Wang Xin, Qin Yuxin, Huang Yong, Hu Kun, Zhao Shulin, Tian Jianniao
School of Chemistry and Pharmaceutical Science of Guangxi Normal University, State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guilin 541004, China.
School of Chemistry and Pharmaceutical Science of Guangxi Normal University, State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guilin 541004, China.
Spectrochim Acta A Mol Biomol Spectrosc. 2022 Oct 15;279:121476. doi: 10.1016/j.saa.2022.121476. Epub 2022 Jun 6.
MicroRNAs (miRNAs) are important biomarkers that are closely associated with certain diseases. The detection of miRNA is critical because it provides the necessary information for Disease Diagnosis. In this study, we achieved miRNA determination by coupling the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated) system with strand displacement amplification (SDA). In the experiment, miRNA was used as the initiator of SDA, and the activator of Cas12a nuclease activity was amplified by SDA. Subsequently, the unique nuclease activity of Cas12a was exploited to carry out trans cleaving on the ssDNA reporting probe modified with carboxyfluorescein(FAM) and BHQ1(dark Quencher: 480-580 nm) to achieve a signal output. In addition to chain design and reaction simplification, this method is lofty sensitive and selective for the determination of miRNA with a good linear range of 250 fmol·L ∼ 40 pmol·L, the detection limit of 150 fmol·L (S/N = 3), and the method showed good recovery in spiked human serum. Overall, this method is expected to be applied to diagnosis with miRNA biomarkers because of its rapidity, high sensitivity, and high selectivity.
微小RNA（miRNA）是与某些疾病密切相关的重要生物标志物。miRNA的检测至关重要，因为它为疾病诊断提供了必要信息。在本研究中，我们通过将CRISPR-Cas（成簇规律间隔短回文重复序列-CRISPR相关蛋白）系统与链置换扩增（SDA）相结合实现了miRNA的测定。在实验中，miRNA用作SDA的引发剂，通过SDA扩增Cas12a核酸酶活性的激活剂。随后，利用Cas12a独特的核酸酶活性对用羧基荧光素（FAM）和BHQ1（暗猝灭剂：480-580nm）修饰的单链DNA报告探针进行反式切割以实现信号输出。除了链设计和反应简化外，该方法对miRNA的测定具有高灵敏度和高选择性，线性范围良好，为250fmol·L至40pmol·L，检测限为150fmol·L（S/N=3），并且该方法在加标的人血清中显示出良好的回收率。总体而言，由于其快速性、高灵敏度和高选择性，该方法有望应用于基于miRNA生物标志物的诊断。
