Various signal amplification strategies have been developed for microRNA (miRNA) detection, but most of these amplification strategies always need some enzymes. In this work, we have constructed an enzyme-free signal amplification method for miRNA determination via target-triggered catalytic hairpin assembly (CHA). Two hairpin probes (H1 and H2) were ingeniously designed, and fluorescein (FAM)-labeled H1 (as a signal reporter) was conjugated on the gold nanoparticles (AuNPs) surface. In the presence of target miRNA, the cyclic self-assembly took place between H1 and H2 on the AuNPs, resulting in amplification of the fluorescence signal. Using miRNA-21 as a model analyte, the linear concentration range for miRNA-21 detection was from 0.1 nM-10 nM under the optimized experimental conditions (25 μL AuNPs (3 nM), 100 nM H2, 25 °C, pH 7.4). The fluorescence method exhibited high sensitivity to with a 10 pM detection limit. The recoveries in 2% normal human serum were in the range of 96.4%-103.6%. What's more, it was found that the addition of ten thymine bases (T10) as spacer between H1 and AuNPs could significantly accelerate the CHA reaction. A complete analysis for the determination of miRNA could be accomplished within 45 min, which is faster than a number of previous reports.
Keywords: Catalytic hairpin assembly; Enzyme-free; Fluorescence; Gold nanoparticles; microRNA.
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