Aims
Salivary cortisol is an established biomarker of hypothalamic-pituitary-adrenal axis function with important applications in the diagnosis and monitoring of endocrine disorders including Cushing syndrome and adrenal insufficiency (1–4). Current laboratory methods require specialised instrumentation and limit rapid clinical decision-making (3,5). This study aimed to develop and optimise a dual-mode gold nanoparticle (AuNP) biosensor integrating colourimetric detection and exploratory surface-enhanced Raman spectroscopy (SERS) for future point-of-care salivary cortisol testing.
Methods
Citrate-stabilised AuNPs were synthesised and functionalised with the Raman reporter 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA) and anti-cortisol monoclonal antibodies using 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP). Nanoparticle synthesis, conjugation strategy, antibody loading, buffer composition and salt-induced aggregation conditions were systematically investigated using ultraviolet-visible spectroscopy, visual colourimetric assessment and exploratory Raman analysis (6,7). A prototype portable workflow incorporating colourimetric detection and smartphone-based analysis was also established.
Results
Self-synthesised citrate-stabilised AuNPs demonstrated superior stability and retained salt responsiveness following TFMBA functionalisation compared with commercial nanoparticles. Antibody pre-activation before conjugation improved colloidal stability; however, antibody-functionalised nanoparticles became resistant to salt-induced aggregation, preventing reproducible cortisol-dependent colourimetric responses. Reduced antibody loading partially restored salt responsiveness, identifying antibody surface coverage as a key determinant of assay performance. These findings establish critical optimisation parameters while demonstrating the feasibility of the dual-mode biosensing platform. Laboratory optimisation remains ongoing and is currently focused on systematic antibody-density optimisation, alternative salt systems, competitive assay formats incorporating multivalent cortisol conjugates, and evaluation in saliva matrices.
Conclusion
This work establishes a promising foundation for development of a portable dual-mode cortisol biosensor while identifying the principal physicochemical barriers limiting antibody-functionalised AuNP assays. Ongoing optimisation and validation are expected to support translation towards rapid, inexpensive point-of-care cortisol testing for endocrine practice, with potential applications in assessment of hypothalamic-pituitary-adrenal axis disorders and longitudinal patient monitoring (5–7).