To achieve single-molecule detection based on enzyme-free and isothermal amplification techniques, we have developed a strategy employing coupled catalytic hairpin assembly reactions and investigated several parameters influencing its yield. We employed avidin-biotin assay using biotin-modified trigger DNA and surface plasmon resonance to excite fluorescence-labeled hairpin DNA captured a surface. Our investigation of association and dissociation of fluorescence-labeled hairpin DNA allows to determine the reaction yield and provide guide to design the involved oligonucleotides.
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