A three-line lateral flow biosensor for logic detection of microRNA based on Y-shaped junction DNA and target recycling amplification

A rapid, sensitive, and accurate detection strategy for microRNA 16 (miR-16) was developed, which combined the convenience of lateral flow biosensors (LFBs), the design flexibility of Y-shaped junction DNA probe, and the enhancement ability of endonuclease-assisted target recycling amplification. The system is composed of a molecular beacon (MB) probe, an assistant probe, and endonuclease Nt.BbvCI, which plays the role of signal translation and amplification. In the presence of the target microRNAs (miRNAs), three chains of nucleic acid could hybridize with each other to form a Y-shaped junction structure, which could be recognized by the endonuclease Nt.BbvCI. The MB probe was efficiently cleaved by endonuclease and produced two new DNA fragments, while the regenerated assistant probe and target were hybridized to another MB probe and entered into the next cycle of the amplification. In this way, the detection of the readily biodegradable miRNA was turned into the detection of two DNA fragments in the LFB. Meanwhile, the detection of two different DNAs would improve the accuracy and effectively avoid false results. The amplified products containing DNA fragments were then applied to the lateral flow nucleic acid biosensor (LFNAB) with two test zones, on which specific DNA probes were designed. The formed DNA-DNA/gold nanoparticle (GNP) conjugates were captured and accumulated to produce two red bands in two test zones. The logic judgment of the two test zones provided more accurate and convincing results. Under optimal conditions, the visual detection limit of miR-16 in aqueous solutions was 0.1 pM, which is 100-1000 times lower than that of visual or colorimetric methods in the literature. It could be used for on-field and point-of-care testing and meet the urgent demand of sensitive and selective miRNA detection in remote rural areas without costly equipment. The system displayed good universality, compatibility, high specificity, and stability of miRNA detection, which revealed significant potentiality in biomedical diagnosis.