Short-lived calcium carbonate precursors observed in situ via Bullet-dynamic nuclear polarization

The classic nucleation-and-growth theory (CNT) describes crystallization as a two-step process: nucleation followed by crystal growth. However, the discovery of metastable pre-nucleation clusters (PNC) has challenged this paradigm. PNC are transient, metastable ionic assemblies that form in solution before the onset of crystallization. While PNC have been observed for various systems including calcium phosphates and carbonates under conditions of mild oversaturation, their characterization under high oversaturation remains challenging due to their extremely short lifetimes (<<5s). This study aims to address this gap by utilizing Bullet-DNP, a technique that enhances NMR signals sufficiently to enable the detection of these short-lived species. Understanding the early stages of calcium carbonate (CaC) formation is crucial for fundamental material science and biomaterial design, as CaC is a major component of many biological materials. The high-oversaturation regime is particularly relevant as it may reveal undocumented precipitation pathways and non-classical crystallization phenomena. Previous attempts using dissolution dynamic nuclear polarization (DDNP) were limited by the pressure sensitivity of carbonate, leading to its loss during sample transfer. This study overcomes this limitation using Bullet-DNP, which allows for the in situ detection of short-lived precursors.

Extensive research has explored pre-nucleation clusters (PNCs) in various systems. Studies have shown the existence of PNCs in calcium phosphates and carbonates, crucial components of biominerals. These studies highlight the importance of understanding PNCs for both fundamental material science and biomaterial design. Researchers have reported the coalescence and dehydration of PNCs, leading to the formation of solid phases. This mechanism challenges the classical nucleation-and-growth theory (CNT). The debate on reconciling PNC observations with CNT is hampered by a lack of experimental evidence, particularly from the high-oversaturation regime. Most previous studies focused on mild oversaturation conditions, where PNCs exhibit longer lifetimes and are experimentally accessible. However, high-concentration regimes, where lifetimes are drastically reduced, have remained largely unstudied. Recent work proposed using NMR enhanced by DDNP to access these short-lived precursors, but this proved challenging for CaC due to the pressure sensitivity of carbonate during sample transfer.

This study employed Bullet-DNP, a technique that overcomes the limitations of traditional DDNP. In Bullet-DNP, a hyperpolarized sample (50 µL of 500 mM ¹³C-enriched sodium carbonate) is prepared and frozen. This frozen pellet is then ‘shot’ into an NMR spectrometer where it is dissolved in a calcium chloride solution (500 µL of 20 mM CaCl2 in 50 mM HEPES buffer), creating a highly supersaturated solution. The rapid mixing and subsequent NMR detection allow for the observation of very short-lived species. The experimental setup includes a custom-designed injector that prevents degassing of the carbonate during transfer and dissolution. Three resonances were observed: free carbonate at δ(¹³C) = 160.4 ppm, and two additional ¹³C resonances centered around δ(¹³C) = 162.3 ppm and δ(¹³C) = 168.9 ppm. A weak CO₂ resonance was also observed at 125.9 ppm. The data was corrected for convection using hyperpolarized glycerol as an internal standard. Linewidths were analyzed to determine the size of the PNS. The time-dependent signal intensities revealed the effective relaxation rates (<i>R</i>_eff) of the different species and provided insight into their conversion kinetics. The experiments were repeated at pH 7 and 8 to investigate the pH dependence of the process. Powder X-ray diffraction was used to characterize the final solid product. Signal enhancement factors were determined by comparing hyperpolarized and thermal equilibrium spectra. Methodological considerations focused on potential gas bubble formation and sample homogeneity issues.

Bullet-DNP successfully enabled the characterization of short-lived CaC precursors (lifetimes ≤5 s) under high oversaturation conditions. Two distinct CaC PNS were identified based on their chemical shifts and linewidths, suggesting differences in size and composition. The species at δ(¹³C) ≈ 162.3 ppm was attributed to a smaller PNS, possibly bicarbonate-based, while the broader species at δ(¹³C) ≈ 168.9 ppm was assigned to larger clusters with reduced rotational mobility. The effective relaxation rates (<i>R</i>_eff) for the three observed species (free carbonate and the two PNS) were determined, revealing faster relaxation rates for the Ca²⁺-bound species. Experiments at pH 6, 7, and 8 showed that higher pH resulted in a faster conversion of small clusters into larger aggregates. Powder X-ray diffraction indicated a pH-dependent formation of vaterite and calcite. The observed PNS did not lead to one specific solid product. A significant signal enhancement of ≈52,700 was achieved for the free carbonate and the narrow PNS resonance at pH 6. Control experiments using traditional DDNP systems resulted in the complete loss of carbonate due to degassing, highlighting the advantage of Bullet-DNP. The results show that the formation mechanism observed under mild oversaturation extends to the high-oversaturation regime but occurs at much faster kinetics.

The findings demonstrate the feasibility of using Bullet-DNP to study very fast material formation processes. The identification of two distinct PNS strongly supports the non-classical crystallization pathways previously observed under mild oversaturation. These pathways seem to be relevant even at high concentrations, although the faster kinetics might lead to some events falling outside the time resolution of the technique. The observed pH-dependent ratios of the two PNS and the final product (vaterite/calcite) suggest complex interactions between pH, precursor species, and final solid morphology. The results are consistent with existing models suggesting the existence of multiple CaC precursor species. Future research could incorporate molecular dynamics simulations to complement the experimental data, given that MD simulations are well-suited to studying processes at the microsecond to millisecond timescale, and high concentrations reduce computational costs.

This study successfully applied Bullet-DNP to identify and characterize short-lived calcium carbonate pre-nucleation species under high oversaturation. Two distinct PNS were observed, confirming non-classical crystallization pathways extend to this previously inaccessible regime. This advancement in methodology expands the possibilities for studying a range of pressure-sensitive systems and may lead to novel designs of CaC-based materials with tailored morphologies and properties. Future work should focus on integrating molecular dynamics simulations and applying advanced NMR techniques such as ultrafast exchange and diffusion spectroscopy.

The rapid precipitation of the sample complicated the accurate determination of signal enhancement factors and introduced potential biases due to concentration gradients. The presence of glycerol as an internal standard might influence the CaC formation kinetics. The time resolution of the Bullet-DNP technique may limit the complete observation of all steps in the precipitation process, particularly the very early stages. The exact nature and composition of the two identified PNS could benefit from further investigation.