Ultrasensitive duplex lateral flow immunoassay for enhanced detection of viral antigens using hybridization chain reaction amplification

The global spread of viral infections, particularly highlighted by the COVID-19 pandemic, has underscored the critical demand for rapid, sensitive, and widely accessible diagnostic tools. While polymerase chain reaction (PCR) remains the gold standard for virus detection due to its high sensitivity and specificity, its reliance on complex instrumentation and lengthy processing times makes it unsuitable for point-of-care testing (POCT). In contrast, lateral flow immunoassays (LFIA) are commonly used in POCT for their simplicity and rapid results, yet they often exhibit low sensitivity and limited accuracy, particularly when detecting low concentrations of viral antigens. In this study, we reported an ultrasensitive duplex lateral flow immunoassay (UdLFIA) that integrated hybridization chain reaction (HCR) amplification and dual-functionalized gold nanoparticles (AuNPs) to achieve both high sensitivity and accuracy. To demonstrate its capabilities, we applied the UdLFIA for the simultaneous detection of two SARS-CoV-2 antigens-the nucleocapsid (N) protein and the receptor-binding domain (RBD) of the spike protein. This model confirmed UdLFIA's benefits, enhancing diagnostic accuracy by reducing false negatives when targeting a single antigen. The UdLFIA achieved a limit of detection (LOD) of 0.65 ng/mL for the N protein and 0.99 ng/mL for the RBD protein, representing a 10–20 folds improvement in sensitivity with HCR amplification. The good selectivity of the UdLFIA was demonstrated by comparing the measurement with closely related viruses. Additionally, the 20 min measurement time makes it well-suited for large-scale screening and POCT settings. The UdLFIA platform represents a step toward bridging between high-sensitivity laboratory diagnostics and accessible point-of-care testing. By combining the simplicity and speed of LFIA with enhanced sensitivity and accuracy, UdLFIA may support scalable and cost-effective testing after further validation in clinical and real-world settings for both pandemic response and routine viral screening, addressing the limitations of current POCT technologies.