AND-gated ratiometric fluorescence biosensing of dual-emissive Ag nanoclusters based on transient recycling amplification of bivariate targeting DNA

Development of logic AND-gated multivariate assay systems of DNA-scaffolded green- and red-fluorescent Ag nanoclusters (g-AgNC and r-AgNC) is promising and remains challenging. By using two DNA segments (To and Tr) as dual-targeting inputs, herein we report an innovative strategy that utilizes targets-recycled amplification to create a sensitive AND-gated light-up ratiometric sensor with reversely changed fluorescence of g-AgNC and r-AgNC. The design is based on that: two hairpins (Ho and Hr) are individually recognizable to To and Tr, and two hybridized duplexes are resultantly implemented for transient dissipative base pairing, i.e. Ho·Hr, releasing To and Tr for repeated recycling. In an original stem-loop hairpin reporter (HR), one exposed overhang is to host g-AgNC. When HR is unfolded by the cooperative sticky toeholds in Ho∙Hr, its liberated stem merging with the g-AgNC template is for r-AgNC clustering. Only when meeting simultaneously the overexpressed To and Tr in SARS-CoV-2, the AND-gated ratio fluorescence of r-AgNC over g-AgNC can be lighted-up in a typical assay, enabling highly specific-inputs binding and sensitive dose-dependence down to 0.29 pM. This enzyme-free and label-free AND-logic ratiometirc strategy illustrates the potential of using multicolor AgNCs emitters for more accurate multivariate assay.

Modulating the Fluorescence of Silver Nanoclusters Wrapped in DNA Hairpin Loops via Confined Strand Displacement and Transient Concatenate Ligation for Amplifiable Biosensing.

It is intriguing to modulate the fluorescence emission of DNA-scaffolded silver nanoclusters (AgNCs) via confined strand displacement and transient concatenate ligation for amplifiable biosensing of a DNA segment related to SARS-CoV-2 (s2DNA). Herein, three stem-loop structural hairpins for signaling, recognizing, and assisting are designed to assemble a variant three-way DNA device (3WDD) with the aid of two linkers, in which orange-emitting AgNC (oAgNC) is stably clustered and populated in the closed loop of a hairpin reporter. The presence of s2DNA initiates the toehold-mediated strand displacement that is confined in this 3WDD for repeatable recycling amplification, outputting numerous hybrid DNA-duplex conformers that are implemented for a transient "head-tail-head" tandem ligation one by one. As a result, the oAgNC-hosted hairpin loops are quickly opened in loose coil motifs, bringing a significant fluorescence decay of multiple clusters dependent on s2DNA. Demonstrations and understanding of the tunable spectral performance of a hairpin loop-wrapped AgNC via switching 3WDD conformation would be highly beneficial to open a new avenue for applicable biosensing, bioanalysis, or clinical diagnostics.

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 (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 (TOC) 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 TOC, its affinity ligating with yH is further implemented to unfold rH and induce the DNA construct switching into closed conformation, causing TOC-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 TOC 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.