Ultrasensitive SERS-Active Silver/Graphene Oxide Nanocomposites for Label-Free Detection of Lung Cancer-Associated miRNA-21

This study reports an ultrasensitive Ag/graphene oxide (Ag/GO) hybrid substrate for label-free SERS detection of lung cancer-associated miRNA-21. Three Ag precursor loadings were evaluated to clarify how metal loading controls nanoparticle density, interparticle spacing, and hot-spot formation. The intermediate formulation, Ag/GO-2, provided the most favorable balance between morphological uniformity and plasmonic coupling, yielding an average Ag crystallite size of about 18.5 nm and an Ag loading of 35.6 wt% by ICP-MS, in good agreement with TGA. A 532 nm laser and 0.5 mW power were selected from a preliminary optimization study to maximize signal stability while minimizing photothermal perturbation of RNA. Using rhodamine 6G as a probe, Ag/GO-2 exhibited an enhancement factor of 2.4 x 107. For miRNA-21, the adenine band at 732 cm-1 showed a linear response from 10-7 to 10-13 M (R2 = 0.948) with a limit of detection of 15 fM. Specificity was verified against a single-base mutant, miR-21-3p, let-7a, miR-141, and BSA using full spectral fingerprints and multivariate PCA. The platform showed good spot-to-spot reproducibility (12.4% RSD), acceptable inter-batch reproducibility (<11% RSD), retained more than 85% of its initial activity after 30 days under dark sealed storage, and delivered 92.0-108.0% recovery in 1:10 diluted serum while maintaining detectable signals in 1:2 and undiluted serum. These results clarify the Ag-loading/structure/performance relationship and support the practical potential of Ag/GO substrates for liquid-biopsy-oriented nucleic-acid sensing.