title: Target-triggered cyclic assembly of DNA-protein hybrid nanowires for dual-amplified fluorescence anisotropy assay of small molecules.
abstract: Aptamer-based fluorescence anisotropy (FA) assays have attracted great interest in recent years. However, a key factor that determines FA value is molar mass, thus limiting the utility of this assay for the detection of small molecules. To solve this problem, streptavidin, as a molar mass amplifier, was used in a hybridization chain reaction (HCR) to construct a target-triggered cyclic assembly of DNA-protein hybrid nanowires for highly sensitive detection of small molecules by fluorescence anisotropy. In this assay, one blocking DNA strand is released by target-aptamer recognition. The DNA then serves as an initiator to trigger enzyme-free autonomous cross-opening of hairpin probes via HCR to form a DNA nanowire for further assembly of streptavidin. Using adenosine triphosphate (ATP) as the small molecule target, this novel dual-amplified, aptamer-based FA assay affords high sensitivity with a detection limit of 100 nM. This limit of detection (LOD) is much lower than that of the disassembly approach without HCR amplification or the assembly strategy without streptavidin. In contrast to the previous turn-off disassembly approaches based on nonspecific interactions between the aptamer probe and amplification moieties, the proposed aptamer-based FA assay method exhibits a turn-on response to ATP, which can increase sensing reliability and reduce the risk of false hits. Moreover, because of its resistance to environmental interferences, this FA assay has been successfully applied for direct detection of 0.5 μM ATP in complex biological samples, including cell media, human urine, and human serum, demonstrating its practicality in real complex biological systems.
authors.affiliation: Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University , Changsha, Hunan 410082, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Zhang
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Kang
authors.forename: Guo-Li
authors.initials: GL
authors.lastname: Shen
authors.forename: Ru-Qin
authors.initials: RQ
authors.lastname: Yu
authors.forename: Weihong
authors.initials: W
authors.lastname: Tan
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D005454:Fluorescence Polarization / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D053770:Nanowires / Q000737:chemistry*; D009841:Oligonucleotides / Q000032:analysis / Q000235:genetics
chemical_list: D009841:Oligonucleotides; D004247:DNA