Dual-Competitive Turn-On SERS-Lateral Flow Immunoassay for Ultrasensitive Cortisol Detection in Saliva

Fluorescent "turn-on" lateral flow immunoassays (LFIAs) provide higher sensitivity than conventional "turn-off" formats, but their broader application is often hindered by the potential toxicity and background interference of fluorescent nanomaterials. To address these limitations, we developed a novel "dual-competitive turn-on" LFIA (DCTO-LFIA), in which competitive binding between the capture antigen and goat antimouse antibody to the immunoprobe activates signal generation upon target recognition. By further integrating surface-enhanced Raman scattering (SERS) nanoprobes, the assay enabled ultrasensitive detection of salivary cortisol (COR). Under optimized conditions, the method achieved a visual limit of detection (vLOD) of 0.05 ng/mL, representing an over 10-fold enhancement compared with a conventional colorimetric LFIA. In the SERS detection mode, the assay reached a quantitative limit of detection (LOD) as low as 0.003 ng/mL, offering markedly higher sensitivity than the colloidal gold nanoparticle-based quantitative LFIA. Moreover, the platform successfully monitored circadian COR rhythms and detected exercise-induced fluctuations, demonstrating sensitivity to both physiological and stress-related variations. With its innovative design and robust performance, the SERS-based DCTO-LFIA holds considerable promise for highly sensitive detection of small-molecule biomarkers in clinical and point-of-care applications.