A dual-channel self-amplifying lateral flow immunoassay for the on-site ultrasensitive simultaneous detection of salivary cortisol and melatonin

Abstract

The on-site simultaneous measurement of cortisol and melatonin is valuable for comprehensively assessing circadian rhythms and understanding their complex interplay under physiological conditions. However, no commercial point-of-care test currently enables the concurrent detection of these salivary hormones. Consequently, there is a significant demand for an on-site, user-friendly diagnostic platform. While the lateral flow immunoassay (LFI) is widely used in point-of-care testing, its sensitivity is insufficient for detecting salivary cortisol and melatonin. To overcome this limitation, the self-amplifying LFI (saLFI) developed previously for salivary cortisol detection, is extended to enable salivary melatonin detection in combination with a gold enhancement method. Two saLFIs are integrated into a single device using a dual-channel sample pad, creating a dual-channel self-amplifying lateral flow immunoassay (2ch-saLFI). This integrated dual-channel sample pad provides an equal distribution of the sample solution to two strips and two self-amplifying complexes. Consequently, the novel 2ch-saLFI enables the simultaneous on-site detection of both hormones from a single sample injection, delivering results within 30 min. The 2ch-saLFI demonstrates a high sensitivity, with detection limits of 5.39 and 0.476 pg mL⁻¹ for cortisol and melatonin, respectively (i.e., below typical physiological levels). Validation using 20 clinical samples provides a strong correlation with standard ELISA methods (R² = 0.9017 for cortisol and 0.9101 for melatonin). Overall, the 2ch-saLFI represents the first point-of-care test enabling the on-site, simultaneous, and highly sensitive measurement of salivary cortisol and melatonin via a single sample injection. This development allows an affordable clinical assessment of circadian biology for advanced healthcare applications.