43Ca freeze-quenched MAS-DNP for the characterization of transient phases involved in the non-classical formation of hydroxyapatite

Calcium orthophosphates, such as hydroxyapatite (HAP), have been shown to follow non-classical nucleation and crystallization mechanisms involving intermediate species, including solute prenucleation species (PNS) that precede solid transient phases. Characterizing these transient soluble and solid phases in situ remains a challenge due to their short-lived nature. In this study, we combine 43Ca isotopic enrichment with dynamic nuclear polarization (MAS-DNP) under cryogenic conditions to enhance the 43Ca sensitivity and capture transient calcium species in their native state in the course of HAP formation. Fine calibration of the Hartmann-Hahn cross-polarization conditions (low vs high radio-frequency powers) enables to distinguish different calcium-based phases based on the strength of their 43Ca quadrupolar couplings. Hence, we can record the 43Ca NMR fingerprint of sub-millimolar concentrations of PNS and two dimensional 1H-43Ca correlation NMR spectra of calcium phosphate solid phases can be recorded within hours. Thanks to this 2D analysis, the first nucleating solid phase is identified as an amorphous calcium phosphate (ACP) rich in acidic orthophosphate ions and in water molecules. Moreover, the core-layer organization of the final HAP phase is also evidenced allowing to distinguish the 43Ca spectral fingerprints of the crystalline hydroxyapatite core from the amorphous hydrated layer. This work demonstrates the potential of ⁴³Ca MAS-DNP for characterizing the complex nucleation and growth mechanisms of calcium-containing materials, thus offering new opportunities for studying similar systems in environmental and biological contexts.