Millisecond-time-scale controlled freeze-quench for solute-intermediate analysis by solid-state NMR

Crystallization is a fundamental process in biomineralization, yet its complexity increases significantly in non-classical pathways of nucleation and growth, where numerous intermediates exist between free ions in solution and the final crystalline phase. Characterization of such intermediates can be delicate, as some processes can proceed very fast, inducing a short temporal window for their characterization. The problem is particularly true for the so-called prenucleation species, for which this difficulty is made worse due to their solubility, nanometric size and highly dynamic nature. In this communication, we introduce an innovative approach based on the "trapping" of reactive intermediates in a vitreous matrix, inspired by cryo-TEM sample preparation but adapted specifically to solid-state NMR characterization. This approach enables time-resolved analysis, since the aging time of the mineralization reaction is controlled on the millisecond time-scale using a dedicated stopped-flow device. The cryo-fixation is achieved by spraying the reacting solution into cold liquid isopentane at -145 °C, and the NMR rotors are filled with specifically designed packing tools, ensuring the control of the low temperature during the whole process. Finally, the cryo-fixed solution can be studied via low-temperature solid-state NMR by acquiring time-resolved NMR spectra as snapshots of the ongoing reaction. First, we show that phosphate solutions can be efficiently vitrified using this protocol and studied via low-temperature ³¹P solid-state NMR at -120 °C. We demonstrate that the ³¹P chemical-shift interaction of cryo-quenched solutions varies with the pH, allowing us to distinguish the different phosphate species coexisting in solution (PO₄^3-, HPO₄^2-, H₂PO₄^-and H₃PO₄) based on their chemical shift anisotropy patterns, which are characteristic of their protonation degree. The determination of the proportion of each species at varying pH levels enables us to construct a speciation diagram under our experimental conditions. We observe that the "apparent pK_a" values, i.e., the pH values for each chemical equilibrium, are slightly influenced by the low temperature, and possibly by the preparation conditions. Finally, we demonstrate that our method can be applied to study fast calcium phosphate crystallization, revealing the early stages of amorphous calcium phosphate (ACP) nucleation within just 20 ms of reaction time, as shown by ³¹P ssNMR. Importantly, the described methodology is the first step towards studying fast out-of-equilibrium solutions through trapping and then studying transient intermediate species via solid-state NMR. Indeed, proper freeze-quenching prevents the transient species from transforming and preserves their native environment, such as hydration, pH, or ionic strength. We demonstrated applications for biomineralization-relevant reactions, but in principle, any aqueous reaction can be studied.