hydrophilic amino acid residues that spontaneously fold into a D-Pro-Gly-induced β-hairpin conformation (Scheme 1) at the air–water interface not only by interfacial assembly but also by inter-strand hydrogen bonding interactions.35–37 Artificial amphiphilic hairpin peptides represent a novel class of functional model peptides that form insoluble monolayers. The film-forming properties of these molecules as yet have not been studied in depth with respect to different in-film molecular packing arrangements. The possibility to adjust the monolayer's surface charge density in conjunction with different molecular packing arrangements provides additional degrees of experimental freedom if compared to single strand model peptides comprised of alternating hydrophobic/hydrophilic residues. From an experimental point of view, the latter peptides, although structurally more closely related to natural proteins, are very difficult to prepare by standard solid-phase synthesis owing to their high propensity to form insoluble aggregates, which furthermore renders them difficult to spread as monolayer films in a controlled and reproducible fashion. Calcium carbonate crystallization beneath β-hairpin peptide monolayers having different amino acid sequences will be described in the following. Although these peptides have similar primary structure, it is found that CaCO3 nucleation and crystallization beneath each monolayer they form follow distinct pathways, which might be ascribed to the different peptide packing arrangements at the air–water interface.
MALDI-ToF mass spectra were measured on a Voyager DE Instrument (PE Biosystems, Weiterstadt, Germany) mounted with a 1.2 m flight tube. 2,5-Dihydroxybenzoic acid was used as the matrix. Depending on the mass range the ions were accelerated at 15 to 25 kV with the option of detecting positive or negative ions. The instrument default calibration was used for calibrating the mass axis.
For peptide elongation the corresponding Fmoc-amino acid (3–4 equiv. with respect to resin loading) was dissolved in a minimum volume of DMF (1 mmol amino acid mL−1) and pre-activated for up to 5 min with 3–4 equiv. of TBTU, dissolved in a minimum volume of DMF, and 6 equiv. DIPEA were added to the reaction mixture. The pre-activated Fmoc-amino acid was then added under argon to the resin loaded with the first amino acid or peptide and agitated for 45 minutes at room temperature. The reaction progress result was monitored by MALDI-ToF MS or Kaiser test.
For capping of unreacted amino groups 3 equiv. Ac2O (based on resin loading) and 3 equiv. Et3N, dissolved in DMF, were added and the mixture was agitated at room temperature for 30 min. Then the resin was washed with CH2Cl2 (4×), DMF (4×), CH2Cl2 (4×).
After assembly of the protected peptide on the resin, it was cleaved by adding 5 mL TFA in CH2Cl2 (1%, v/v). After 5 min the resin was filtered and the filtrate was neutralized with pyridine. The cleavage was repeated once and the combined filtrates were evaporated to dryness under reduced pressure. The crude fully protected peptides were lyophilized.
Deprotection of the protected peptides was achieved by treatment with the cleavage cocktail TFA–H2O–TIS (95:
2.5
:
2.5) for several hours at ambient temperature. The crude products were purified by preparative HPLC using acetonitrile–water–TFA gradients. The pure peptides were obtained in low yields because of their high tendency towards aggregation and precipitation.
MS (MALDI-ToF): m/z = 1978.37 [M + H]+, 1999.42 [M + Na]+, 2015.59 [M + K]+.
Calculated Mass (monoisotopic): [M + H]+ = 1977.73, [M + Na]+ = 1999.71, [M + K]+ = 2015.69.
MS (MALDI-ToF): m/z = 2086.92 [M + H]+, 2108.32 [M + Na]+, 2124.35 [M + K]+.
Calculated Mass (monoisotopic): [M + H]+ = 2087.84, [M + Na]+ = 2109.83, [M + K]+ = 2125.80.
MS (MALDI-ToF): m/z = 1886.83 [M + Na]+; calculated mass (monoisotopic): [M + Na]+ = 1885.72.
MS (MALDI-ToF): m/z = 1941.36 [M + Na]+, 1957.61 [M + K]+; calculated mass (monoisotopic): [M + Na]+ = 1941.78, [M + K]+ = 1957.76.
The surface pressure of the monolayers was measured using a filter paper Wilhelmy plate. The Langmuir film was formed by spreading a chloroform solution of peptides (0.2 mg mL−1, with 10% DMF) onto Milli-Q purified water (ρ > 18 MΩ cm). After evaporation of the solvent (approx. 15 min), π-A isotherms of the spread films were recorded at room temperature (approx. 22 °C) by compressing or expanding the Langmuir film at a barrier speed of 15 cm2 min−1. Surface potential measurements were performed with a Trek electrometer. A vibrating electrode was placed about 2 mm above the air–water interface and a stainless steel plate as the reference electrode was immersed in the subphase. The potential solubilisation of peptides in the aqueous subphase was tested by measuring at least 3 repeated compression/decompression cycles of all monolayer samples (pure water or 9 mM CaCl2). In no case any shift of the limiting area up to a maximum compression of 20 mN m−1 was found.
Monolayers and multilayers of peptides were transferred vertically at 15 mN m−1 on freshly cleaved mica, CaF2, and silicon substrates for AFM, FT-IR and XPS measurements, respectively. The transfer speed was 2 mm min−1 for both upwards and downwards strokes.
The peptides were designed as β-hairpins and their folded conformation is based on three stabilizing factors: (i) hydrogen bonds between backbone amide moieties across the strands, (ii) side chain interactions across two β-strands, and (iii) the presence of a reverse turn (β-turn). In particular, the interaction between aromatic side chains forming hydrophobic clusters as a stabilizing effect has been shown for a number of β-hairpin peptides and it is apparent as an aromatic contribution in the CD curve.39
The CD spectrum of 1a in HFIP indicates the predominance of β-turns, e.g. βI–III type with random coil contributions, characterized by negative bands at 195 nm, a broader one centralized at 212 nm (see Fig. 1a) and a weak one at 230 nm. Solvents like TFE or HFIP in principle promote sheet or helix formation by reducing the water activity.40 However, the overall influence depends on the structure propensity of the sequence. Fluorinated alcohols like TFE or HFIP have been found to attenuate hydrophobic interactions between residues that are distant in the peptide sequence, like e.g. across strands in some β-sheets, and, therefore, are able to disrupt these structures.38 In contrast to peptide 1a, the CD spectrum of 1b in HFIP indicates the predominance of a β-hairpin, composed of β-sheet and β-turn conformations, with the typical positive band at 194 nm and the negative band at 221 nm, as shown in Fig. 1b. The positive shoulder at 202 nm that increased with concentration (data not shown) is characteristic of the β-turn present in the β-hairpin conformation.
A conformational change was observed for both peptides upon addition of water to the HFIP solutions. The CD spectrum of the Asp-containing peptide 1a indicated increasingly ordered structural units, e.g. with β-hairpin/β-turn (mainly βI–III) characteristics as well as some aromatic contributions of the Phe residues (positive band at 194 nm, increase in negative intensity at 215 nm) with increasing water contents (Fig. 1a). Likewise, the CD-spectrum of the Glu-containing peptide 1b showed a positive band at 190 nm and a negative band at 220 nm, also indicating β-hairpin conformation (Fig. 1b). However, for both peptides 1a and 1b the positive CD effects at 190–194 nm are accompanied by negative bands at 200–203 nm and additional positive bands at 207–208 nm. This unusual pattern in aqueous solutions can be assigned to an interaction of aromatic chromophores. Such effects on hairpin stability have in particular been described for specific Trp/Trp, Trp/Tyr, and Tyr/Tyr interactions.39 It has been suggested that they efficiently stabilize the secondary structure and can be employed to monitor the cross-strand interaction between the aromatic side chains. Moreover, such orthogonal arrangements of aromatic rings have been observed in proteins and have been suggested as a source of tertiary structure stabilization.41 This shows that the increase of the water concentration stabilizes the β-hairpin conformation especially of peptide 1b, presumably by hydrophobic interactions between the phenylalanine side chains.
Molecular modelling of hairpin peptides assuming an ordered head-to-head β-hairpin packing arrangement42 suggests an occupied area of 259 Å2 for one peptide (Scheme 2), which closely matches the limiting area of peptide 1b on the water subphase. This result indicates that 1b organizes in a head-to-head packing arrangement on the water surface as shown in Scheme 2, while a relatively smaller limiting area of 1a suggests a less ordered peptide packing arrangement on the water surface.
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Scheme 2 β-Hairpin packing arrangement of the peptides at air–water interface. The interstrand distance and the distance between two adjacent phenyl group was taken from ref. 42. |
The surface pressure increases after the phase transition of both peptides 1a and 1b on the water surface, and no obvious collapse pressure was observed within the range of the compression, demonstrating a strong flexibility of the peptide monolayers, i.e. the monolayers are highly compressible at very low surface area. In addition, the limiting area of the second liquid condensed phase is about half the area of the first liquid expanded limiting area for peptide 1a and 1b on the water surface, which indicates that the peptides overlap to form a double layer during the phase transition. For peptide 2a, a significantly higher surface pressure was observed compared to 1a or 1b at the same molecular area, suggesting higher flexibility of peptide 2a on the water surface. The isotherms of peptide 2b have a shape similar to the isotherms of 1b except for a smaller limiting area, which points at a tighter packing of 2b on the water surface than 1b.
On the subphase of CaCl2, the isotherms of the peptides 1a and 1b both show significant shifts towards larger molecular area. The limiting areas both are about 30 Å2 larger than the ones on pure water surface, while the limiting areas for the peptides 2a and 2b change to a smaller value. The results indicate that the packing arrangement of the peptides on the Ca2+ ion subphase is closely related with the density of charged carboxyl groups.
The ordered head to head packing arrangement of peptide 1b is considered to be a result of the subtle balance between hydrophobic interactions and carboxyl group interactions. However, on a subphase with calcium ions, this interaction balance is broken due to Ca2+ ion binding. Ion binding weakens intermolecular hydrogen bonding interactions and thus alters the inter- and intra-peptide distances. This explains the increase of the limiting areas of the peptides on Ca2+ ion subphase. Calculations that imply the modification of the distances between adjacent β-strands revealed that an interstrand distance change from 4.7 Å to 5.1 Å can explain the limiting area change of peptide 1b on a CaCl2 subphase.43
BAM investigations of the peptides spread on water and on an aqueous CaCl2 subphase were performed in order to elucidate monolayer properties in depth. Typical BAM images are shown for 1b and 2b in Fig. 3, because the monolayer morphologies of 1a and 1b as well as the structures of 2a and 2b are similar both on water and CaCl2 subphase. The BAM images of the peptides 1a and 1b on water show large domains with small holes inside (Fig. 3a). Upon further compression, a uniform film was observed within the resolution of BAM (not shown). However, on the subphase of CaCl2, the monolayer consists of many small domains at the onset pressure (Fig. 3b). This monolayer morphology change suggests that the intermolecular interactions change in the monolayer after binding with Ca2+ ions, which is consistent with the results of surface pressure-area isotherms.
Peptides 2a and 2b form larger domains on the water subphase at the onset of the surface pressure, while the film splits into many small domains on the subphase of CaCl2. These domains merge and form a homogeneous monolayer at high pressure. The morphology difference of 2a or 2b on water and CaCl2 subphase also suggests a strong binding interaction between Ca2+ ion and peptide monolayers.
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Fig. 4 AFM images of peptide 1b monolayer on mica transferred from a pure water subphase at π = 15 mN m−1. (a) scanning area 1 μm × 1 μm. (b) scanning area 0.2 μm × 0.2 μm. |
For the LB films with peptide 1b, a distinctive arrangement was observed (Fig. 4a). The molecules were seen to form a highly regular stripe pattern with no preferential orientation, indicating lack of correlation between the peptide arrangement and the crystalline orientation of mica. Thus it can be excluded that the substrate itself has an impact on the structure of the peptide film. The measured film height is about 1 nm, confirming that only one monolayer has been transferred to the mica substrate. Phase images of the LB films (providing higher lateral resolution than the topographies) are shown in Fig. 4b and reveal a double stripe pattern with a separation of 3.1 nm ± 0.1 nm. A few single strands are also observed. As the phase of an oscillating AFM tip depends on the specimen properties (such as elasticity and adhesion), the strong contrast in the AFM phase images may result from the tip interaction with hydrophobic and hydrophilic areas of the sample, or more specifically, with the peptide molecules (hydrophobic sidechains and hydrophilic termini) and mica (in the film gaps) or partially adsorbed water molecules. The observed stripe pattern therefore confirms the assumption that the hairpins form β-sheets containing opposing rows of equally oriented molecules (each with a width of about 3 nm).
AFM scans were repeated on monolayers formed from different subphases from peptide 1b. The LB films transferred from CaCl2 (0.01 M) subphase exhibit shapes similar to the ones discussed above. The film height is 1.00 nm ± 0.03 nm, indicating that the subphase ions have not altered the dimensionality of the peptide film.
The secondary structures of the peptides were analyzed by FT-IR spectroscopy. Multilayers (i.e. 40 layers) of the peptides 1a and 1b were transferred onto CaF2 substrates and analysed, as shown in Fig. 5. Transfer ratios were recorded during multilayer transfer on CaF2 substrates and they were found to be close to unity during all cycles and for all peptides being transferred. The IR absorption band between 1600–1700 cm−1 is assigned to the amide I stretching band, while the peak position in this range is characteristic for the secondary structure of peptides. The amide I vibrational band around 1630 cm−1 and 1696 cm−1 existing in all peptide spectra indicate the presence of similar β-pleated sheet structure motifs of all peptides at the air–water interface.44 FT-IR spectra were also recorded for peptide 1b on a CaCl2 subphase, as shown in Fig. 5b. While the amide I band at 1630 cm−1 is still visible, the relative absorption intensity was significantly reduced due to the interactions with calcium ion stemming from the aqueous subphase.
Calcite crystals with a <01.2> orientation were dominating the surface for 1b peptide monolayers after 4 hours of growth time (Fig. 7d). The immature crystals isolated after 10 minutes growth time showed a triangular morphology (Fig. 7a). Some particles had a rectangular morphology (Fig. 7b) with inhomogeneous electron density distribution, indicating that these particles are composed of even smaller particles. HR-TEM showed that they are composed of amorphous calcium carbonate (ACC) with crystalline interior domains (Fig. 7c). This indicates that the crystals were formed via transformation from an amorphous phase into a crystalline one (Fig. 7c).
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Fig. 7 Crystallization of CaCO3 beneath peptide 1a monolayer at 0.1 mN m−1. (a–c) TEM images of crystals separated after 10 min growth time. (d) SEM image of crystals separated after 4 hours. |
Crystallization experiments beneath peptide 2a and 2b monolayers resulted in each case in {10.4} rhombohedral crystals with a concave pyramidal-shaped face. These crystals were predominant beneath the monolayer of peptide 2a, but were less prominent underneath peptide 2b. Here calcite crystals with <01.2> orientation could be additionally found (Fig. 8).
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Fig. 8 SEM images of CaCO3 crystals grown beneath peptide 2a (a, b) and 2b (c, d) monolayer at 0.1 mN m−1. Crystals were separated after 4 hours (a, b) and 16 hours (c, d). |
Immediately after spreading the 1b peptide monolayer on Ca(HCO3)2 solution, domains with holes were observed with BAM (Fig. 9). Bright spots appeared simultaneously, indicating calcium carbonate nucleation under these domains. The nucleation sites multiplied and grew with time, while the peptide domains became more and more tightly packed until they appeared completely homogenous (after approximately 7 h). Similar monolayer domain changes were also observed for other peptides during CaCO3 crystallization (see Fig. S4 and S5 in the ESI†). As far as it is possible to judge from BAM images the growth of calcium carbonate seems to take place mainly at the edges of the peptide monolayer domains. Typical images of the crystallization process beneath 2b monolayers are shown in Fig. 10. Peptide 2b forms irregular monolayer domains on an aqueous Ca(HCO3)2 solution right after spreading (Fig. 10a). After a few minutes, some CaCO3 particles could be seen at the edge of the domains (Fig. 10b). Within 1 hour their number and size increased considerably. Simultaneously, the peptide domains were pushed together and formed a tightly packed monolayer within few hours of crystallization, as shown in Fig. 10c and d.
In the present study, different CaCO3 crystal morphologies and crystal orientations were found to evolve underneath different hairpin peptide monolayers, suggesting that structural or geometrical factors of the monolayers might have some influence at different stages of crystal formation. As a feature common to all peptide monolayers we observed formation of an ACC precursor within the first minutes, thus pointing to a general crystallization pathway being operative under Langmuir monolayers at the early stage of crystal nucleation.
Scheme 3 highlights the likely succession of events accompanying the formation of CaCO3 crystals beneath peptide monolayers. First, ACC primary particles (small black circles) form preferentially at the boundary of separate monolayer domains (blue patches), as indicated by time-resolved in situ BAM investigations. These nucleation sites might serve two different purposes: the edges of the monolayer are structurally flexible, being able to co-adapt to virtually any spatial arrangement of ions constituting the nascent ACC primary particles. Moreover, the non-covered water surface allows for efficient CO2 escape from the aqueous subphase, leading to a local supersaturation of CaCO3 at close proximity to the edges of the freely floating monolayer domains. The LC phase of the monolayer itself is characterized by a dense packing arrangement of amphiphilic molecules, and thus by a high surface charge density. The cumulative electrostatic effects should lead to preferential accumulation of Ca2+ and HCO3– ions underneath patches of the monolayer. However, the evaporation of CO2 and thus the transformation of bicarbonate into carbonate should preferentially occur at the edges of monolayer domains, which might serve to rationalize the preferred formation of CaCO3 particles at this location.
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Scheme 3 Subsequent stages of calcium carbonate nucleation and aggregation, and changes of peptide monolayer domains during crystallization. |
Where monolayer domains approach each other, larger aggregates of ACC particles can thus form via fusion of smaller particles being attached to edges of the monolayer domains (Scheme 3). According to results from TEM investigations CaCO3 crystals start to grow from the inside of larger ACC particles, giving rise to CaCO3 single crystals. This statement gleans (indirect) support from the fact that orientations, sizes and shapes of CaCO3 crystals observed in the present and similar model systems often show broad distributions, which lack a specific and direct correlation with regard to the structure of the templating monolayer.
ACC phases have been observed in natural nacre52 and artificial model systems.53–59 It is reported that these ACC phases are mostly stabilized by polyanions such as poly(aspartic acid)60 or poly(acrylic acid)61 in solution. It is thus reasonable to assume that a thin layer of ACC can also form beneath monolayers of acidic peptides owing to their polyanionic character. Hence, CaCO3 crystallization starts subsequent to the accumulation of amorphous particles, and there is no experimental evidence for a direct epitaxy of CaCO3 crystals emanating from a two-dimensional patch of the peptide monolayer. Our findings seem to support recent studies of multistep crystallization which in a colloidal model system demonstrated the formation of crystal nuclei of subcritical size inside an amorphous precursor phase and their subsequent fusion and transformation into single crystals.62
Subsequent to the formation of ACC particles, vaterite crystals have been observed in the early stage of all crystallization experiments performed with peptide monolayers. After 4 hours the proportion of vaterite crystals decreased significantly for peptide 1a monolayers, and the number of calcite crystals increased at the same time. We conclude that vaterite is a transient polymorph which transforms into calcite during the later stage of the crystallization process. The relatively slow transformation of vaterite into calcite suggests, however, that this process might be influenced by the monolayer. However, for peptide 1b monolayers, vaterite and calcite crystals occurred simultaneously and their relative ratio did not change significantly within the experimental period. Since a less ordered β-hairpin packing arrangement for peptide 1a is indicated by surface pressure-area isotherms, FTIR and AFM investigations, we might tentatively suggest that the more flexible nature of peptide 1a compared with peptide 1b, leads to rapid formation of vaterite nuclei under 1a monolayers and their subsequent transformation into <10.0> oriented calcite crystals. Similar results have been suggested by Kuther et al.63 for a self-assembled monolayer showing a low degree of structural organisation, which promoted the formation of aragonite over calcite under experimental conditions different to ours.
Various orientations and morphologies of calcite crystals were observed for the different peptide monolayers at the late stages of crystallization, i.e. after a few hours. For peptides 1a and 1b, XPS results show that individual peptide molecules on average bind about 4 calcium ions if being spread on an aqueous subphase containing calcium acetate (ESI S6†). Therefore, the different orientations of calcite crystals grown under 1a and 1b peptide monolayers cannot be attributed to different monolayer charge densities. AFM investigations indicate that 1b assumes a highly ordered head-to-head packing arrangement (cf. Scheme 2), while 1a self-assembles into a less well-organized packing arrangement. Based on these results, we conclude that monolayer structure flexibility, i.e. the ability of the monolayer to co-adapt to structural changes imposed by the aggregating and transforming CaCO3 particles is a crucial factor in controlling the orientations and shapes of the mature calcite single crystals.
Footnote |
† Electronic supplementary information (ESI) available: Additional AFM topography images of Langmuir–Blodgett films of peptide monolayer transferred from water subphase, BAM images of CaCO3 crystallization under monolayers of peptide 1a and 2a. Raman spectrum of crystals grown under the monolayer of peptide 1a. See DOI: 10.1039/c4dt01154f |
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