Spatially confined silver nanoparticles on macroporous microspheres as a three-dimensional SERS substrate for ultrasensitive label-free hormone analysis

Surface-enhanced Raman scattering (SERS) is a powerful analytical technique for ultrasensitive detection; however, its practical application is often limited by poor substrate stability and insufficient signal reproducibility. In this work, a robust three-dimensional (3D) SERS substrate based on macroporous polymer microspheres decorated with silver nanoparticles (AgNP@PM) was developed for trace-level analysis. The substrate was fabricated via atom transfer radical polymerization combined with a solution-phase deposition method, enabling Ag nanoparticle loading within a confined polymer framework. The resulting 3D architecture generated a high density of electromagnetic hotspots and facilitated analyte enrichment through surface functional groups, leading to enhanced and reproducible SERS signals. Under optimized conditions, the AgNP@PM substrate exhibited an enhancement factor of 1.2 × 10 11 and an ultralow detection limit of 5.0 × 10 −13  M for Rhodamine 6G. Excellent signal reproducibility and long-term stability were achieved, with a relative standard deviation of 8.97% after 180 days of storage. Importantly, the analytical applicability of the proposed substrate was demonstrated by the direct, label-free detection of β-estradiol and testosterone in serum samples at nanomolar concentrations (5.0 × 10 −9  M). These results indicate that the AgNP@PM substrate provides a reliable and sensitive SERS platform for hormone analysis in complex biological matrices, showing potential for practical applications in bioanalysis and food safety.