Controlled Assembly of Gold Nanoparticles on Nanopillar Arrays for Improved Sensitivity and Reproducibility in Molecular Diagnostics

Surface-enhanced Raman scattering (SERS) platforms using nanostructured substrates enable ultrasensitive biomolecular detection through strong, reproducible signals in sub-10 nm nanogaps. In this study, we developed a robust SERS bioassay platform with rigid gold nanopillar substrates, functionalized with gold nanoparticles (AuNPs) via DNA hybridization. DNA-mediated internalization provided higher efficiency and retention compared to electrostatic methods, especially for 60 nm AuNPs, optimizing plasmonic coupling and SERS enhancement. The created nanogaps yielded dense, uniform hot spots, enabling sensitive and reproducible SERS detection of cancer biomarker miRNA-21 down to attomolar concentrations─without the need for amplification as in qRT-PCR. Incorporating digital SERS analysis improved measurement reliability and achieved a lower limit of detection. These results suggest AuNP-internalized nanopillar substrates are highly promising for sensitive, amplification-free genetic diagnostics, with significant potential in clinical and point-of-care early disease detection.