Abstract：
A novel functional nucleic acid (FNA) nanomaterial based on hybrid chain reaction (HCR) nanoscaffolds is proposed to solve the problem of time superposition and repeated primer design in sensitive miRND detection using cascade amplification technique. Rolling circle amplification (RCA) was cascaded with the prepared FNA nanomaterials for miRNA let-7a (as a model target) sensitive detection by lateral flow assay (LFA). Under the optimal conditions, the proposed RCA-FNA-LFA assay demonstrated the specificity and accuracy for miRNA let-7a detection with a detection limit of 1.07 pM, which increased sensitivity by nearly 20 times compared with that of RCA -LFA assay. It is worth noting that the non-target-dependent self-assembly process of HCR nanoscaffolds does not take up the whole detection time, thus, less time is taken than that of the conventional cascaded method. Moreover, the proposed assay does not need to consider the system compatibility between two kinds of isothermal amplification techniques. As for detection of different miRNAs, only the homologous arm of the padlock probe of RCA needs to be changed, while the FNA nanomaterial does not need any change, which greatly simplifies the primer design of the cascaded amplification techniques. With further development, the proposed RCA-FNA-LFA assay might achieve more sensitive and faster results to better satisfy the requirements of clinical diagnosis combing with more sensitive labels or small strip reader.
摘要：
为了解决级联扩增技术在miRND灵敏检测中存在的时间叠加和重复引物设计问题,提出了一种基于混合链式反应(HCR)纳米支架的新型功能核酸(FNA)纳米材料。将滚环扩增（RCA）与制备的FNA纳米材料级联，通过侧流测定（LFA）对miRNAlet-7a（作为模型靶标）进行灵敏检测。在最优条件下,拟议的RCA-FNA-LFA分析证明了miRNAlet-7a检测的特异性和准确性，检测限为1.07pM，与RCA-LFA测定相比，灵敏度提高了近20倍。值得注意的是，HCR纳米树体的非靶标依赖自组装过程并不占用整个检测时间，因此，比传统的级联方法花费更少的时间。此外，该方法不需要考虑两种等温扩增技术之间的系统兼容性。至于不同miRNA的检测，只需要改变RCA的挂锁探针的同源臂，虽然FNA纳米材料不需要任何改变，大大简化了级联扩增技术的引物设计。随着进一步发展,拟议的RCA-FNA-LFA测定可能会获得更灵敏和更快的结果，从而更好地满足临床诊断与更灵敏的标签或小型条检仪梳理的要求。
