Electron spin resonance (ESR) spectroscopy is employed in many fields of science to study paramagnetic materials (i.e., having unpaired electron spins).  One of ESR’s most prominent application field is structural biology. This research focused on the development of three new methodological approaches to enhance the capabilities of ESR in this field of science.

The first approach is related to ESR and electron nuclear double resonance (ENDOR) measurements of single crystal proteins, which are powerful tools for characterizing their structure. However, conventional ESR systems lack sensitivity and require large single crystals (~1mm) with many (>1012) spins.  In this work, we developed a method to obtain ESR signal from very small micro-crystals (20-200 μm), using specially-tailored miniature microwave resonators.  Resonator design are optimized for a specific sample geometry. The resonators’ microwave mode were characterized by high resolution ESR imaging and compared to theoretical calculation. These novel resonators could be used to measure proteins that cannot be crystallized to a large mm-size single crystal, hopefully leading to a more complete picture of biological phenomena and to the development of new drugs and bio-inspired molecular machines.

The second approach involves the measurement of liquid samples with small volume using microfluidics. Microfluidics is a well-established technique to synthesize, process, and analyze small amounts of materials for chemical, biological, medical, and environmental applications. Typically, it involves the use of reagents with a volume smaller than ~1 µl ideally even nano or picoliters. When the sample of interest contains paramagnetic species, it can in principle be quantified and analyzed by ESR spectroscopy. However, conventional ESR is typically carried out with a sample volume of ~1 ml, thereby making it incompatible with most microfluidics applications. In this work we show that ESR can be applied to measure small liquid samples, down to picoliter volumes, without considerably sacrificing concentration sensitivity. Our experiments, carried out with resonators whose mode volumes range from ~1 to 3.6 nL, showed that with a sample volume of ~0.25 nL good signals could be obtained from solutions with spin concentrations of less than 0.1 μM.

The third approach involves measurement of molecular diffusion using pulsed magnetic field gradients. To better understand chemical and biological systems the diffusion coefficient can be measured by several methods including nuclear magnetic resonance (NMR), dynamic light scattering (DLS) and pulsed gradient spin echo (PGSE) ESR. Previously, the capabilities of PGSE ESR were extended by our group for typical diffusion distance of ~300nm and typical diffusion time of 50µs. In this work we demonstrate measurement of shorter distances ~70nm and timescale ~2µs using miniature surface resonators and more powerful magnetic gradients ~1000 T/m.