Rapid and enzyme-free signal amplification for fluorescent detection of microRNA via localized catalytic hairpin assembly on gold nanoparticles

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.