基于多组份DNA酶介导滚环扩增对microRNA的超灵敏电化学检测

发布时间：2018-01-16 13:21

本文关键词：基于多组份DNA酶介导滚环扩增对microRNA的超灵敏电化学检测　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：Micro RNAs(miRNAs)是一类调节基因表达的短RNA序列,通过连续内切酶剪切过程从长的前体RNA释放出来。Mi RNA异常表达与人类肿瘤,神经性疾病以及病毒感染等有关。定性定量分析miRNA对疾病早期诊断和治疗起到十分重要的作用。因此,发展灵敏、特异的检测方法已成为miRNA检测分析的迫切需求。然而,miRNA序列高度同源、尺寸小和自然丰度低。现应用最多的技术,例如聚合酶链反应(PCR)、微阵列和Northern印迹法不能灵敏和特异地检测miRNA。所以发展高灵敏和有效的方法对miRNA检测尤为重要。本研究基于多组份DNA酶(MNAzyme)放大和滚环扩增(RCA),构建了一个电化学策略来超灵敏和特异地检测miRNA。在靶miRNAs存在的情况下,A部分酶(partzyme A)和B部分酶(partzyme B)组装形成有酶活性的多组份DNA酶。多组份DNA酶形成之后开始剪切发夹状底物而释放出生物素标记的片段,该片段与金电极上的捕获探针互补结合。然后在电极上触发滚环扩增形成重复串联的扩增产物并结合大量的检测探针。最后,通过亲和素化的碱性磷酸酶进行酶促反应得到电流信号。该生物传感器检测线可达到1.66 f M,线性范围为10 f M到1 n M。同时具有很好的重现性和特异性,可区分单个碱基错配。而且可对人乳腺癌MCF-7细胞总RNA提取样本中的miRNA进行检测。
[Abstract]:Micro RNAsmiRNAs) is a kind of short RNA sequence that regulates gene expression. The abnormal expression of Mi RNA was released from the long precursor RNA by continuous endonuclease shearing process. Qualitative and quantitative analysis of miRNA plays a very important role in the early diagnosis and treatment of the disease. Therefore, the development of sensitivity. Specific detection methods have become an urgent need for miRNA detection and analysis. However, miRNA sequences are highly homologous, small in size and low in natural abundance. For example, polymerase chain reaction (PCR). Microarrays and Northern blotting can not detect miRNAs sensitively and specifically. Therefore, it is very important to develop highly sensitive and effective methods for miRNA detection. MNAzyme) amplification and rolling loop amplification (RCA). An electrochemical strategy was constructed for the detection of miRNA in a hypersensitive and specific manner in the presence of target miRNAs. Partzyme A (A) and partzyme B (B). Assemble to form a multicomponent DNA enzyme with enzyme activity. After the formation of a multicomponent DNA enzyme, the hairpin substrate is cut and biotin labeled fragments are released. The fragment is complementary to the capture probe on the gold electrode. Then the rolling loop amplification is triggered on the electrode to form a repeated tandem amplification product and a large number of detection probes are combined. Finally. The current signal was obtained by enzymatic reaction of affinity alkaline phosphatase. The detection line of the biosensor can reach 1.66 FM. The linear range is from 10 FM to 1 n M. at the same time, it has good reproducibility and specificity. Single base mismatch can be distinguished and miRNA in total RNA samples of human breast cancer MCF-7 cells can be detected.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R440;TP212.3

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