Quantitative SERS-Based Sandwich-Hybridization Assay for Nucleic Acid Detection.

Surface-enhanced Raman spectroscopy (SERS) has great potential for quantifying biological molecules, particularly DNA. In this study, we developed a SERS-based sandwich-hybridization assay for detecting DNA corresponding to viral (SARS-CoV-2) RNA using commercially available components and standard bioconjugation procedures; hence, no sophisticated synthesis steps were required. The assay employs TAMRA-labeled DNA conjugated to gold nanoparticles as nanoprobes and biotin-labeled surface DNA strands. Upon hybridization with the target analyte, the complete sandwich structure forms and is captured on a neutravidin-coated glass via the avidin-biotin interaction. The observed Raman signals originate from the TAMRA reporter dye, leveraging signal enhancement from both resonance and plasmonic effects, enabling the sensitive detection of the target analyte. Quantification of target DNA was performed using both a conventional single-point peak-height spectral analysis and a binary image-based analysis, with the latter providing a reliable evaluation of the assay's performance. Additionally, atomic force microscopy (AFM) was employed to characterize the samples, and the strong correlation between AFM and SERS results confirms the accuracy and reliability of this approach.