Simulated body fluids (SBFs) that mimic human blood plasma are widely used media for in vitro studies in an extensive array of research fields, from biomineralization to surface and corrosion sciences. We show that these solutions undergo dynamic nanoscopic conformational rearrangements on the timescale of minutes to hours, even though they are commonly considered stable or metastable. In particular, we find and characterize nanoscale inhomogeneities made of calcium phosphate (CaP) aggregates that emerge from homogeneous SBFs within a few hours and evolve into prenucleation species (PNS) that act as precursors in CaP crystallization processes. These ionic clusters consist of ∼2 nm large spherical building units that can aggregate into suprastructures with sizes of over 200 nm. We show that the residence times of phosphate ions in the PNS depend critically on the total PNS surface. These findings are particularly relevant for understanding nonclassical crystallization phenomena, in which PNS are assumed to act as building blocks for the final crystal structure.
Calcium carbonate (CaCO3) is one of the most significant biominerals in nature. Living organisms are able to control its biomineralization by means of an organic matrix to tailor a myriad of hybrid functional materials. The soluble organic components are often proteins rich in acidic amino-acids such as l-aspartic acid. While several studies have demonstrated the influence of amino acids on the crystallization of calcium carbonate, nanoscopic insight of their impact on CaCO3 mineralization, in particular at the early stages, is still lacking. Herein, we implement liquid phase-transmission electron microscopy (LP-TEM) in order to visualize in real-time and at the nanoscale the prenucleation stages of CaCO3 formation. We observe that l-aspartic acid favors the formation of individual and aggregated prenucleation clusters which are found stable for several minutes before the transformation into amorphous nanoparticles. Combination with hyperpolarized solid state nuclear magnetic resonance (DNP NMR) and density functional theory (DFT) calculations allow shedding light on the underlying mechanism at the prenucleation stage. The promoting nature of l-aspartic acid with respect to prenucleation clusters is explained by specific interactions with both Ca2+ and carbonates and the stabilization of the Ca2+–CO3 2–/HCO3 – ion pairs favoring the formation and stabilization of the CaCO3 transient precursors. The study of prenucleation stages of mineral formation by the combination of in situ LP-TEM, advanced analytical techniques (including hyperpolarized solid-state NMR), and numerical modeling allows the real-time monitoring of prenucleation species formation and evolution and the comprehension of their relative stability.
Barnacles are convenient model organisms to study both biomineralization and bioadhesion phenomena. They secrete a proteinaceous adhesive from cement proteins (CPs) to strongly attach to solid immersed substrates. More recently, it has been suggested that some CPs also play a key role in regulating the calcification of barnacles’ protective shells. In this study, combining both solution and solid-state NMR spectroscopy, Raman and infrared spectroscopy studies, and atomic force microscopy (AFM) and transmission electron microscopy (TEM) imaging, we have explored the CaCO3 mineralization pathway regulated by Megabalanus rosa CP 20 (MrCP20). Our data show that MrCP20 can sequestrate inorganic calcium and carbonate ions from the solution state, which quickly coarsen into liquid-like microdroplets and subsequently form protovaterite amorphous CaCO3 (ACC) particles. This pathway leads to the stabilization of the metastable vaterite polymorphism of CaCO3. Simultaneously, AFM and TEM investigations show that MrCP20 undergoes fibrillization triggered by a pH drift arising during CaCO3 mineralization, leading to amyloid-like nanofibrils. Based on protein NMR, this mechanism appears to be stabilized by the reduction of intramolecular disulfide bonds. Collectively, our results demonstrate that MrCP20 plays a synergistic role of regulating CaCO3 biomineralization while concomitantly self-assembling into adhesive nanofibrils.
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