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基于磁珠(MBs)的分离富集和双链特异性核酸酶(DSN)选择性切割DNA单链的特性,建立了信号增强型荧光生物传感器用于microRNA-21(miR-21)的检测。荧光素(FAM)修饰的捕获探针(Cps),通过亲和素-生物素的特异识别作用固定在磁珠表面。当miR-21存在时,Cps与其杂交形成DNA/RNA双螺旋结构,DSN能特异性水解杂合双链中的DNA,同时释放出荧光标记片段和完整的miR-21。被释放出来的miR-21与另一Cp再次杂交并被DSN酶切,如此循环,从而实现恒温条件下一个miR-21与多个Cp杂交、酶切,释放出大量的荧光标记片段的循环过程,最终使体系的荧光强度明显变大。相反,当miRNA-21不存在时,Cps无法形成双螺旋结构,DSN对单链DNA无酶切作用,不能水解Cps,经磁分离,上清液没有荧光标记片段,所以检测不到荧光信号。最佳条件下,miR-21浓度在100~5×104fmol/L范围内,荧光强度与其浓度呈良好的线性关系,检测限达80 fmol/L。该传感器可以识别单碱基错配序列,有望为肿瘤早期诊断提供新思路。
Based on the separation and enrichment of magnetic beads (MBs) and the selective cleavage of single strand DNA by double strand specific nucleases (DSNs), a signal-enhanced fluorescent biosensor was developed for the detection of microRNA-21 (miR-21). Fluorescein (FAM) -modified capture probe (Cps) was immobilized on the surface of magnetic beads by specific recognition of avidin-biotin. When miR-21 is present, Cps hybridizes with it to form a DNA / RNA double helix structure. DSN can specifically hydrolyze DNA in the hybrid double stranded DNA while releasing the fluorescently labeled fragment and intact miR-21. The released miR-21 hybridizes again with another Cp and is cleaved by DSN, so as to circulate so that a miR-21 hybridizes with multiple Cps at a constant temperature, digesting and releasing a large number of fluorescently labeled fragments , Eventually the system significantly increased the fluorescence intensity. On the contrary, when miRNA-21 is absent, Cps can not form double-helix structure. DSN can not digest single-stranded DNA and can not hydrolyze Cps. After magnetic separation, there is no fluorescent labeled fragment in supernatant, so no fluorescence signal can be detected. Under optimal conditions, the concentration of miR-21 was in the range of 100 ~ 5 × 104fmol / L, and the fluorescence intensity showed a good linear relationship with the concentration, with the detection limit of 80 fmol / L. The sensor can recognize single-base mismatch sequences and is expected to provide new ideas for the early diagnosis of tumors.