title: An ultrasensitive label-free biosensor for assaying of sequence-specific DNA-binding protein based on amplifying fluorescent conjugated polymer.
abstract: Sensitive, reliable, and simple detection of sequence-specific DNA-binding proteins (DBP) is of paramount importance in the area of proteomics, genomics, and biomedicine. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, visual, quantitative, and "turn-on" detection of DBP. A Förster resonance energy transfer (FRET) assay utilizing a cationic conjugated polymer (CCP) and an intercalating dye was designed to detect a key transcription factor, nuclear factor-kappa B (NF-κB), the model target. A series of label-free DNA probes bearing one or two protein-binding sites (PBS) were used to identify the target protein specifically. The binding DBP protects the probe from digestion by exonuclease III, resulting in high efficient FRET due to the high affinity between the intercalating dye and duplex DNA, as well as strong electrostatic interactions between the CCP and DNA probe. By using label-free hairpin DNA or double-stranded DNA containing two PBS as probe, we could detect as low as 1 pg/μL of NF-κB in HeLa nuclear extracts, which is 10000-fold more sensitive than the previously reported methods. The approach also allows naked-eye detection by observing fluorescent color of solutions with the assistance of a hand-held UV lamp. Additionally, a less than 10% relative standard deviation was obtained, which offers a new platform for superior precision, low-cost, and simple detection of DBP. The features of our optical biosensor shows promising potential for early diagnosis of many diseases and high-throughput screening of new drugs targeted to DNA-binding proteins.
authors.affiliation: Key Laboratory for Organic Electronics & Information Displays (KLOEID), Institute of Advanced Materials (IAM), School of Materials Science and Engineering, Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210046, China. iamxfliu@njupt.edu.cn
authors.forename: Xingfen
authors.initials: X
authors.lastname: Liu
authors.forename: Lan
authors.initials: L
authors.lastname: Ouyang
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Cai
authors.forename: Yanqin
authors.initials: Y
authors.lastname: Huang
authors.forename: Xiaomiao
authors.initials: X
authors.lastname: Feng
authors.forename: Quli
authors.initials: Q
authors.lastname: Fan
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
mesh_terms: D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes