Nanoimprint Lithography-Derived Scalable Periodic Au Metasurfaces as Reproducible SERS Substrates for Cotinine Biomarker Detection in Real Biological Matrices

Abstract

Passive exposure to tobacco smoke can cause severe chronic medical conditions, including respiratory and cardiovascular diseases and cancer. Cotinine, a primary metabolite of nicotine, serves as a reliable biomarker for investigating tobacco exposure and potential health risks. The development of strategies for rapid, sensitive, and selective detection of cotinine at trace levels remains critically important for clinical diagnostics. In this context, surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful label-free analytical technique owing to its high sensitivity and molecular specificity. However, achieving uniform, reproducible SERS-active substrates with high enhancement is still challenging, particularly for the selective detection of target analytes such as cotinine. Therefore, it is essential to design and fabricate robust nanostructured platforms with controlled morphology and optimized plasmonic properties for sensitive detection. In this work, we report the development of a sensitive SERS-active substrate using thermal nanoimprint lithography (NIL), nanopatterns coated with nanostructured Au films with thicknesses from 10 nm to 50 nm, deposited by magnetic radio frequency (RF) sputtering. We observed a significant contribution from the nanopatterned substrate, with a calculated enhancement factor (EF) of up to 10⁹ for R6G using a 25 nm Au coating over the nanopattern. The high SERS enhancement is attributed to the periodic structure of the nanopattern. The developed substrate exhibits a limit of detection (LOD) of 2.31 ng/mL, with high uniformity and reproducibility for cotinine detection in synthetic saliva. Furthermore, the SERS results for the developed metasurface were applied to real urine samples from smoker and non-smoker patients, and the derived concentrations were validated using a commercial ELISA assay. The SERS results showed a good agreement with ELISA measurements, providing a scalable, reproducible, and clinically relevant SERS platform for trace-level cotinine detection.