A one-pot strategy for the detection of proteins based on sterically and allosterically tunable hybridization chain reaction

In this work, we report a facile one-pot strategy for protein detection based on sterically and allosterically tunable hybridization chain reaction (HCR). In our strategy, DNA hairpins H1 and H2 are dual-labeled with pyrene moieties through a six-carbon-atom spacer at each end; and a single-stranded DNA primer is designed to contain two small molecules near each end. In the absence of target protein, the primer can trigger HCR events between alternating H1 and H2 hairpins to form a nicked double-helix. As a result, the pyrene excimers are formed to emit at approximately 485 nm. On the contrary, upon binding of the specific target protein onto the primer through the protein-small molecule interaction, the HCR will be inhibited due to the steric and allosteric effect. The changes of the fluorescent signals of pyrene excimers are in response to the concentration of target protein, so that the detection of protein can be realized. We have demonstrated the feasibility of this strategy by using streptavidin (SA) and folate receptor (FR) as model targets. Results show that both of them can be well detected with a detection limit of 1.07 nM and 2.7 nM, respectively. The developed method for protein assay is flexible, so we infer that the one-pot strategy holds great potential for the detection of other proteins.