Automatically Signal-Enhanced Lateral Flow Immunoassay for Ultrasensitive Salivary Cortisol Detection

Competitive lateral flow immunoassay (LFI) is a commonly used platform for detecting cortisol and other small molecules. However, its low sensitivity is a critical limitation for application in point-of-care tests. Thus, various signal enhancement methods have been developed, requiring complex operational steps and specialized detectors. In this study, we propose an automatic signal-enhanced LFI (asLFI), providing 100 times improved colorimetric sensitivity by applying 10,000-fold fewer AuNP conjugates compared to conventional competitive LFI, involving a single operational step. The incorporation of fewer AuNP conjugates improves the sensitivity of competitive LFI but does not produce a visible signal spontaneously. asLFI facilitates automatic signal enhancement without requiring additional operational steps, affording visible colorimetric signals. asLFI demonstrated high sensitivity in detecting cortisol (limit of detection = 3.8 pg mL-1) across a detection range of 0.01-10 ng mL-1 with linearity correlation (R 2 = 0.9159). Furthermore, a strong correlation with ELISA was observed, validating the performance of the asLFI sensor with 18 human saliva samples (R 2 = 0.9763). This novel asLFI sensor offers simple operation, affording ultrasensitive point-of-care tests for the circadian monitoring of cortisol.