Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction

Abstract

MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3’ end modified with biotin) for a target miRNA of miR-21 and capture sequence (5’ end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

Extracellular Vesicles: Current Analytical Techniques for Detection and Quantification

“…Thus, LFIA is a great tool for cost-effective on-site detection. Since the microRNA (miRNA) expression in EV may be altered in some diseases [ 30 , 31 ], the development of nucleic acid lateral flow systems that enable miRNA detection [ 32 , 33 ] confirms the potential of this technique.…”

Recent advances in sensitivity enhancement for lateral flow assay

“…Different from protein biomarkers present at the level of nanomolar or picomolar, the amounts of pathogenic bacteria that cause disease can be as low as a few CFU/mL [ 34 ]. In order to enhance the sensitivity of LFA for nucleic acid detection, various isothermal amplification methods are used, including rolling circle amplification (RCA) [ 35 ], loop-mediated isothermal amplification (LAMP) [ 36 ], recombinase polymerase amplification (RPA) [ 37 ], nucleic acid sequence–based amplification (NASBA) [ 38 ], helicase-dependent isothermal DNA amplification (HDA) [ 39 ], and hybridization chain reaction (HCR) [ 40 ].…”

Potential role of micro ribonucleic acids in screening for anal cancer in human papilloma virus and human immunodeficiency virus related malignancies

“…In addition to the conventional miRNA detection platforms which include Northern blotting, in situ hybridization, next generation sequencing, reverse transcription qPCR, and microarrays[ 138 ], new miRNA extraction and detection platforms have emerged to compensate for the limitations of conventional assays[ 139 ]. These technologies are referred to as point-of-care (PoC) technologies and include isothermal amplification-based assays[ 140 ], lateral flow assay-based systems[ 141 ], nanobead-based[ 142 ], electro-chemical-based[ 143 ], and microfluidic chip-based[ 144 ] strategies. The latter, which is also known as Lab-on-a-chip or microchip, is highly specific, cost-effective, and a quick approach for the multiplexed detection of miRNAs[ 139 ].…”