Detection of biotin-streptavidin interactions based on poly(thymine)-templated copper nanoparticles coupled with Exo Ⅲ-aided DNA recycling amplification

摘要

The recognition and analysis of small molecule–protein interactions plays an important role in chemical genetics, clinical diagnostics, and drug development. However, various established highly sensitive fluorescence strategies still involve the use of expensive fluorescence dye-labelled nucleic acids. Herein, we constructed a highly sensitive and label-free fluorescent assay for small molecule–protein interactions on the basis of exonuclease III (Exo III)-assisted signal amplification and poly(thymine)-templated fluorescent copper nanoparticles (CuNPs) as signal probes. Biotin–streptavidin system was taken as a model. It was designed that biotinylated double-strand DNA served as the binding probe. Biotin was labelled at the 3′-end of the trigger strand. One kind of stem-loop DNA, which consisted of the thymine rich sequence and the trigger DNA recognition domain, was also designed. Once streptavidin bind to the binding probe, with the help of Exo III digestion, the trigger strand was able to reach the cycle for multiple poly(thymine) sequences generation. The produced poly(thymine) sequences can be used as a template for the formation of CuNPs. Comparing with no Exo III-aided DNA recycling amplification, significantly improved signals for the quantitative determination of streptavidin were obtained.