A nanoparticle-based sensor for visual detection of multiple mutations

Abstract

Disposable dipstick-type DNA biosensors in the form of lateral flow strips are particularlyuseful for genotyping in a small laboratory or for field testing due to their simplicity, lowcost and portability. Their unique advantage is that they enable visual detection in minuteswithout the use of instruments. In addition, the dry-reagent format minimizes thepipetting, incubation and washing steps. In this work, we significantly enhance themultiplexing capabilities of lateral flow strip biosensors without compromising theirsimplicity. Multiplex genotyping is carried out by polymerase chain reaction (PCR)followed by a single primer extension reaction for all target alleles, in which a primer isextended and biotin is incorporated only if it is perfectly complementary to the target.Multiallele detection is achieved by multiple test spots on the membrane of thesensor, each comprising a suspension of polystyrene microspheres functionalizedwith capture probes. The products of the primer extension reaction hybridize,through specific sequence tags, to the capture probes and are visualized by usingantibiotin-conjugated gold nanoparticles. This design enables accommodation ofmultiple spots in a small area because the microspheres are trapped in the fibresof the membrane and remain fixed in site without any diffusion. Furthermore,the detectability is improved because the hybrids are exposed on the surfaceof the trapped microspheres rather than inside the pores of the membrane. Wedemonstrate the specificity and performance of the biosensor for multiallele genotyping.