Highly Sensitive Resonators for EPR Spectroscopy of Submicroliter/Submicromolar Biomacromolecular Samples

Abstract

We report ‘planar inverse anapole’ microresonators with high quality factors (Q-factors) and nanoliter active volumes, that increase both absolute sensitivity and concentration sensitivity of inductive-detection electron paramagnetic resonance (EPR) spectroscopy at room temperature. Due to its inherently high sensitivity, ability to interrogate specific sites by site-directed spin-labeling, and advances in pulsed measurements, EPR spectroscopy has gained importance as a technique to elucidate protein structure and dynamics. However, the limit of detection of state-of-the-art EPR spectrometers (approximately 50 uL of a 50 uM solution) is still too high compared to the amounts that are easily obtainable for some biomacromolecules - for example, membrane proteins, which are notoriously difficult to express and purify in sufficient amounts for macromolecular structural determination. To further decrease the detection limit, an advantageous approach is to miniaturize the resonator, thereby decreasing the interrogated sample volume. This miniaturization can be easily accomplished using standard photolithographic techniques to create resonant devices with micron-sized features and nanoliter active volumes.[1], [2] However, such miniaturization also increases radiation losses from the resonators, resulting in poor Q-factors and offsetting the gains in sensitivity achieved by decreasing the active volume. Here, we report an innovative planar microresonator that is designed to have minimal radiation losses at room temperature,[3] thereby ensuring high Q-factors without the need to operate at cryogenic temperatures. Using this combination of a small active volume and high Q-factor, we project an increase in the sensitivity of room-temperature EPR spectroscopy (and by extension, low-temperature EPR spectroscopy) of two orders of magnitude.