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基于双链特异性核酸酶(Duplex-specific nuclease,DSN)选择性切割DNA单链和DNA保护的银纳米簇的特性,建立了一种新型的荧光传感器用于MicroRNA(MiR-21)的高灵敏检测。当MiR-21存在时,与Cp杂交形成DNA/RNA双链结构,此时在DSN的水解下,释放出探针DNA部分片段和完整的MiR-21。释放出来的MiR-21可再次发生与Cp杂交、DSN酶切等反应,如此反复,可实现一条MiR-21分子与多条探针杂交、酶切的循环过程。经磁分离,上清液中的探针DNA部分片段与Ag+结合并在硼氢化钠(Na BH4)的还原下产生银簇,检测到较强的荧光信号。相反,当MiR-21不存在时,DSN对单链探针无酶切作用,不能水解Cp,经磁分离,上清液中检测不到荧光信号。在最佳条件下,该传感器检测MiR-21的线性范围为50 fmol/L~10 pmol/L,检测限达6 fmol/L,而且具有较好的选择性,有望用于临床实际样本中微量MiR-21的检测。
Based on the characteristic of single-strand DNA and DNA-protected silver nanoclusters selectively cleaved by Duplex-specific nuclease (DSN), a novel fluorescence sensor was developed for the highly sensitive microRNA (MiR-21) Detection. When MiR-21 is present, it hybridizes to Cp to form a double-stranded DNA / RNA structure, where part of the probe DNA and complete MiR-21 are released by hydrolysis of DSN. The released MiR-21 may repeat the reaction with Cp hybridization and DSN digestion, etc. In this way, a MiR-21 molecule can be hybridized with a plurality of probes to digest and circulate. After magnetic separation, part of the probe DNA in the supernatant bound to Ag + and produced silver clusters under the reduction of sodium borohydride (Na BH4). Strong fluorescence signals were detected. In contrast, DSN did not cleave single-stranded probes without MiR-21, did not hydrolyze Cp, and did not detect fluorescent signals in the supernatant by magnetic separation. Under the optimal conditions, the detection limit of MiR-21 was 50 fmol / L ~ 10 pmol / L and the detection limit was 6 fmol / L, and the selectivity of MiR-21 was good. MiR-21 testing.