Abnormal microRNA (miRNA) expression levels are confirmed as diagnostic biomarkers of the emergence and development of diseases. In this study, we developed a fluorescence biosensor for detecting miRNAs based on double amplification reactions with the primer exchange reaction (PER) and CRISPR/Cas12a. In the absence of target miRNA-21, PER hairpins remained locked by the protector strands and the primers did not extend. In the presence of target miRNA-21, the miRNA-21 bound to the guard sequence and exposed primer binding sites. Also, the closed PER hairpin was unlocked to specifically extend primers into single-stranded DNA (ssDNA) of unequal lengths. These ssDNAs of unequal lengths could activate the cleavage of a reporter by Cas12a, leading to an increase in detectable fluorescence signals. A large number of short nucleic acid fragments were amplified by PER-CRISPR multiple cycle cleavage fluorescent probes. Based on PER-combined CRISPR/Cas12a established dual signal amplification method was characterized by a low limit of detection of 10fM. The fluorescent biosensor for miRNA detection had the advantages of low detection cost, simple operation, and mobility, providing a very promising platform for the point-of-care testing of miRNA-21.

异常的 microRNA (miRNA) 表达水平被确认为疾病出现和发展的诊断生物标志物。在本研究中，我们开发了一种基于引物交换反应 (PER) 和 CRISPR/Cas12a 的双扩增反应检测 miRNA 的荧光生物传感器。在没有目标 miRNA-21 的情况下，每个发夹仍然被保护链锁定，并且引物没有延伸。在存在目标 miRNA-21 的情况下，miRNA-21 与保护序列结合并暴露引物结合位点。此外，封闭的 PER 发夹被解锁以专门将引物延伸到长度不等的单链 DNA (ssDNA) 中。这些长度不等的 ssDNA 可以激活 Cas12a 对报告基因的切割，从而导致可检测荧光信号的增加。PER-CRISPR多循环切割荧光探针扩增出大量短核酸片段。基于PER-combined CRISPR/Cas12a建立的双信号放大方法的特点是检测下限为10fM。用于miRNA检测的荧光生物传感器具有检测成本低、操作简单、移动方便等优点，为miRNA-21的即时检测提供了一个非常有前景的平台。