Lateral flow assays (LFA) have been used for several decades to detect chemicals, biologically relevant proteins, toxins, metals, and specific disease-related analytes (either bacterial or viral). The low cost and ease of use make these assays ideal for point-of-care diagnostics. One of the disadvantages of LFAs has been that they are prone to false positive when used for detecting more than one analyte on a strip, e.g., multiplexing the assay. Nanomaterials are being used to overcome the disadvantages of LFAs. A review on multiplexing nanoparticle-based LFAs, Yahaya, Zakaria, Noordin, and Abdul Razak (2018)1Yahaya, M., Zakaria, N., Noordin, R., & Abdul Razak, K. (2018). Multiplexing of nanoparticles-based lateral flow immunochromatographic strip: A review. Advanced Materials and Their Appli-cations—Micro to Nano Scale; Ahmad, I., Di Sia, P., Raza, R., Eds, 112-139. detailed several nanomaterials which have been used as labels, such as colloidal gold (AuNPs), silver (AgNPs), carbon (CNPs), selenium (SNPs), quantum dots (QDs), up-converting phosphors (UCPs), dye-doped and magnetic nanoparticles (NPs). This report will detail the use of a three color multiplex assay using AgNPs.

Detecting more than one analyte in a single assay reduces the time and cost for detecting multiple biological agents. Yen et al. (2015)2 designed such an assay for detecting dengue, Yellow Fever, and Ebola viruses in a single assay. The researchers used the size-dependent optical properties of silver nanoparticles (AgNPs) to develop a multiplexed LFA. Researchers conjugated triangular plateshaped AgNPs of varying sizes to antibodies that bind to specific biomarkers. Triangular plate-shaped AgNPs have narrow absorbances that are “tunable” through the visible spectrum. The growth of large AgNPs resulted in color changes based on the morphology change from spherical particles to triangular nanoplates. The AgNP colors were evident and distinguishable from one another when applied to paper and dried. These AgNPs were prepared for LFA by conjugating monoclonal antibodies to the NPs. It is combining antibodies with AgNP in a solution that results in antibody binding to the AgNP by electrostatic adsorption. The virus-specific antibodies recognized the dengue virus (DENV) NS1 protein (green), Yellow Fever virus (YFV) NS1 protein (orange), and Ebola virus, Zaire strain (ZEBOV) glycoprotein GP (red). A sandwich assay formed to capture the viral protein ligand (NS1 or GP) bound by both the antibody conjugated to the AgNP and the capture antibody loaded onto the test areas of the nitrocellulose strip. In a typical run, you load into the sample pad, the sample solution containing the antigen (NS1 protein of either DENV or YFV; GP of ZEBOV). The liquid migrates through the conjugate pad, where the antigen binds to the AgNP-Ab. The AgNP-Ab/ antigen complex then wicks through the strip, and they are captured by the antibodies (specific for each viral protein) “printed” at each test line. The reaction creates a colored band at the test detection area. For a positive result, the single test area was orange if YFV NS1 was present, green for DENV NS1, and red if ZEBOV GP was present. A positive control detection area, which is brown when all three AgNPs are present, is essential to demonstrate a complete test run and that the reagents worked as expected. In the absence of antigen, the test line was blank, indicating that the AgNP-Abantigen binding is specific, and the non-specific adsorption of the AgNP-Ab to the test line was undetectable.

Yen et al. (2015)2Yen, C. W., de Puig, H., Tam, J. O., Gómez-Márquez, J., Bosch, I., Hamad- Schifferli, K., & Gehrke, L. (2015). Multicolored silver nanoparticles for multiplexed disease diagnostics: distin-guishing dengue, yellow fever, and Ebola viruses. Lab on a Chip, 15(7), 1638-1641. c5lc00055f demonstrated the ability to utilize the optical properties of AgNPs for multiplexing point of care diagnostics for infectious disease using their size-tunable absorption spectra. Results showed a capacity for three test lines, each with a different color based on which AgNP-Ab bound to a specific viral protein. The limit of detection of the biomarkers for each virus was 150ng/mL. A three test line approach may apply to multiplexing other biological analytes for developing new, improved multiplexed LFAs.