Exploring Non-Classical Crystallization Pathways—Cryo-Stopped Flow Combined to Solid-State NMR for the Analysis of Calcium Phosphate Pre-Nucleation Species

The crystallization phenomenon is a ubiquitous physiochemical process covering many natural and synthetic activities in our everyday lives, impacting many scientific areas, from organic to inorganic chemistry and material science.¹ Description of crystallization has relied on the classical nucleation and growth theory (CNT) for many decades. However, CNT has been challenged in recent years by the observation of transient soluble species formed before the nucleation event, suggesting the existence of an alternative route, the so-called non-classical nucleation pathway (NCP). This pathway depends on the formation of stable and soluble pre-nucleation species (PNS) in the form of droplets, clusters, or oligomers. In a biomineralization context, these PNS have been described and reported for calcium phosphates,² carbonates³ and oxalates phases.⁴ Characterisation of such species is challenging due to their solubility, nanometric size, dynamic nature, and short lifespan, necessitating innovative real-time monitoring techniques.<br/>To address this challenge, we introduce a novel approach where solid-state Nuclear Magnetic Resonance (ssNMR) analysis is coupled with a cryo-stopped flow methodology to track the evolution of PNS of calcium phosphate over time, providing insights into their structure and composition. This approach involves two steps: 1) the precise, controlled addition and mixing of calcium and phosphate solutions enabled by a stopped-flow device; 2) followed by rapid freezing to halt the reaction at specific time-points on the milliseconds time scale. This crucial step is allowed by spraying the aqueous solution into cold iso-pentane (-145°C), leading to cryofixed matrices where PNS are stabilized. Subsequent analysis using low-temperature ssNMR enables the interrogation of transient PNS at the atomic/molecular level. Here, we detail our original setup and optimization of experimental conditions to investigate calcium phosphate PNS during the early stages of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) formation, a key component of bone and teeth. We also used low-temperature XRD and optical microscopy to characterize the vitrified micrometric droplets produced by the cryo-stopped flow device. This novel methodology offers a powerful tool for unraveling the complexities of pre-nucleation species, shedding light on fundamental processes underlying crystallization processes. Recently, we successfully used this approach to investigate amino-acid stabilized pre-nucleation clusters and dense liquid phases of CaCO_3, although the vitrification process was not automatized.⁵<br/><br/><b>References:</b><br/>[1] J. J. De Yoreo, <i>et. al.</i>, <i>Science</i>, 2015, <b>349 </b>(6247)<br/>[2] A. Dey, <i>et.al.</i>, <i>Nat. Mater.</i>, 2010, <b>9</b>, 1010–1014.<br/>[3] D. Gebauer, <i>et.al</i>., <i>Science</i>, 2008, <b>322</b>, 1819–1822.<br/>[4] E. Ruiz-Agudo, <i>et.al.</i>, <i>Nat. Commun.</i>, 2017, <b>8</b>, 768.<br/>[5] V. Ramnarain, <i>et.al.</i>, <i>J. Am. Chem. Soc.</i>, 2022, <b>144</b>, 15236–15251.