In this study, a biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) was proposed for the highly sensitive detection of cancer-related let-7a microRNA (miRNA). The sensing system consists of a padlock probe (PP), which contains a target recognition sequence and two binding sites for nicking endonuclease (NEase), and molecular beacon (MB) as reporting molecule. Upon hybridization with let-7a, the PP can be circularized by ligase. Then, the miRNA acted as polymerization primer to initiate rolling circle amplification (RCA). With the assistance of NEase, RCA products can be nicked on the cyclized PP and are displaced during the subsequent duplication process, generating numerous nicked fragments (NFs). These NFs not only induce another RCA reaction but also open the molecular beacons (MBs) via hybridization, leading to significantly amplified fluorescence signal. Under the optimized conditions, this method exhibits high sensitivity toward target miRNA let-7a with a detection limit of as low as 10 pM, a dynamic range of three orders of magnitude is achieved, and its family member is easily distinguished even with only one mismatched base. Meanwhile, it displays good recovery and satisfactory reproducibility in fetal bovine serum (FBS). Therefore, these merits endow the newly proposed N-RCA strategy with powerful implications for miRNA detection.

Open image in new window

在这项研究中，提出了一种基于切口增强滚环扩增（N-RCA）的生物传感系统，用于高灵敏度检测与癌症相关的let-7a microRNA（miRNA）。传感系统由一个挂锁探针（PP）组成，该探针包含一个靶标识别序列和两个用于切割核酸内切酶（NEase）的结合位点，以及作为报告分子的分子信标（MB）。与let-7a杂交后，可通过连接酶将PP环化。然后，miRNA充当聚合引物以启动滚环扩增（RCA）。在NEase的帮助下，RCA产品可以在环化的PP上形成缺口，并在随后的复制过程中移位，从而产生大量的缺口碎片（NF）。这些NF不仅诱发了另一个RCA反应，还通过杂交打开了分子信标（MBs），导致荧光信号明显放大。在优化的条件下，该方法对目标miRNA let-7a表现出高灵敏度，检测限低至10 pM，动态范围达到三个数量级，并且即使只有一个不匹配的情况，其家族成员也易于区分根据。同时，它在胎牛血清（FBS）中显示出良好的回收率和令人满意的重现性。因此，这些优点赋予了新提出的N-RCA策略对miRNA检测强大的意义。而且即使只有一个不匹配的基数，也很容易区分其家庭成员。同时，它在胎牛血清（FBS）中显示出良好的回收率和令人满意的重现性。因此，这些优点赋予了新提出的N-RCA策略对miRNA检测强大的意义。而且即使只有一个不匹配的基数，也很容易区分其家庭成员。同时，它在胎牛血清（FBS）中显示出良好的回收率和令人满意的重现性。因此，这些优点赋予了新提出的N-RCA策略对miRNA检测强大的意义。

在新窗口中打开图像