title: Enzyme-free detection of sequence-specific microRNAs based on nanoparticle-assisted signal amplification strategy.
abstract: Developing direct and convenient methods for microRNAs (miRNAs) analysis is of great significance in understanding biological functions of miRNAs, and early diagnosis of cancers. We have developed a rapid, enzyme-free method for miRNA detection based on nanoparticle-assisted signal amplification coupling fluorescent metal nanoclusters as signal output. The proposed method involves two processes: target miRNA-mediated nanoparticle capture, which consists of magnetic microparticle (MMP) probe and CuO nanoparticle (NP) probe, and nanoparticle-mediated amplification for signal generation, which consists of fluorescent DNA-Cu/Ag nanocluster (NC) and 3-mercaptopropionic acid (MPA). In the presence of target miRNA, MMP probe and NP probe sandwich-capture the target miRNA via their respective complementary sequence. The resultant sandwich complex (MMP probe-miRNA-CuO NP probe) is separated using a magnetic field and further dissolved by acidolysis to turn CuO NP into a great amount of copper (II) ions (Cu(2+)). Cu(2+) could disrupt the interactions between thiol moiety of MPA and the fluorescent Cu/Ag NCs by preferentially reacting with MPA to form a disulfide compound as intermediate. By this way, the fluorescence emission of the DNA-Cu/Ag NCs in the presence of MPA increases upon the increasing concentration of Cu(2+), which is directly proportional to the amount of target miRNA. The proposed method allows quantitative detection of a liver-specific miR-221-5p in the range of 5 pM to 1000 pM with a detection limit of ~0.73 pM, and shows a good ability to discriminate single-base difference. Moreover, the detection assay can be applied to detect miRNA in cancerous cell lysates in excellent agreement with that from a commercial miRNA detection kit.
authors.affiliation: Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China.
authors.forename: Ru-Dong
authors.initials: RD
authors.lastname: Li
authors.affiliation: Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China. Electronic address: binchengyin@ecust.edu.cn.
authors.forename: Bin-Cheng
authors.initials: BC
authors.lastname: Yin
authors.affiliation: Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China. Electronic address: bcye@ecust.edu.cn.
authors.forename: Bang-Ce
authors.initials: BC
authors.lastname: Ye
mesh_terms: D001483:Base Sequence; D003300:Copper / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004798:Enzymes; D004867:Equipment Design; D019544:Equipment Failure Analysis; D058185:Magnetite Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D053768:Metal Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000295:instrumentation*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation
chemical_list: D004798:Enzymes; D058185:Magnetite Nanoparticles; D035683:MicroRNAs; D003300:Copper; D004247:DNA; C030973:cupric oxide
keywords: CuO nanoparticle; Fluorescent DNA–Cu/Ag nanoclusters; Magnetic microparticle; MicroRNA detection; Signal amplification