Development of Self-amplifying Lateral Flow Immunoassay for Ultrasensitive and User-friendly Detection of Analytes

Abstract

Competitive lateral flow immunoassay (LFI) is a commonly used platform for detecting cortisol and other small molecules. However, the low sensitivity of competitive LFI is a critical limitation for point-of-care tests. Thus, various signal enhancement methods have been developed while 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 10000-fold fewer AuNP conjugates from conventional competitive LFI, with a single operational step. The incorporation of fewer AuNP conjugates improves the sensitivity of competitive LFI but does not produce a visible signal spontaneously. The asLFI facilitates automatic signal enhancement without requiring additional operational steps, affording visible colorimetric signals. The 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 (R2 = 0.9159). Furthermore, a strong correlation with an ELISA was observed, validating the performance of the asLFI sensor with 18 human saliva samples (R2 = 0.9763). This novel asLFI sensor offers simple operation, affording ultra-sensitive point-of-care tests for the circadian monitoring of cortisol.