Effect of report probe concentration on response of the strip biosensor.

<p>P_T and P_C are peak areas of test and control lines, respectively. The histograms represent P_T/P_C values in the presence of 100 pM miR-21 (blue) and without miR-21 (purple), respectively. Error bars are standard deviations from three repetitive experiments.</p

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

<div><p>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 <i>duplex specific nuclease</i> (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.</p></div

Selectivity of the strip biosensor for miR-21 over competing sequences.

<p>Concentrations were 10 nM for miR-21 and 10 nM for other sequences.</p

Schematic representation of the dual target-recycling amplification strategy for sensitive detection of microRNAs based on duplex-specific nuclease and hybridization chain reaction with lateral flow assay.

<p>The explanations of all acronyms were listed as follows: H1-bio, Hairpin probe1-biotin; SA-MB, Streptavidin-magnetic bead; AuNPs, Au nanoparticles; SA-AuNPs, Streptavidin-Au nanoparticles; BSA, bovine serum albumin; DSN, duplex specific nuclease; FAM, fluorescein isothiocyanates; anti-Fam mAb, anti-FAM- monoclonal antibody.</p

Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction - Fig 5

<p><b>(A) Photographs of detection results of strips with different miR-21 concentrations. (Left) Images were recorded with a scanner. (Right) Corresponding optical responses of red bands on the strip. Peak areas were analyzed with the Image J software. (B) Calibration curve of miR-21 sensing system.</b> The curve was plotted as P_T/P_C vs logarithmic value of miR-21 concentration. Inset shows a linear relationship between P_T/P_C and the logarithm of miR-21 concentration. P_T and P_C are peak areas of test and control lines, respectively. Error bars are standard deviations from three independent measurements.</p

Effect of reaction temperature on response of the strip biosensor.

<p>The histograms represent P_T/P_C values in the presence of 100 pM miR-21 (blue) and without miR-21 (green), respectively. P_T and P_C are peak areas of test and control lines, respectively. Error bars are standard deviations from three independent measurements.</p

Effect of incubation time on response of the strip biosensor.

<p>PT/PC values were obtained by detecting 100 pM miR-21 (blue) and 10 pM miR-21 (green), respectively. Error bars are standard deviations from three independent measurements.</p