title: Fluorescence Detection of Single DNA Molecules.
abstract: Single-molecule detection (SMD) and single-molecule fluorescence resonance energy transfer (smFRET) were conducted using Cy3- and Cy5-labeled single-strand DNAs (ssDNAs) either immobilized on substrates or encapsulated in microdroplets. High-quality fluorescent images were obtained using a total internal reflection fluorescence microscope (TIRFM). In the substrate system, deposition of a low concentration of fluorescence molecules on substrates through electrostatic adsorption showed that most of the fluorescence spots were single molecules, and the mean value of signal to noise ratio (S/N) reached 6.9 ± 0.34. smFRET analysis was conducted through immobilization of donor- and acceptor-labeled oligonucleotides on substrates. In the droplet system, fluorophor-labeled oligonucleotides were injected into T-type microfluidics. Single and double fluorophor-labeled DNA molecules encapsulated in droplets were detected, the FRET efficiency and inter-dye distance of a single donor-acceptor pair were measured accurately. smFRET was conducted detailedly in the tortuous channel for the first time.
authors.affiliation: School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Weidong
authors.initials: W
authors.lastname: Huang
authors.affiliation: School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Wang
authors.affiliation: School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China. zmwang@sjtu.edu.cn.
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Wang
mesh_terms: D004247:DNA / Q000032:analysis* / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005898:Glass / Q000737:chemistry; D059629:Signal-To-Noise Ratio
chemical_list: D005456:Fluorescent Dyes; D004247:DNA
keywords: Microdroplet; Signal to noise ratios; Single-molecule detection; TIRFM; smFRET