Focused Ion Beam Tomography Reveals the Presence of Micro‑, Meso‑, and Macroporous Intracrystalline Regions Introduced into Calcite Crystals by the Gastropod Nacre Protein AP7

Intracrystalline modification of calcium carbonates by macromolecules is a fascinating process that offers insights into potential pathways for modifying the material properties of inorganic solids. Recently, we reported on the induction of intracrystalline nanoporosities within calcite by the nacre layer intracrystalline protein, AP7 (Haliotis rufescens). In this report we revisited this AP7-mediated phenomenon and tracked time-dependent intracrystalline porosity formation during in vitro mineralization using FIB/SEM serial milling. We find that AP7 induces intracrystalline nanoporosities as early as 1 min of elapsed assay time. Quantitation of pore regions confirms that average cross-sectional volume (ACSV), average void volume (AVV), and percent porosity parameters increase over time, leading to the formation of porous calcite crystals with a high surface-to-volume ratio. FIB serial milling, SEM imaging, and 3-D tomography revealed the presence of unexpected semicontinuous channels and cavities in the subsurface regions of a representative 60 min assay crystal. The random locations of these intracrystalline features are limited to the top and sides of the calcite crystal, which correspond to the sites of AP7 protein phase deposition during mineral formation. This random porosity distribution was also documented for protein-containing voids within nacre aragonite tablets in situ. In some instances we observed geometric relationships between adjacent channels and cavities. Interestingly, all three IUPAC-defined material porosity categories (micro-, meso-, and macro-) were represented in the AP7-treated crystals. Thus, the deposition of AP7 protein phases onto calcite surfaces induces surface nanoparticle nucleation and subsurface multiscale intracrystalline porosities and interconnected channels.