Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification
Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification
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DOI：
10.1007/s00216-015-8629-y
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发表时间：
2015-05-01
影响因子：
4.3
通讯作者：
Oishi, Motoi
中科院分区：
文献类型：
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作者：
Oishi, Motoi
建立了一种基于磁珠上点击化学连接辅助杂交和杂交链反应（HCR）的无酶等温检测microRNA（miRNA）的方法。叠氮化物修饰的探针DNA和二苯并环辛炔修饰的探针DNA之间的点击化学连接通过靶miRNA（miR-141）的杂交发生。通过添加DNA发夹单体（H1和H2）对MB进行HCR。在磁性分离和HCR产物的变性/再杂交（[H1/H2]（n））之后，通过从嵌入染料发射的荧光分析所得HCR产物，允许荧光信号的放大。所提出的测定具有0.55 fmol的检测限，这是230倍以上的灵敏度比HCR上的MB耦合与常规的夹心杂交测定（无点击化学连接）（检测限127 fmol）。此外，所提出的测定可以区分miR-141和其他miR-200家族成员。与使用酶和热循环的定量逆转录聚合酶链反应技术相比，这是一种无酶测定法，可在等温条件下进行，并可特异性检测胎牛血清中的miR-141。
An enzyme-free and isothermal microRNA (miRNA) detection method has been developed based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction (HCR) on magnetic beads (MBs). The click-chemical ligation between an azide-modified probe DNA and a dibenzocyclooctyne-modified probe DNA occurred through the hybridization of target miRNA (miR-141). HCR on MBs was performed by the addition of DNA hairpin monomers (H1 and H2). After magnetic separation and denaturation/rehybridization of HCR products ([H1/H2] (n) ), the resulting HCR products were analyzed by the fluorescence emitted from an intercalative dye, allowing amplification of the fluorescent signal. The proposed assay had a limit of detection of 0.55 fmol, which was 230-fold more sensitive than that of the HCR on the MBs coupled with a conventional sandwich hybridization assay (without click-chemical ligation) (limit of detection 127 fmol). Additionally, the proposed assay could discriminate between miR-141 and other miR-200 family members. In contrast to quantitative reverse transcription polymerase chain reaction techniques using enzymes and thermal cycling, this is an enzyme-free assay that can be conducted under isothermal conditions and can specifically detect miR-141 in fetal bovine serum.