An emerging technology in point of care assays, vertical flow assays are founded on the same nanoparticle-analyte-recognition element sandwich principle as lateral flow assays. Notable and unique features of vertical flow include multiplex detection: the ability to check for the presence of up to four targets simultaneously, and their extra-rapid test time (<5 min). Notably, by conjugating different probe molecules to differently coloured nanoparticles (e.g. gold spheres vs nanourchins), spots of different colour based on analyte can be achieved.
Important considerations for vertical flow devices include nitrocellulose membrane thickness and running buffer properties. The running buffer must block non-specific binding sites on the membrane (e.g. with BSA/casein) before application of the conjugate sample. As well, the viscosity of the running buffer affects sample flow-through time, impacting sensitivity through time available for the conjugate and analyte/recognition element to interact.