Amplifiable ratiometric fluorescence biosensing of nanosilver multiclusters populated in three-way-junction DNA branches

To explore the fluorescence bio-responsiveness of emissive silver nanoclusters (AgNCs) populated in DNA-branched scaffolds is intriguing yet challenging. In response to a desired targeting model (T*) as a vehicle, herein a customized three-way-junction DNA construct (TWJDC) is assembled via competitive hybridizing cascade among three stem-loop hairpins with specific base sequences, where the repeated recycling of T* enables the exponentially amplifiable output of rigid TWJDC. As designed, these stable hybridization products are highly T*-stimulated responsive and constructing-directional. In the three branched-arms, the unpaired sticky ends provide isotropic binding sites for a signaling hairpin encoded with two C-rich templates of green-and red-AgNCs clustering. The identical ligation of signal probe with three arms of TWJDC liberates its locked stem, enabling the separate growth of red-clusters in three branches. As demonstrated, three clusters of red-AgNCs possess ad-vantageous self-enhancing fluorescent performance relative to single or two cluster(s), good biocompatibility and low cytotoxicity. Utilizing the bicolor AgNCs as dual-emitters with reversely changed emission intensity, we developed an innovative ratiometric strategy displaying sensitively linear dose-dependence on variable T* down to 1.9 pM, which can afford a promising platform for biosensing, bioanalysis, cell imaging, or even clinical theranostics.