title: Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification.
abstract: An enzyme-free and isothermal microRNA (miRNA) detection method has been developed based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction (HCR) on magnetic beads (MBs). The click-chemical ligation between an azide-modified probe DNA and a dibenzocyclooctyne-modified probe DNA occurred through the hybridization of target miRNA (miR-141). HCR on MBs was performed by the addition of DNA hairpin monomers (H1 and H2). After magnetic separation and denaturation/rehybridization of HCR products ([H1/H2] n ), the resulting HCR products were analyzed by the fluorescence emitted from an intercalative dye, allowing amplification of the fluorescent signal. The proposed assay had a limit of detection of 0.55 fmol, which was 230-fold more sensitive than that of the HCR on the MBs coupled with a conventional sandwich hybridization assay (without click-chemical ligation) (limit of detection 127 fmol). Additionally, the proposed assay could discriminate between miR-141 and other miR-200 family members. In contrast to quantitative reverse transcription polymerase chain reaction techniques using enzymes and thermal cycling, this is an enzyme-free assay that can be conducted under isothermal conditions and can specifically detect miR-141 in fetal bovine serum.
authors.affiliation: Division of Materials Science, Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, 305-8573, Japan, oishi@ims.tsukuba.ac.jp.
authors.forename: Motoi
authors.initials: M
authors.lastname: Oishi
mesh_terms: D057930:Click Chemistry; D004247:DNA / Q000737:chemistry*; D004798:Enzymes / Q000737:chemistry; D035683:MicroRNAs / Q000737:chemistry*
chemical_list: D004798:Enzymes; D035683:MicroRNAs; D004247:DNA