Ultra-sensitive detection of protein thermal unfolding and refolding using near-zone microwaves

We use planar slot antennas proximal to proteins in solution to detect changes in conformation. The antennas are attached to fused-quartz or glass sample holders and the cuvette/antenna assembly is placed in the sample holder of an optical spectrophotomer (either UV/VIS or fluorescence polarization), allowing simultaneous dielectric and optical measurements. Return loss is recorded using a vector network analyzer. This system was used to study the equilibrium thermal unfolding and refolding of a small globular protein, as well as the binding of small hormones to a receptor. Good agreement between optical and microwave measurements was obtained for all systems studied. We show that microwave measurements can be made at protein concentrations as low as 0.3 ng/mL (19 pM), several orders of magnitude lower than that required for optical spectroscopy. The results from these experiments demonstrate that resonant slot antennas can be used to detect changes in protein conformation and the presence of microwave radiation does not perturb the system under study.