High-sensitivity method for detecting dielectric changes in water driven by biomolecular hydration

We previously demonstrated that sub-terahertz irradiation significantly accelerates the formation of hydration structures in protein aqueous solutions under nonequilibrium conditions, immediately after mixing. To monitor this phenomenon, we developed a microwave dielectric measurement technique capable of sensitively detecting time-dependent changes in hydration under sub-terahertz irradiation. This method utilizes the dielectric-dependent modulation of multiply reflected signals in short-path-length samples (Sugiyama et al., Nat. Commun. 14: 2825, 2023). However, the physical origin of the observed signal remained unclear, limiting its broader applicability. In this study, we identify the origin as destructive interference between reflections at the probe-sample interface and the bottom surface of the sample container, which arises uniquely under a short-path-length condition satisfying <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi>d</mml:mi> <mml:mo>=</mml:mo> <mml:mi>λ</mml:mi> <mml:mi>/</mml:mi> <mml:mn>4</mml:mn></mml:mrow> </mml:math> . This finding establishes a clear measurement principle and enables direct evaluation of dielectric changes in biomolecular hydration from raw reflection data without converting to complex permittivity.