A Quasi-Time-Resolved CryoTEM Study of the Nucleation of CaCO₃under Langmuir Monolayers

Abstract

Calcium carbonate biomineralization uses complex assemblies of macromolecules that control the nucleation, growth, and positioning of the mineral with great detail. To investigate the mechanisms involved in these processes, for many years Langmuir monolayers have been used as model systems. Here, we descibe the use of cryogenic transmission electron microscopy in combination with selected area electron diffraction as a quasi-time-resolved technique to study the very early stages of this process. In this way, we assess the evolution of morphology, polymorphic type, and crystallographic orientation of the calcium carbonate formed. For this, we used a self-assembled Langmuir monolayer of a valine-based bisureido surfactant (1) spread on a CaCl2-containing subphase and deposited on a holey carbon TEM grid. In a controlled environment, the grid is exposed to an atmosphere containing NH3 and CO2 (the (NH4)2CO3 diffusion method) for precisely determined periods of time (reaction times 30-1800 s) before it was plunged into melting ethane. This procedure allows us to observe amorphous calcium carbonate (ACC) particles growing from a few tens of nanometers to hundreds of nanometers and then crystallizing to form [00.1] oriented vaterite. The vaterite in turn transforms to yield [10.0] oriented calcite. We also performed the reaction in the absence of monolayer or in the presence of a nondirective monolayer of surfactant containing an oligo(ethylene oxide) 2 head group. Both experiments also showed the formation of a transient amorphous phase followed by a direct conversion into randomly oriented calcite crystals. These results imply the specific though temporary stabilization of the (00.1) vaterite by the monolayer. However, experiments performed at higher CaCl2 concentrations show the direct conversion of ACC into [10.0] oriented calcite. Moreover, prolonged exposure to the electron beam shows that this transformation can take place as a topotactic process. The formation of the (100) calcite as final product under different conditions shows that the surfactant is very effective in directing the formation of this crystal plane. In addition, we present evidence that more than one type of ACC is involved in the processes described.

Mechanism of Calcite Co-Orientation in the Sea Urchin Tooth

“…28 Our results in the sea urchin tooth are in agreement with the Sommerdijk group's results from synthetic calcite growth, templated by an organic Langmuir monolayer, and observed with time resolution during the transition. 29 In both systems, ACC-to-calcite is a solid-state transformation, more than one type of ACC is involved, and no bonds are broken in the process.…”

Solid‐State Transformation of Amorphous Calcium Carbonate to Aragonite Captured by CryoTEM

“…Here, we used a water and alcohol mixture to stabilize the formation of aragonite to study the crystallization mechanisms using cryo‐transmission electron microscopy (cryoTEM). This technique allows the direct visualization of the early stages of the crystallization process while maintaining intermediate reaction products in their native, hydrated state 8. Utilizing this method, we demonstrate that aragonite formation in this highly supersaturated system progresses via amorphous precursor particles that aggregate to form a crystal, going through an intermediate in which semi‐oriented crystalline domains co‐exist with the amorphous phase.…”

A Biomimetic Nano-Scale Aggregation Route for the Formation of Submicron-Size Colloidal Calcite Particles

“…Some of them have been focused on exploring the promoting effect of matrices (templates) on the crystals' nucleation and growth (Popescu et al, 2007;Tremel et al, 2007). Several procedures have been developed, depending on the structural complexity of the templates used, such as self-assembled monolayers (Aizenberg et al, 1999;An and Cao, 2008), Langmuir monolayers (Heywood & Mann, 1994;Pichon et al, 2008), and gelatin films (Martinez-Rubi et al, 2008). Several studies have also shown that the formation of biogenic calcium carbonate structures is controlled by organic macromolecules (matrix proteins), mostly peptides and proteins, which are directly involved in regulating the nucleation, growth, and morphology of the precipitates.…”