Scalable GLAD silver nanorod substrates with enhanced fluorescence and improved spot uniformity for microarray sensing

Metal-enhanced fluorescence (MEF) is a powerful approach for improving detection sensitivity in fluorescence-based biosensing by utilizing localized surface plasmon resonance near metallic nanostructures. In this study, large-area (75 × 25 mm²) silver nanostructured substrates were fabricated using a scalable and cost-effective glancing-angle deposition (GLAD) process and evaluated for fluorescence microarray applications. Vertically aligned and isolated silver nanorod arrays with controlled lengths were grown on standard glass slides by electron-beam evaporation under tilted flux conditions. Surface morphology and nanorod geometry were analyzed by field-emission scanning electron microscopy, while wettability changes with nanorod length were verified through static contact angle measurements. Fluorescence performance was assessed using Cy5-conjugated streptavidin over a wide concentration range (100 ng/mL to 100 µg/mL) and measured with a microarray scanner. Fluorescence intensity increased systematically with nanorod length, providing up to ~ 17-fold signal enhancement compared with glass in the non-saturated regime. Although viscosity-controlled spotting with a 50% glycerol buffer reduced droplet spreading, residual spot variation remained due to capillary effects on the nanorod surface. To address this, lithographically defined photoresist wells were introduced around the active regions, enabling effective droplet confinement and highly uniform spot geometry. The combined control of nanorod structure, surface wettability, buffer composition, and patterned confinement produced strong, reproducible fluorescence signals, demonstrating that PR-patterned GLAD nanorod substrates are well suited for sensitive and quantitative protein and DNA microarray assays.