Liu Lin, Deng Dehua, Wu Daohong, Hou Weilin, Wang Lu, Li Ning, Sun Zhifang
Henan Province of Key Laboratory of New Optoelectronic Functional Materials, Anyang Normal University, Anyang, Henan, 455000, PR China.
Henan Province of Key Laboratory of New Optoelectronic Functional Materials, Anyang Normal University, Anyang, Henan, 455000, PR China.
Anal Chim Acta. 2021 Mar 8;1149:338199. doi: 10.1016/j.aca.2021.338199. Epub 2021 Jan 7.
We proposed a simple and sensitive strategy for the detection of microRNAs (miRNAs) by converting homogeneous assay into surface-tethered electrochemical analysis. Specifically, the biotinylated detection probes (biotin-DNA-biotin) can trigger the in-situ assembly of tetrameric streptavidin (SA) proteins on an electrode surface via the SA-biotin interactions. The (SA-biotin-DNA-biotin) assemblies electrically insulated the electrode interface, thereby blocking the electron transfer of [Fe(CN)]. When the probe was hybridized with the target miRNA, it would be cleaved into small fragments (denoted as biotin-DNA) by duplex-specific nuclease (DSN). The released target miRNA can enter into the next hybridization-enzymolysis cycle, thus leading to the generation of considerable amounts of biotin-DNA fragments. The released biotin-DNA competed with the detection probe to bind SA, thus limiting the in-situ formation of (SA-biotin-DNA-biotin) assemblies. The surface-tethered electrochemical analysis by the dual signal amplification of DSN and (SA-biotin-DNA-biotin) assemblies has been used for the determination of miRNAs in cell lysate with a satisfactory result. The method showed a detection limit down to 10 aM. The "one-step" immobilization-free strategy can be used to design novel biosensors for the detection of other biomarkers.
我们提出了一种简单且灵敏的策略，通过将均相分析转化为表面 tethered 电化学分析来检测微小 RNA（miRNA）。具体而言，生物素化的检测探针（生物素 - DNA - 生物素）可通过链霉亲和素（SA）-生物素相互作用触发电极表面四聚体链霉亲和素（SA）蛋白的原位组装。（SA - 生物素 - DNA - 生物素）组装体使电极界面电绝缘，从而阻断[Fe(CN)]的电子转移。当探针与靶 miRNA 杂交时，它会被双链特异性核酸酶（DSN）切割成小片段（记为生物素 - DNA）。释放的靶 miRNA 可进入下一个杂交 - 酶解循环，从而导致产生大量生物素 - DNA 片段。释放的生物素 - DNA 与检测探针竞争结合 SA，从而限制（SA - 生物素 - DNA - 生物素）组装体的原位形成。通过 DSN 和（SA - 生物素 - DNA - 生物素）组装体的双重信号放大进行的表面 tethered 电化学分析已用于测定细胞裂解物中的 miRNA，结果令人满意。该方法的检测限低至 10 aM。这种“一步法”免固定策略可用于设计用于检测其他生物标志物的新型生物传感器。
