Multi‐Color Au/Ag Nanoparticles for Multiplexed Lateral Flow Assay Based on Spatial Separation and Color Co‐Localization

Paper or membrane‐based lateral flow assay (LFA) is a well‐established platform widely used in point‐of‐care diagnostics. The multiplexing on LFA platform is mainly realized by spatially separating test lines along the test strip, and it has limited multiplexing capability due to space restriction. In this report, a new strategy for multiplexing on LFA platform is presented. In addition to spatial separation, morphology and composition‐controlled Au/Ag nanoparticles of three primary colors are also employed to add another parameter for multiplexing. The co‐localization of primary particles would result in an array of secondary colors as an indication of various target combinations. This two‐parameter multiplexing strategy significantly improves the multiplexing capability of LFA, and extends the applicability of LFA to more complicated analysis. Using the proposed two‐parameter multiplexing LFA, all 32 combinations of two protein and three DNA targets are successfully identified. Subtyping of three herpes viruses is also performed by amplifying the viral DNA with loop‐mediated isothermal amplification, and detecting the amplicons using the two‐parameter multiplexing LFA. The co‐localization of Au/Ag nanoparticles of three primary colors results in many secondary colors as an indication of various target combinations in lateral flow assays (LFAs). When combined with multiplexing by spatial separation, this two‐parameter multiplexing strategy significantly improves the multiplexing capability of LFA, and extends the applicability of LFA to more complicated analysis.