Monitoring of CaCO₃ Nanoscale Structuration through Real-Time Liquid Phase Transmission Electron Microscopy and Hyperpolarized NMR

Abstract

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.

43Ca MAS-DNP NMR of Frozen Solutions for the Investigation of Calcium Ion Complexation

“…We investigated two different molar ratios in solution: [ l -Asp]/[Ca 2+ ] = 1:2 (Ca-LAsp-50) and 1:1 (Ca-LAsp-100). We have recently shown that in solution, one calcium ion binds with one aspartic acid molecule with a supposed preferential complexation site on the C 1 from aspartic acid . The 43 Ca MAS-DNP NMR spectra (HP CP condition) of the three samples (Ca-LAsp-50, Ca-LAsp-100 and Ca–H 2 O) were found to be very similar (Figure A) with a 43 Ca chemical shift of −9.6 ppm and a line width of 20.2 ppm for Ca-LAsp-50 and Ca-LAsp-100.…”

“…We have recently shown that in solution, one calcium ion binds with one aspartic acid molecule with a supposed preferential complexation site on the C 1 from aspartic acid. 64 The 43 Ca MAS-DNP NMR spectra (HP CP condition) of the three samples (Ca-LAsp-50, Ca- LAsp-100 and Ca−H 2 O) were found to be very similar (Figure 5A) with a 43 Ca chemical shift of −9.6 ppm and a line width of 20.2 ppm for Ca-LAsp-50 and Ca-LAsp-100. Only a slight increase of the 43 Ca chemical shift (<1 ppm, Figure S7) is observed when Ca 2+ is complexed to L-Asp when compared to water molecules.…”

Characterization of Nanomaterials Using In Situ Liquid-Cell Transmission Electron Microscopy: A Review

“…With the development of liquid-phase TEM technology, it has been extended to more emerging fields, such as real-time observations of nanostructure synthesis in organic solvents − and mineralization processes in biomimetic media. − However, limited by more advanced characterization techniques, few relevant studies and references have been reported, and these complicated mechanisms remain poorly understood on the nanoscale. Other examples may include imaging unlabeled biostructures, soft materials, dynamical phenomena, and photocatalysis behavior. ,− ,− In the future, an increasing number of research groups will engage in related research of in situ liquid-cell TEM.…”

Shedding Light on the Birth of Hybrid Perovskites: A Correlative Study by In Situ Electron Microscopy and Synchrotron-Based X-ray Scattering

“…The possibility of using these techniques as in situ characterization tools is now considered a major step forward for monitoring, with high spatial resolution, chemical and physical phenomena using TEM. A wide range of processes can be now directly visualized using in situ TEM, to cite a few: biomineralization of calcium carbonate, 41 morphological evolution of catalysts during electrochemical reaction, 42,43 nucleation and growth of particles, 44−46 etc. Regarding the perovskite field and the thousands of studies published on this topic each year, only a handful reported the use of this tool, and their focus is mainly on monitoring the degradation of the material exposed to different factors such as moisture, light, and electron beam radiation.…”

“…The possibility of using these techniques as in situ characterization tools is now considered a major step forward for monitoring, with high spatial resolution, chemical and physical phenomena using TEM. A wide range of processes can be now directly visualized using in situ TEM, to cite a few: biomineralization of calcium carbonate, morphological evolution of catalysts during electrochemical reaction, , nucleation and growth of particles, – etc. Regarding the perovskite field and the thousands of studies published on this topic each year, only a handful reported the use of this tool, and their focus is mainly on monitoring the degradation of the material exposed to different factors such as moisture, light, and electron beam radiation. – To the best of our knowledge, to date, only two articles focused on the growth of LHPs NCs using in situ TEM as the main tool: Wang et al studied the growth dynamic of MAPI films with the influence of urea in the precursor solution, whereas Qin et al reported the first observations of nucleation and growth of MAPI NCs using the electron-beam assisted solvent evaporation approach.…”