Biomineralization is a process in nature by which proteins regulate the crystallization of inorganic minerals by controlling nucleation pathways, attachment dynamics, and growth kinetics to produce nanostructured protein-mineral composites endowed with remarkable function. Unfortunately, controlling the synthesis of similarly endowed hierarchical nanostructures in the laboratory remains a great challenge due to limited mechanistic understandings of how proteins direct multi-step nucleation pathways and particle assembly. In an effort to develop that understanding, we combined in situ liquid-phase transmission electron microscopy (LP-TEM) with in situ liquid-phase attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and ex situ TEM to investigate the nucleation and growth of CaCO3 in the presence of de novo designed acidic proteins that present arrays of negatively charged carboxylate groups in patterns expected to mimic the patterns of calcium sites on various faces of the CaCO3 polymorphs. While protein-free solutions generate vaterite and addition of less ordered acidic proteins and polymers, like bovine serum albumin and poly acrylic acid, respectively, produces amorphous CaCO3 followed by vaterite, our results show that the introduction of the designed proteins drives direct nucleation of calcite nanocrystals with a size of ~3.3 nm. LP-TEM results show that the formation of Ca-protein assemblies provide an interface to direct calcite nanocrystals nucleation, that is interface-driven nucleation, followed by their assembly into larger (110) facets dominant crystals (~10-20 nm) and final micron-scale rhombohedral calcite through oriented attachment of the primary calcite crystals. These investigations address the significant roles of protein in the nucleation through a cooperative interaction: concentrated Ca2+ by proteins binding to increase local supersaturation and enhanced assembly of protein by Ca2+ to provide an interface that promote calcite nanocrystal nucleation, and protein is critical to later growth through stabilizing the calcite nanocrystals and inhibiting their direct growth into large faceted rhombohedral calcite as well as directing their oriented attachment. This work defines a novel approach both for understanding how proteins affect CaCO3 nucleation and growth to achieve controllable crystal polymorphs and for achieving biomimetic synthesis of functional nanomaterials.

Understanding regulation of CaCO3 crystallization by de novo designed proteins through in situ TEM imaging and spectroscopy

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