Quantitative immunoassay of prostate-specific antigen dependent on SERS substrate of polymer-silver nanocubes modified with 4-mercaptobenzoic acid: the crucial effect of thiol molecule as internal standard

The early and reliable detection of prostate cancer remains a significant challenge in clinical diagnostics. This study presents a quantitative immunoassay for prostate-specific antigen (PSA) using a surface-enhanced Raman scattering (SERS) substrate comprised of polymer-silver nanocubes (Ag NCs) modified with 4-mercaptobenzoic acid (4MBA). We investigated the impact of thiol-containing internal standards (IS) on the quantitative accuracy of SERS, highlighting the superior stability of 4MBA compared to non-thiol molecules like crystal violet (CV). In contrast to CV, 4MBA exhibited significantly greater stability, with only a 9.3% decrease in characteristic peak intensity after prolonged storage. Furthermore, the quantitative detection of PSA was realized using an IS-mediated sandwich immunostructure. Particularly, the coefficient of determination (R 2 ) for the PSA standard calibration curve demonstrated remarkable enhancement from 0.987 to 0.994, highlighting the advantage of thiol-anchored IS correction strategy. Moreover, the limit of detection (LOD) for PSA was refined to 5.6 × 10 -10 mg/mL after a perfect linear fitting was facilitated using 4MBA-based IS correction, which was superior to standard enzyme-linked immunosorbent assay (ELISA) and comparable SERS techniques. In addition, the specificity of the assay was confirmed by the lack of signal for non-target antigens. These findings demonstrate that the strategic selection of robust IS molecules can significantly enhance the quantitative capabilities of SERS technology, paving the way for its application in clinical diagnostics.