Quantitative detection of tumor necrosis factor-α by single molecule counting based on a hybridization chain reaction

• The method was based on single molecule counting and hybridization chain reaction.
• The proposed method was suitable for quantification of low concentration biomarker.
• The strategy exhibited a reliable result for TNF-α detection in real serum.

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.