Ligand-induced minimal strand proximity hybridization to illuminate biemissive Ag nanocluster for ratiometric fluorescence biosensing

To better balance background leakage and disassociation kinetics in traditional ligand-aptamer competition-based transduction, herein we explore the ratiometric fluorescence ability of red- and green-emissive Ag cluster ( r AgC and g AgC) that is illuminated by the adenosine triphosphate (ATP)-induced proximity hybridization. For proof-of-concept, an ATP-recognizable strand (AS) encoding the aptamer sequence (aS) in the middle and a stem-loop bi-template hairpin ( b TH) are introduced. Originally, the partial sequence for r AgC is blocked in the stem of hairpin-structured b TH, while tethering an exposed g AgC-clustering toehold for ‘green-on’ emission . Upon inputting ATP, the specific ATP-aptamer binding induces the dynamic switch of random-coil AS into stable hairpin structure (off-to-on transition) with a short-paired stem, in which two split invaders are oriented closely as a parent invader to interfere the unpaired loop of b TH. Through perfect proximity hybridization, rapid orthogonal DNA assembly urges b TH to lose its hairpin conformation geometry, thereby connecting the contiguous stem for preferable development of r AgC with ‘red-on’ emitting signal. Thus, ligand ATP and aptamer AS are like a logic ‘key/switch’ pair to execute an input-output connection. As predicted, the fluorescence intensity of r AgC and g AgC reversely changes in a tailored ATP-responsive route, obtaining the dose-dependent ratiometric response for sensing ATP. The conformation alteration by ligand-aptamer recognition would be more beneficial to guide precise proximity hybridization between two reactive species, constructing an applicable biosensing platform with simplification, specificity, low background and rapid reaction kinetics.