title: Quantitative detection of tumor necrosis factor-α by single molecule counting based on a hybridization chain reaction.
abstract: This work reports a novel and sensitive quantitative method for detection of tumor necrosis factor-α (TNF-α) based on single molecule counting and hybridization chain reaction (HCR). In the presence of TNF-α, sandwich-type immunocomplex was formed on the surface of glass substrate. The streptavidin acted as a bridge bounded to the biotinylated immunocomplex, which provided three sites to fixate the biotinylated initiator strands. The initiator strands triggered the chain reaction of hybridization to form a long double-helix polymer and SYBR Green I, acted as the fluorescence label, intercalated into the grooves of the long dsDNA polymer. Then, the quantitative detection of TNF-α was realized by single molecule counting. Under the optimal conditions, HCR-based single molecule counting quantitative method could successfully detect TNF-α in the range of 50 fM to 1 pM, and it revealed a reliable result for TNF-α detection in real serum. Moreover, the proposed immunosensor exhibited excellent specificity. These results greatly demonstrated that the proposed method possessed the potentiality in clinical application and it was suitable for quantification of biomarker under low concentration.
authors.affiliation: Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Dai
authors.affiliation: Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China.
authors.forename: Chunjing
authors.initials: C
authors.lastname: Feng
authors.affiliation: Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China. Electronic address: wjiang@sdu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China. Electronic address: wangl-sdu@sdu.edu.cn.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation; D004247:DNA / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007118:Immunoassay / Q000295:instrumentation*; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation*; D008832:Microchemistry / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D057054:Molecular Imaging / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000097:blood* / Q000235:genetics
chemical_list: D014409:Tumor Necrosis Factor-alpha; D004247:DNA
keywords: Enzyme-free; Hybridization chain reaction; Single molecule counting; TNF-α