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 ( T o and T r) 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 ( H o and H r) are individually recognizable to T o and T r, and two hybridized duplexes are resultantly implemented for transient dissipative base pairing, i.e. H o· H r, releasing T o and T r 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 H o∙ H r, its liberated stem merging with the g -AgNC template is for r- AgNC clustering. Only when meeting simultaneously the overexpressed T o and T r 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.