Label‐Free Sensing of Coronaviral Sequences via Kretschmann‐Configuration Reflectance Spectroscopic Ellipsometry: Sensitivity, Specificity, and Interfacial Effects

ABSTRACT We employ Spectroscopic Ellipsometry (SE) in the Kretschmann—Raether (KR) configuration (KRSE) to characterize a DNA‐functionalized platform designed for detecting specific viral oligonucleotide sequences. KRSE merges SE with Surface Plasmon Resonance, improving sensitivity to subtle interfacial phenomena. The sensor consists of a self‐assembled monolayer of thiolated DNA probes mixed with mercaptohexanol (MCH) spacers to promote upright probe orientation. The system is evaluated for its ability to selectively recognize the RdRp‐Helicase sequence of SARS‐CoV‐2, effectively discriminating it from the still circulating SARS‐CoV HKU strain. KRSE spectra exhibit pronounced redshifts (δλ) of the resonance as a function of target concentration. The formation of the probe layer and, in particular, the deposition of MCH, induce a deeper minimum in Ψ vs λ data at resonance. No further variation of the value of Ψ at resonance is observed in sensing/regeneration/sensing cycles. Optical modelling indicates that δλ redshifts are determined by increases in optical thickness of the adsorbed layer, while the Ψ changes at resonance derive from the formation of a sub‐nanometric Au‐thiol interfacial layer. This interface is modelled using a Bruggeman effective medium approximation, following previous standard SE experiments. Analysis of δλ shifts allows to derive a calibration curve with a nanomolar detection limit, and enables precise assessment of sensor specificity, outperforming previous conventional SE measurements on the same system.