Target Deoxyribonucleic Acid-Recycled Lighting-Up Amplifiable Ratiometric Fluorescence Biosensing of Bicolor Silver Nanoclusters Hosted in a Switchable Deoxyribonucleic Acid Construct.

Ratiometric assays of label-free dual-signaling reporters with enzyme-free amplification are intriguing yet challenging. Herein, yellow- and red-silver nanocluster ( y H-AgNC and r H-AgNC) acting as bicolor ratiometric emitters are guided to site-specifically cluster in two template signaling hairpins ( y H and r H), respectively, and originally, both of them are almost non-fluorescent. The predesigned complement tethered in y H is recognizable to a DNA trigger ( T OC ) related to SARS-CoV-2. With the help of an enhancer strand (G 15 E) tethering G-rich bases (G 15 ) and a linker strand (LS), a switchable DNA construct is assembled via their complementary hybridizing with y H and r H, in which the harbored y H-AgNC close to G 15 is lighted-up. Upon introducing T OC , its affinity ligating with y H is further implemented to unfold r H and induce the DNA construct switching into closed conformation, causing T OC -repeatable recycling amplification through competitive strand displacement. Consequently, the harbored r H-AgNC is also placed adjacent to G 15 for turning on its red fluorescence, while the y H-AgNC is retainable. As demonstrated, the intensity ratio dependent on varying T OC is reliable with high sensitivity down to 0.27 pM. By lighting-up dual-cluster emitters using one G 15 enhancer, it would be promising to exploit a simpler ratiometric biosensing format for bioassays or clinical theranostics.