Application of Electron Spin Resonance in biophysics:: from rapid mixing stopped-flow to high-hydrostatic pressure ESR

We report a novel technological approach that enables one to combine Electron Spin Resonance (ESR) spectroscopy with rapid mixing and stopped-flow techniques for performing measurements of kinetics of chemical reactions and biophysical processes. This technology involves implementation of the recently developed miniature microwave resonant structures that are designed around commercially available high dielectric ceramic resonators (DRs). For aqueous samples measured in stopped-flow conditions this approach resulted in a considerably better sensitivity and time resolution than that obtained with traditional ESR probe heads equipped with voluminous flow flat-cells. The DR-based systems made it possible to follow kinetics of chemical reactions and biophysical processes with micromolar sample concentrations and temporal resolution < 1 ms. Similar technological approach involving implementation of the DR-based resonant structures having large side-access enabled us to perform sensitive ESR measurements under high-hydrostatic pressures. A combination of Diamond-Anvil Cell (DAC) or inherently cheaper Sapphire-Anvil Cell (SAC) with our wide-stretched DR-based resonant microwave structures resulted in simpler overall design and higher sensitivity as compared with traditional high-pressure ESR probe heads that were built around conventional microwave cavities. The DR-based high-pressure ESR system that contains a miniature SAC operates now at pressures up to 2 GPa and in the temperature range of 25 - 320 K. This newly developed high-pressure ESR probe found its applications in spin resonance study of crystalline and polycrystalline solids, as well as of paramagnetic liquids, like aqueous solutions of free radicals and spin-labeled proteins.