Sandwich SERS Chip with Raman‑Silent Reporters for Ultrasensitive Serum Cortisol Detection and Depression Differentiation

Cortisol is a critical stress hormone, and its abnormal levels are closely linked to diseases such as depression and Cushing’s syndrome. Cortisol has only a single binding site, which precludes conventional sandwich immunoassays, and the large antibody size often masks its SERS signal. Existing silent-region SERS strategies are limited by low signal intensity and poor reproducibility, arising from small-molecule reporters with low scattering cross-sections and randomly distributed hot spots. To address these challenges, we develop a silent-region SERS platform based on plasmonic superlattice enhancement combined with a top–bottom nanogap cavity. The bottom layer is a superlattice film of gold octahedral nanoparticles that generate uniform hot spots and is functionalized with anticortisol antibodies. The top layer uses Prussian blue@Ag core–shell nanoparticles as integrated reporters that exhibit characteristic Raman signals in the 1800–2400 cm–1 silent region and are functionalized with anticortisol-binding globulin antibodies. Because serum cortisol predominantly exists as a complex with cortisol-binding globulin, we designed a SERS chip with a dual-antibody sandwich configuration. This configuration enables efficient target capture and signal transduction within the three-dimensional nanogap hot spots. This sensing chip achieves a detection limit of 17.43 pM in serum samples from 93 patients with depression, with a linear range of 10 pM–10 μM and high reproducibility. It also exhibits excellent selectivity toward common serum interferents and distinguishes depressed patients from healthy controls. This work provides a robust approach for highly sensitive and specific SERS detection of single-binding-site small-molecule hormones and holds promise for objective molecular assessment of depression.