Liposome-enhanced lateral-flow assays for the sandwich-hybridization detection of RNA

Clinical and environmental analyses frequently necessitate rapid, simple, and inexpensive point-of-care or field tests. These semiquantitative tests may be later followed up by confirmatory laboratory-based assays, but can provide an initial scenario assessment important for resource mobilization and threat confinement. Lateral-flow assays (LFAs) and dip-stick assays, which are typically antibody-based and yield a visually detectable signal, provide an assay format suiting these applications extremely well. Signal generation is commonly obtained through the use of colloidal gold or latex beads, which yield a colored band either directly proportional or inversely proportional to the concentration of the analyte of interest. Here, dye-encapsulating liposomes as an alternative are discussed. The LFA biosensors described in this chapter rely on the sandwich-hybridization of a nucleic acid sequence-based amplified (NASBA) mRNA target between a membrane immobilized capture probe and a visible dye (sulforhodamine B)-encapsulating liposome conjugated reporter probe. Although the methodology of this chapter is focused on LFAs for the detection of RNA through sandwich hybridization, the information within can be readily adapted for sandwich and competitive immunoassays. Included are an introduction and application notes toward this end. These include notes ranging from the detection of nonamplified RNA and single-stranded DNA, conjugation protocols for antibodies and other proteins to liposomes, and universal assay formats.