title: Electrochemical biosensor for microRNA detection based on poly(U) polymerase mediated isothermal signal amplification.
abstract: 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 of p-nitrophenyl phosphate disodium salt hexahydrate can be hydrolyzed to produce 4-nitrophenol. According to the relationship between the electrochemical signal of p-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.
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, PR China.
authors.forename: Yunlei
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, PR China; State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, PR China. Electronic address: yinhs@sdau.edu.cn.
authors.forename: Huanshun
authors.initials: H
authors.lastname: Yin
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, PR China.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, PR China.
authors.forename: Bingchen
authors.initials: B
authors.lastname: Li
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, PR China.
authors.forename: Xue
authors.initials: X
authors.lastname: Li
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, PR China.
authors.forename: Shiyun
authors.initials: S
authors.lastname: Ai
authors.affiliation: State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, PR China. Electronic address: zhangxs@sdau.edu.cn.
authors.forename: Xiansheng
authors.initials: X
authors.lastname: Zhang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D055664:Electrochemical Techniques / Q000379:methods*; D035683:MicroRNAs / Q000032:analysis*; D012275:Oryza / Q000737:chemistry*; D018749:RNA, Plant / Q000032:analysis*; D014544:Uridine Triphosphate / Q000737:chemistry
chemical_list: D035683:MicroRNAs; D018749:RNA, Plant; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate
keywords: Electrochemical biosensors; MicroRNA detection; Phytohormone; Poly(U) polymerase; Rice seedlings