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Confinement effects promote the formation of aragonite

Related publication: Zeng M., Meldrum F. C., Kim Y.-Y., Laundy D., Frontera C., Anduix-Canto C., Kapur N. & Christenson H. K. Confinement generates single-crystal aragonite rods at room temperature. Proc. Natl. Acad. Sci. 115, 7670–7675 (2018). DOI: 10.1073/ pnas.1718926115

Publication keywords: Calcium carbonate; Biomineralisation; Bio-inspired crystallisation; Biomimetic; Confinement

Aragonite is a common crystalline form (polymorph) of calcium carbonate, an important biomineral found, for example, in seashells. Outside of the natural environment, aragonite usually only crystallises from solution at high temperatures, or in the presence of magnesium ions. However, although organisms can readily form aragonite, the way in which they do so remains unclear.

A team of researchers from the University of Leeds, the Institut de Ciencia de Materials de Barcelona, and Diamond Light Source explored whether the confined volumes in which all biomineralisation occurs allows organisms to control calcium carbonate production.

To investigate how confined volumes affect the precipitation of calcium carbonate, they precipitated calcium carbonate in the cylindrical pores of commercially-available filtration membranes. As these membranes are available with a wide range of pore diameters, the researchers were able to systematically investigate the relationship between the pore diameter and the calcium carbonate polymorph formed.

The team only had small volumes of sample material to work with, and so used the micro-focus capability of the Test beamline (B16) to obtain powder X-ray diffraction patterns. They found that oriented single crystals of aragonite were the sole product from additive-free solutions in 25 nm pores, and significant quantities of aragonite formed in pores as large as 200 nm in the presence of low concentration of magnesium and sulphate ions. This study shows that confinement effects can promote the formation of aragonite, and suggests that organisms may exploit confinement effects to control calcium carbonate formation.

: Figure 1: Calcium carbonate rods precipitated within pores of diameter 200 nm.

Organisms exhibit remarkable control over mineralisation processes, generating biominerals with complex morphologies, hierarchical structures, and outstanding mechanical properties¹. However, while many of nature’s strategies for controlling biomineralisation are known, the mechanisms by which organisms control crystal structure are poorly understood. While it was long believed that the proteins entrapped within biominerals would hold the key, precipitation of calcium carbonate in the presence of proteins extracted from calcite or aragonite biominerals (where these are “polymorphs” of calcium carbonate, having the same composition but different crystal structure) failed to deliver polymorph control. The work described here explores an alternative explanation. It is well known that biominerals invariably form within small volumes, and that these can strongly influence crystallisation processes^2,3. A simple system – crystallisation within the cylindrical pores of commercial filtration membranes – was therefore used to systematically investigate how confinement influences calcium carbonate polymorph.

Experiments were conducted in the absence of additives, and in the presence of low concentrations of sulphate and magnesium ions. These are significant components of the seawater in which many organisms live, and can promote aragonite formation. It would therefore be surprising if they do not contribute to aragonite formation in vivo. Calcium carbonate was precipitated within track-etched membranes by placing membranes between two half U-tube arms, one which was filled with a solution containing calcium and magnesium ions, and the other with a solution containing carbonate and sulphate ions. Calcium carbonate is precipitated when the calcium and carbonate ions meet in the pores⁴.

Reaction conditions containing low concentrations of magnesium and sulphate ions were used, where these produced only a few percent aragonite in bulk solution. Rod-shaped crystals with lengths comparable to the membrane thickness formed in the pores (Fig. 1). The structure of these crystals was then investigated. For crystals formed in the larger pores, it was possible to extract sufficient material to carry out powder X-ray diffraction – a method that fingerprints the structure of crystals – using a laboratory instrument. This showed that the crystals formed within the 1,200 nm pores were almost entirely calcite, while those in the 800 nm pores were 81% calcite and 19% aragonite. Further reduction in the pore size to 200 nm increased the proportion of aragonite to 69%.

: Figure 2: Synchrotron micro-beam pXRD pattern of aragonite crystals precipitated in 25 nm
pores in the absence of additives.

In order to characterise the polymorphs of the crystals formed in smaller pores, however, it was necessary to use synchrotron micro-beam XRD, where this was conducted at beamline B16. The analysis showed that aragonite was the only polymorph present in the 50 nm and 25 nm pores (Fig. 2). Individual rods grown in the 200, 50 and 25 nm pores were also investigated using electron diffraction (performed in a transmission electron microscope) to gain further information on their structures, and to identify if they were oriented with respect to the pore. The aragonite crystals grown in the 200 nm pores were polycrystalline and showed no preferential orientation. Those in the 50 nm pores, in contrast, were almost single crystals, while the 25 nm rods were single crystals (Fig. 3). These aragonite crystals were also preferentially oriented with their crystallographic c-direction parallel to the pore axis, such that 50% and 100% of rods in the 50 and 25 nm pores were oriented in this way.

Finally, experiments were conducted in the absence of magnesium and sulphate ions. Very little aragonite formed in the 1,200 and 800 nm pores, just 8% in the 200 nm pores, and a significant increase to 47% was recorded in the 50 nm pores. Precipitation within the 25 nm pores, however, yielded a most unexpected result – aragonite single crystals formed in the absence of any additives in these very small pores.

These results therefore demonstrate that confinement can promote the formation of aragonite such that all of the crystals precipitated in 25 nm pores were aragonite under additive-free conditions. This effect is enhanced in the presence of low concentrations of magnesium and sulphate ions. But what are the origins of these effects? Calcite/aragonite polymorphism is a complex problem that has challenged researchers for decades. The production of these mineral phases from solution is dependent on kinetics as well as thermodynamics, and thus on variables including temperature, concentration, and the presence of additives. While aragonite is only slightly less thermodynamically stable than calcite at room temperature, it typically only appears as a minor product on precipitation from additive-free solutions at room temperature.

$Figure 3: Transmission Electron Microscope (TEM) image and electron diffraction pattern <br/>(inset) of an aragonite rod precipitated within a 50 nm pore.$: Figure 3: Transmission Electron Microscope (TEM) image and electron diffraction pattern
(inset) of an aragonite rod precipitated within a 50 nm pore.

The possibility that the small pores affect the transport of ions to growing crystals – which may influence polymorph⁵– was explored by modelling the diffusion of ions through the membrane pores. This showed that the calcium and carbonate solutions were fully mixed in under 0.1 sec, where mixing is rapid due to the short length of the pores. This cannot then be the origin of the polymorph change. The membrane surface may also have a significant influence on crystal formation. This was explored by analysing our data to explore the relationship between the aragonite fraction and the pore diameter. A graph of the aragonite fraction versus the inverse of the pore diameter revealed a roughly linear relationship between these quantities. This is expected if the number of aragonite nucleation sites is proportional to the surface area. Thus, as the surface increases in importance relative to the bulk with the degree of confinement, the proportion of aragonite increases. The distribution of ions adjacent to a charged membrane surface can also differ significantly in a membrane pore as compared to a planar surface, which may contribute to the effects seen.

This study demonstrates that confinement can promote the formation of aragonite, and that this is intimately linked to the properties of the confining surface. These results are of particular significance to calcium carbonate biomineralisation, which invariably occurs in privileged environments bound by organic matrices. Confinement effects may enhance the influence of such organic frameworks on crystallisation, enabling organisms to achieve superior control over characteristics such as polymorph or orientation.

References:

Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry (2002). ISBA:9780198508823
Stephens C. J. et al. Amorphous calcium carbonate is stabilized in confinement. Adv. Funct. Mater. 20, 2108–2115 (2010). DOI: 10.1002/ adfm.201000248
Anduix-Canto C. et al. Effect of nanoscale confinement on the crystallization of potassium ferrocyanide. Cryst. Growth Des. 16, 5403–5411 (2016). DOI: 10.1021/acs.cgd.6b00894
Loste E. et al. Precipitation of calcium carbonate in confinement. Adv. Funct. Mater. 14, 1211–1220 (2004). DOI: 10.1002/adfm.200400268
Prieto M. Nucleation and supersaturation in porous media (revisited). Mineral. Mag. 78, 1437–1447 (2015). DOI: 10.1180/ minmag.2014.078.6.11

Funding acknowledgement:

Engineering and Physical Sciences Research Council (EPSRC) (EP/N002423/1 and EP/J018589/1); Leverhulme Trust.

Corresponding author:

Fiona Meldrum, University of Leeds, [email protected]

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Confinement effects promote the formation of aragonite