A crosslinked submicro-hydrogel formed by DNA circuit-driven protein aggregation amplified fluorescence anisotropy...

Protein is an excellent molecular mass amplifier without fluorescence quenching effect for fluorescence anisotropy (FA) assay. However, in traditional protein amplified FA methods, the binding ratio between amplifier and dye-modified probe is 1:1 or one target can only induce FA change of one fluorophore on probe, resulting in low sensitivity. Herein, we developed a simple FA strategy with high accuracy and sensitivity by using a crosslinked submicro-hydrogel that was formed through a catalyzed hairpin assembly (CHA) assisted protein aggregation as a novel FA amplifier. In the presence of catalyst, the CHA process was initiated through the toehold-mediated strand exchange reaction, which led to the formation of a dye and biotin-labeled Y-shaped H1-H2 duplex (YHD) and recycling of catalyst. With the introduction of streptavidin, a crosslinked submicro-hydrogel was formed by strong binding affinity between biotin on YHD and streptavidin, resulting in an increased FA of fluorescent dye. After rational design of the catalyst sequence, this method has been utilized for the detection of miRNA-145, staphylococcal enterotoxin B (SEB) and ATP with an LOD of 2.5 nM, 92 pg mL^-1 and 3.6 μM, respectively. Moreover, this FA assay has been successfully applied for direct detection of target in biological samples, demonstrating its practicality in complex biological systems.