Ultrasensitive de novo Protein Sandwich Lateral flow Fluorescence Assay for Cortisol in Saliva and Serum using Quantum dots and Europium Nanoparticles.

Authors

John G Bruno, Ying Chen, Reem Mahrat

Published in

Journal of fluorescence. Jun 27, 2026. Epub Jun 27, 2026.

Abstract

We describe and demonstrate the first lateral flow (LF) sandwich fluorescence assay for the small molecule hormone cortisol, enabled by a pair of de novo-designed cortisol-induced dimerization (CID) proteins. CID proteins C4 and C10, originally characterized in a chemiluminescent format, sandwich a single cortisol molecule between two engineered binding domains, eliminating the inherent sensitivity limitations of traditional competitive small molecule LF assays. In the present assay, C10 was immobilized as the capture reagent on a nitrocellulose membrane, and C4 was conjugated to either streptavidin-coated red quantum dots (QDs) for saliva applications or streptavidin-coated europium chelate-doped polystyrene nanoparticles (Eu NPs) for serum applications. The QD-CID strips produced a visually scored limit of detection (LOD) of 200 pg/mL of cortisol spiked into natural human saliva (20 pg total in a 100 µL sample) and 2 pg/mL in artificial saliva (200 fg total), spanning and extending below the physiological diurnal range of salivary cortisol (approximately 1,000-7,500 pg/mL). A goat anti-avidin control line successfully captured the C4-biotin-streptavidin-QD conjugate after migration, providing flow validation. QDs performed poorly in undiluted human serum, consistent with prior reports of ZnS shell perturbation by serum components. Substituting europium-chelate-doped polystyrene NPs preserved the long Stokes shift advantage while protecting the fluorophore from serum-induced quenching and enabled detection in pooled human serum across a dynamic range of 2-200 ng/mL (200 pg to 20 ng of total cortisol per 100 µL sample), more than spanning the full physiological diurnal range of serum cortisol. The entire LF test is executed in approximately 15 min, requires no cold chain or laboratory instrumentation beyond a handheld UV reader, and is suitable for both point-of-care clinical use and at-home stress and endocrine monitoring.

PMID:
42364004
Bibliographic data and abstract were imported from PubMed on 27 Jun 2026.

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