Dynamic Nuclear Polarization Enhanced Multiple Quantum Spin Counting of Molecular Assemblies in Vitrified Solutions

Crystallization pathways are essential to various industrial, geological, and biolog- ical processes. In nonclassical nucleation theory, prenucleation clusters (PNCs) form, aggregate and crystallize to produce higher order assemblies. Microscopy and X-Ray techniques have limited utility for PNC analysis due to small size (0.5 - 3 nm) and time stability constraints. We present a new approach for analyzing PNC formation based on 31P NMR spin counting of vitrified molecular assemblies. The use of glassing agents en- sures that vitrification generates amorphous aqueous samples, and offers conditions to perform dynamic nuclear polarization (DNP) amplified NMR spectroscopy. We demon- strate that molecular adenosine triphosphate, along with crystalline, amorphous, and clustered calcium phosphate materials formed via a non-classical growth pathway can be differentiated from one another by the number of dipolar coupled 31P spins. We also present an innovative approach to examine spin counting data, demonstrating that a knowledge based fitting of integer multiples of cosine wave functions, instead of the traditional Fourier transform, provides a more physically meaningful retrieval of the ex- isting frequencies. This is the first report of multi-quantum spin counting of assemblies formed in solution as captured under vitrified DNP conditions, which can be useful for future analysis of PNCs and other aqueous molecular clusters.