A Modular Nanoswitch for Mix-and-Detect Protein Assay Based on Binding-Induced Cascade Dissociation of Kissing...

A new modular nanoswitch was described for versatile, rapid (within 1 h), homogeneous, and sensitive protein detection. The system employs two hairpins (HP1 and HP2) that can be reciprocally recognized through the apical loop-loop interaction. HP2 possesses a conformation-switching stem-loop structure, with appended single-stranded tails on each end, which can hybridize with the recognition-element-conjugated DNA strands to construct a protein-responsive HP2 scaffold. It works according to a simple mix-and-detect assay format, with the first formation of a kissing complex between HP1 and HP2 scaffolds for fluorescence quenching, and then cascade propagation from steric strain through protein binding to the dissociation of the kissing complex for fluorescence recovery. The detection universality of such a modular nanoswitch was demonstrated by using three multivalent proteins, including anti-digoxigenin (Anti-Dig) antibody, streptavidin, and thrombin, with detection limits of 0.33, 0.17, and 0.5 nm, respectively.