A metastable liquid precursor phase of calcium carbonate and its interactions with polyaspartate

Abstract

Invertebrate organisms that use calcium carbonate extensively in the formation of their hard tissues have the ability to deposit biominerals with control over crystal size, shape, orientation, phase, texture, and location. It has been proposed by our group that charged polyelectrolytes, like acidic proteins, may be employed by organisms to direct crystal growth through an intermediate liquid phase in a process called the polymer induced liquid precursor (PILP) process. Recently, it has been proposed that calcium carbonate crystallization, even in the absence of any additives, follows a non classical, multi step crystallization process by first associating into a liquid precursor phase before transition into solid amorphous calcium carbonate (ACC) and eventually crystalline calcium carbonates. In order to determine if the PILP process involves the promotion, or stabilization, of a naturally occurring liquid precursor to ACC, we have analyzed the formation of saturated and supersaturated calcium carbonate bicarbonate solutions using Ca 2+ ion selective electrodes, pH electrodes, isothermal titration calorimetry, nanoparticle tracking analysis, 13 C T 2 relaxation measurements, and 13 C PFG STE diffusion NMR measurements. These studies provide evidence that, in the absences of additives, and at near neutral pH (emulating the conditions of biomineralization and biomimetic model systems), a condensed phase of liquid like droplets of calcium carbonate forms at a critical concentration, where it is stabilized intrinsically by bicarbonate ions. In experiments with polymer additive, the data suggests that the polymer is kinetically stabilizing this liquid condensed phase in a distinct and pronounced fashion during the so called PILP process. Verification of this precursor phase and the stabilization that polymer additives provide during the PILP process sheds new light on the mechanism through which biological organisms can exercise such control over deposited CaCO 3 biominerals, and on the potential means to generate in vitro mineral products with features that resemble biominerals seen in nature.

Reconciling disparate views of template-directed nucleation through measurement of calcite nucleation kinetics and binding energies

“…4). Consequently, despite the recent reports of prenucleation clusters (41)(42)(43), liquid-liquid separation (44)(45)(46), and multiphase aggregationbased pathways of calcite formation (41,42,47) in bulk solutions, the results presented here provide strong evidence that the classical theory of nucleation provides a good description of calcite formation on ionized surfaces and that interfacial energy is a useful concept even when critical nuclei are in the nanometer range. Calculations of γ assume rhombohedral nuclei.…”

The multiple effects of amino acids on the early stages of calcium carbonate crystallization

“…Section 3.1). Furthermore, it is worth noting that the influence of aspartate monomers on the initially nucleated phase is to some degree reminiscent of its polymeric analogue, given that polyaspartate is known to stabilize a metastable liquid-like precursor under certain circumstances, commonly referred to as PILP [58][59][60]. Titration experiments performed with pAsp present demonstrated the prolonged occurrence of a highly soluble phase with varying composition (K sp % 2 Á 10 À7 M 2 ), likely corresponding to PILP [46].…”

“…7. We speculate that this intermediate phase relates to a liquid-like (PILP) precursor [58][59][60], which is transient to a degree that it becomes a matter of statistics whether it can be detected or not (leading to the extremely large error bars of the average solubility value shown in Fig. 6).…”

A classical view on nonclassical nucleation

“…Wolf et al (12) performed experiments in acoustically levitated droplets and observed the formation of emulsion-like structures in transmission electron microscopy (TEM), which were proposed to be a dense liquid phase (DLP). Bewernitz et al (13) performed titration experiments at moderate pH levels, in which they supported the proposed emergence of a DLP by 13 C nuclear magnetic resonance (NMR) T 2 relaxation and 13 C pulsed field gradient stimulated-echo self-diffusion NMR measurements. Later, Wallace et al (14) developed the phase stability diagram to include regions for direct nucleation of solid CaCO 3 .…”

“…Attempts have been made to incorporate PNCs into pathways for liquid-liquid separation (13,19); nevertheless, no direct evidence for these mechanisms have been provided. Computer simulations have shown that liquid-liquid separation provides the possibility for a wide distribution of cluster sizes in solution, but no special thermodynamic status for clusters of a well-defined size was demonstrated (14).…”