title: Re-evaluation of biotin-streptavidin conjugation in Förster resonance energy transfer applications.
abstract: Bioaffinity conjugation between streptavidin (SA) and biotin has been widely used to link donors and acceptors for investigating the distance-dependent Förster resonance energy transfer (FRET). When studying a commonly used FRET system of (QD-SA)-(biotin-DNA-dye) [donor: quantum dot (QD); acceptor: small organic fluorescent dye; and linker: deoxyribose nucleic acid (DNA) molecule via SA-biotin conjugation], however, a contradictory finding was recently reported in the literature. It was found that the FRET lost its dependence on the number of DNA base pairs when using a phosphate-buffered saline (PBS) solution. We found that the conflicted results were caused by the ionic strength of the adopted buffer solutions. Our results suggest that the dependent FRET on the number of DNA bases is favorable in a low-ionic-strength buffer, whereas in relatively high-ionic-strength buffers, the FRET loses the DNA length dependence. We propose that the independence is mainly caused by the conformational change of DNA molecules from a stretched to a coiled mode when the cations in the high-ionic-strength buffer neutralize the negatively charged backbone of DNA molecules, thereby bringing the acceptors close to the donors.
authors.affiliation: University of Texas at Arlington, Department of Bioengineering, Ultrasound and Optical Imaging Laboratory, Arlington, 500 UTA Boulevard, Texas 76010, United StatesbUniversity of Texas at Arlington and University of Texas Southwestern Medical Center at Dal.
authors.forename: Bahar
authors.initials: B
authors.lastname: Saremi
authors.affiliation: University of Texas at Arlington, Department of Bioengineering, Ultrasound and Optical Imaging Laboratory, Arlington, 500 UTA Boulevard, Texas 76010, United StatesbUniversity of Texas at Arlington and University of Texas Southwestern Medical Center at Dal.
authors.forename: Ming-Yuan
authors.initials: MY
authors.lastname: Wei
authors.affiliation: University of Texas at Arlington, Department of Bioengineering, Ultrasound and Optical Imaging Laboratory, Arlington, 500 UTA Boulevard, Texas 76010, United StatesbUniversity of Texas at Arlington and University of Texas Southwestern Medical Center at Dal.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: University of Texas at Arlington, Department of Bioengineering, Ultrasound and Optical Imaging Laboratory, Arlington, 500 UTA Boulevard, Texas 76010, United StatesbUniversity of Texas at Arlington and University of Texas Southwestern Medical Center at Dal.
authors.forename: Bingbing
authors.initials: B
authors.lastname: Cheng
authors.affiliation: University of Texas at Arlington, Department of Bioengineering, Ultrasound and Optical Imaging Laboratory, Arlington, 500 UTA Boulevard, Texas 76010, United StatesbUniversity of Texas at Arlington and University of Texas Southwestern Medical Center at Dal.
authors.forename: Baohong
authors.initials: B
authors.lastname: Yuan
mesh_terms: D016477:Artifacts; D001710:Biotin / Q000737:chemistry* / Q000276:immunology; D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods; D045663:Quantum Dots; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D019809:Streptavidin / Q000737:chemistry* / Q000276:immunology
chemical_list: D001710:Biotin; D004247:DNA; D019809:Streptavidin