Electrochemical biosensor for microRNA detection based on poly(U) polymerase mediated isothermal signal amplification.
Electrochemical biosensor for microRNA detection based on poly(U) polymerase mediated isothermal signal amplification.
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DOI：
10.1016/j.bios.2015.12.009
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发表时间：
2016-05
影响因子：
12.6
通讯作者：
Yunlei Zhou;Huanshun Yin;Jiefa Li;Bingchen Li;Xue Li;S. Ai;Xian-sheng Zhang
中科院分区：
文献类型：
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作者：
Yunlei Zhou;Huanshun Yin;Jiefa Li;Bingchen Li;Xue Li;S. Ai;Xian-sheng Zhang
MicroRNA 在动物、植物和病毒基因表达的转录后调控中发挥着至关重要的作用。为了更好地了解microRNA及其功能，开发有效的microRNA检测分析方法非常重要。在此，基于聚（U）聚合酶介导的等温信号放大，制造了一种新型电化学生物传感器，用于灵敏和选择性地检测 microRNA，其中聚（U）聚合酶可以催化 UMP 从 UTP 到 RNA 3' 端的模板独立添加。利用这一活性，可以使用生物素缀合的 UTP（生物素-UTP）作为供体，在目标 microRNA 的 3' 端成功标记生物素缀合的 UMP。然后，基于生物素和亲和素之间的特异性相互作用，亲和素缀合的碱性磷酸酶可以进一步被捕获到目标microRNA的3'端。最后在碱性磷酸酶的催化活性下，底物对硝基苯磷酸二钠盐六水合物水解生成4-硝基苯酚。根据对硝基苯酚的电化学信号与microRNA-319a浓度的关系，可以检测microRNA-319a的含量。这种信号放大方法简单、灵敏。开发的方法可以检测低至 1.7 fM 的 microRNA，并产生 10 至 1000 fM 的精确线性动态范围。所构建的生物传感器进一步应用于检测水稻幼苗与五种不同植物激素孵育后microRNA-319a表达水平的变化。
MicroRNAs play crucial role in post-transcriptional regulation for gene expression in animals, plants, and viruses. For the better understanding of microRNA and its functions, it is very important to develop effectively analytical method for microRNA detection. Herein, a novel electrochemical biosensor was fabricated for sensitive and selective detection of microRNA based on poly(U) polymerase mediated isothermal signal amplification, where poly(U) polymerase can catalyze the template independent addition of UMP from UTP to the 3’ end of RNA. Using this activity, the target microRNA can be successfully labeled with biotin conjugated UMPs at its 3′-end using biotin conjugated UTP (biotin-UTP) as donor. Then, the avidin conjugated alkaline phosphatase can be further captured to the 3′-end of the target microRNA based on the specific interaction between biotin and avidin. Finally, under the catalytic activity of alkaline phosphatase, the substrate ofp-nitrophenyl phosphate disodium salt hexahydrate can be hydrolyzed to produce 4-nitrophenol. According to the relationship between the electrochemical signal ofp-nitrophenol and the concentration of microRNA-319a, the content of microRNA-319a can be detected. This signal amplification method is simple and sensitive. The developed method can detect as low as 1.7 fM microRNA and produce precise and accurate linear dynamic range from 10 to 1000 fM. The fabricated biosensor was further applied to detect the expression level change of microRNA-319a in rice seedlings after incubation with five kinds of different phytohormones.