SERS-based lateral flow immunoassay utilising plasmonic nanoparticle clusters for ultra-sensitive detection of salivary cortisol

Cortisol plays a central role in maintaining physiological homeostasis, and both cortisol excess and deficiency are associated with life-threatening conditions. Accurate diagnosis, adequate treatment and monitoring of disorders of cortisol secretion are essential for good health, normal growth and development. Although commercially available lateral flow immunoassay (LFI) strips can be used to measure cortisol, they have limitations, especially low sensitivity and limited quantitative performance, inhibiting their use in clinical settings. Here, we present a novel LFI platform integrated with surface-enhanced Raman scattering (SERS), employing precisely size-controlled gold nanoparticle clusters functionalised with Raman reporter molecules to overcome these limitations. The approach achieves exceptional sensitivity, covering the relevant therapeutic range in humans, with a limit of detection (LOD) for cortisol of 0.014 pg mL^-1, which is >500 times more sensitive than conventional LFI strips. The platform also showed high specificity for cortisol. The diagnostic potential was confirmed by testing with human saliva samples (<i>n</i> = 28), cross-validated with UPLC-MS/MS, showing excellent correlation (<i>R</i>² = 0.9977). Bland-Altman analysis demonstrated strong agreement, with all samples falling within the 95% limits and yielding a mean bias of -3.5% ± 13.2% relative to UPLC-MS/MS. Given its sensitivity, specificity and simplicity, this LFI-SERS platform offers strong potential for clinical translation to enable convenient cortisol monitoring.