Abstract
A novel fluorescence biosensor based on hyperbranched rolling circle amplication (HRCA) and multi-site strand displacement amplification (SDA) strategy was constructed to realize super-sensitive detection of miRNA-21. The target miRNA-21 was specifically used to trigger HRCA reaction and generated abundant DNA sequences with different lengths. These sequences could initiate the SDA reaction with hairpin HP1 and HP2 to form the fluorescence signal double-stranded bodies (FSDB), and produced the fluorescence resonance energy transfer (FRET) for target assay. The circle template, reverse primer, hairpin HP1 and HP2 were skillfully designed to much improve the assay selectivity. This FRET signal ratio-based strategy not only avoided the false positive signal produced by traditional detection methods, but also minimized the influence of system fluctuation. So the detection limit of the target could reach fM level. In addition, this method could also be applied to the detection of other miRNAs, proteins and biomolecules, and had great potential in biomedical research, environmental detection and clinical diagnostic applications.
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