A multiplex protein-free lateral flow assay for detection of microRNAs based on unmodified molecular beacons

•Unmodified molecular beacons with an ancillary sequence were used for target recognition.•Base stacking hybridization supported high sequence discrimination.•No affinity protein was used at capture lines.•Multiplex assays were developed with a unique gold nanoparticle-DNA probe.

Lateral flow assays (LFAs) have promising potentials for point-of-care applications. Recently, many LFAs have been reported that are based on hybridization of oligonucleotide strands. Mostly, biotinylated capture DNAs are immobilized on the surface of a nitrocellulose membrane via streptavidin interactions. During the assay, stable colorful complexes get formed that are visible by naked eyes. Here, we present an inexpensive and unique design of LFA that applies unmodified oligonucleotides at capture lines. The presented LFA do not utilize streptavidin or any other affinity protein. We employ structural switch of molecular beacons (MB) in combination with base stacking hybridization (BSH) phenomenon. The unique design of the reported LFA provided high selectivity for target oligonucleotides. We validated potential applications of the system for detection of DNA mimics of two microRNAs in multiplex assays.

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