Simultaneous Triplex Detection in a Single-Test-Line Lateral Flow Immunoassay Utilizing Distinct Nanoparticle Colorimetry

Lateral flow immunoassay (LFIA) has become a popular method for the rapid detection of biological molecules, with an emerging need for multiplex detection capabilities. A novel LFIA device capable of simultaneously detecting three different antigens on a single test line was developed, with each antigen identifiable by a unique color. Gold nanoparticles (AuNPs; red), gold nanorods (AuNRs; blue), and silver nanoparticles (AgNPs; yellow) were engineered to flow concurrently within the LFIA device and specifically react with α-fetoprotein (AFP), neuron-specific enolase (NSE), and carcinoembryonic antigen (CEA) on the test line. The device was effective for both individual and simultaneous detection of the analytes, with a limit of detection (LOD) of 50 ng/mL. Given its rapid response, ease of use, and affordability, this multiplex detection LFIA device shows great potential for a wide range of applications, including food quality management, livestock diagnosis, and health and environmental monitoring.