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.

构建了一种基于超支化滚环扩增（HRCA）和多位点链置换扩增（SDA）策略的新型荧光生物传感器，实现对miRNA-21的超灵敏检测。目标 miRNA-21 专门用于触发 HRCA 反应并产生丰富的不同长度的 DNA 序列。这些序列可以启动与发夹HP1和HP2的SDA反应，形成荧光信号双链体（FSDB），并产生用于靶点测定的荧光共振能量转移（FRET）。圆形模板、反向引物、发夹 HP1 和 HP2 经过巧妙设计，大大提高了检测选择性。这种基于 FRET 信号比的策略不仅避免了传统检测方法产生的假阳性信号，同时也将系统波动的影响降到最低。因此目标的检测限可以达到fM级。此外，该方法还可应用于其他miRNA、蛋白质和生物分子的检测，在生物医学研究、环境检测和临床诊断应用等方面具有巨大潜力。