Microscale-patterned SERS substrates with exceptional uniformity: Addressing reproducibility challenges.

Achieving reproducible, quantitative signals remains a critical barrier for the broad adoption of Surface-Enhanced Raman Scattering (SERS) in analytical applications. Here, we introduce an innovative, mask-free laser-lithography fabrication approach to produce uniform arrays of gold-coated nanowells that significantly improve uniformity and reproducibility across large substrate areas compared to conventional methods. The substrates feature precisely controlled periodic arrays of sub-micron wells (500-2000 nm diameter) uniformly coated with a thin gold layer, generating localized and multiplexed plasmonic hot spots. By coupling the substrates to an advanced analytical workflow, we achieve spatial variations below 5% in SERS intensity across large mapping areas on most surfaces, addressing the uniformity challenges inherent in conventional SERS substrates. Using benzenethiol (BT) and Rhodamine 6G (R6G) as model analytes, we demonstrate quantitative detection with analytical enhancement factors of (1.00±0.01)×105 (BT) and (3.98±0.10)×105 (R6G) respectively. Importantly, the reported analytical enhancement factors account for spatial variations and multiple Raman peaks, ensuring reproducible and reliable values unlike conventional EFs based on isolated hot spots and single Raman modes. The developed approach addresses critical challenges in reproducibility and quantitative calibration, establishing a robust and well-characterized SERS substrate platform upon which future application-specific sensing protocols can be developed.