title: Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay.
abstract: This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.
authors: 1] State Key Joint Laboratory of ESPC, School of Environment, Tsinghua University, Beijing 10084, China [2] School of Environmental Science and Engineering, Hebei University of Science and Technology, Shijiazhuang 050000, China.
authors: Yun-Peng
authors: YP
authors: Xing
authors: School of Environmental Science and Engineering, Hebei University of Science and Technology, Shijiazhuang 050000, China.
authors: Chun
authors: C
authors: Liu
authors: State Key Joint Laboratory of ESPC, School of Environment, Tsinghua University, Beijing 10084, China.
authors: Xiao-Hong
authors: XH
authors: Zhou
authors: State Key Joint Laboratory of ESPC, School of Environment, Tsinghua University, Beijing 10084, China.
authors: Han-Chang
authors: HC
authors: Shi
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D015374:Biosensing Techniques; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism*; D054856:G-Quadruplexes; D007364:Intercalating Agents / Q000378:metabolism*; D007612:Kanamycin / Q000032:analysis*; D008892:Milk / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
chemical_list: D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D007612:Kanamycin; D004247:DNA
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