It remains technically challenging to develop a sensitive assay system to isothermally amplify the signal for miRNA detection because of its low abundance in tested sample, sequence similarities and existence in complex biological environments. In this study, using miRNA-21 as target model, a hairpin-inserted cross-shaped DNA nanoprobe (CP) with four functional arms is constructed for the ultrasensitive detection of miRNA via one-step built-in target analogue (BTA) cycle-mediated signal amplification. BTA is pre-locked in one arm of CP probe and inactive. In the presence of target miRNA, BTA can be unlocked and initiate an isothermal amplification process. Utilizing as-designed CP probe, miRNA-21 can be detected to down to 500 fM, and the linear response range spans over five orders of magnitude. The nonspecific signal is less than 1% upon nontarget miRNAs. CP probe exhibits ∼six times enhancement in resistance to nuclease degradation and no obvious degradation-induced fluorescence change is detected during the assay period. The recovery yield ranges from 98.2～105.5% in FBS solution. Because of the high sensitivity, desirable specificity, strong anti-interference ability and substantial increase in nuclease resistance, CP probe is a promising tool for the detection of miRNAs in a complex biological milieu.

由于其在测试样品中的低丰度、序列相似性以及在复杂生物环境中的存在，开发一种灵敏的分析系统来等温放大 miRNA 检测的信号在技术上仍然具有挑战性。本研究以 miRNA-21 为靶标模型，构建了一种具有四个功能臂的发夹插入十字形 DNA 纳米探针 (CP)，用于超灵敏检测 miRNA 。一步内置目标模拟 (BTA) 循环介导的信号放大。BTA 预锁定在 CP 探头的一个臂中并且处于非活动状态。在存在目标 miRNA 的情况下，BTA 可以被解锁并启动等温扩增过程。利用设计的 CP 探针，miRNA-21 可以检测到低至 500 fM，线性响应范围跨越五个数量级。非目标 miRNA 的非特异性信号小于 1%。CP 探针对核酸酶降解的抵抗力提高了 6 倍，并且在测定期间未检测到明显的降解诱导的荧光变化。FBS溶液的回收率在98.2～105.5%之间。由于灵敏度高、特异性好、抗干扰能力强、核酸酶抗性显着提高，