Target Deoxyribonucleic Acid-Recycled Lighting-Up Amplifiable Ratiometric Fluorescence Biosensing of Bicolor Silver...

Ratiometric assays of label-free dual-signaling reporters with enzyme-free amplification are intriguing yet challenging. Herein, yellow- and red-silver nanocluster (yH-AgNC and rH-AgNC) acting as bicolor ratiometric emitters are guided to site-specifically cluster in two template signaling hairpins (yH and rH), respectively, and originally, both of them are almost non-fluorescent. The predesigned complement tethered in yH is recognizable to a DNA trigger (T_OC) related to SARS-CoV-2. With the help of an enhancer strand (G₁₅E) tethering G-rich bases (G₁₅) and a linker strand (LS), a switchable DNA construct is assembled via their complementary hybridizing with yH and rH, in which the harbored yH-AgNC close to G₁₅ is lighted-up. Upon introducing T_OC, its affinity ligating with yH is further implemented to unfold rH and induce the DNA construct switching into closed conformation, causing T_OC-repeatable recycling amplification through competitive strand displacement. Consequently, the harbored rH-AgNC is also placed adjacent to G₁₅ for turning on its red fluorescence, while the yH-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 G₁₅ enhancer, it would be promising to exploit a simpler ratiometric biosensing format for bioassays or clinical theranostics.