title: Direct fluorescence detection of microRNA based on enzymatically engineered primer extension poly-thymine (EPEPT) reaction using copper nanoparticles as nano-dye.
abstract: A new strategy based on enzymatically engineered primer extension poly-thymine (EPEPT) and nanomaterials in situ generation technology is reported for direct detection of microRNA (miRNA) in a fluorescence turn-on format using the sequential and complementary reactions catalyzed by Klenow Fragment exo-(KFexo-) and terminal deoxynucleotidyl transferase (TdTase). The short miRNA can be efficiently converted into long poly-thymine (polyT) sequences, which function as template for in situ formation of fluorescence copper nanoparticles (CuNPs) as nano-dye for detecting miRNA. The polyT-CuNPs can effectively form and emit intense red fluorescence under the 340nm excitation. For the proof of concept, microRNA-21 (miR-21) was selected as the model target to testify this strategy as a versatile assay platform. By directly using miR-21 as the primer, the simple, rapid and sensitive miRNA detection was successfully achieved with a good linearity between 1pM and 1nM and a detection limit of 100fM. Thus, the EPEPT strategy holds great potential in biochemical sensing research as an efficient and universal platform.
authors: College of Chemistry and Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China.
authors: Bao-Zhu
authors: BZ
authors: Chi
authors: College of Chemistry and Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China. Electronic address: rpliang@ncu.edu.cn.
authors: Ru-Ping
authors: RP
authors: Liang
authors: College of Chemistry and Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China.
authors: Wei-Bin
authors: WB
authors: Qiu
authors: College of Chemistry and Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China.
authors: Yan-Hong
authors: YH
authors: Yuan
authors: College of Chemistry and Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, China. Electronic address: jdqiu@ncu.edu.cn.
authors: Jian-Ding
authors: JD
authors: Qiu
mesh_terms: D000072283:A549 Cells; D015374:Biosensing Techniques / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D004253:DNA Nucleotidylexotransferase / Q000737:chemistry; D004256:DNA Polymerase I / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D057230:Limit of Detection; D061986:MCF-7 Cells; D053768:Metal Nanoparticles / Q000737:chemistry*; D035683:MicroRNAs / Q000032:analysis*; D011071:Poly T / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D011071:Poly T; D003300:Copper; D004253:DNA Nucleotidylexotransferase; D004256:DNA Polymerase I
keywords: Fluorescence detection; KFexo(−); PolyT-CuNPs; TdTase; miRNA