Bivalent Ligand-Aptamer Binding-Cooperated Hybridization Assembly for Illuminating Ratiometric Fluorescence Biosensing of Convertible Ag Nanoclusters

To address low affinity and reaction rate in monovalent ligand-aptamer binding, herein, we propose a bivalent binding-cooperated hybridization assembly (bbHA) strategy to light up the ratiometric fluorescence of convertible Ag nanoclusters (cAgNC) for detecting adenosine triphosphate (ATP). Two identical repeats of ATP-recognizable aptamer splits (a1 and a2) are separately programmed in an aptamer templating strand (aT) and an aptamer converter strand (aC), while two pairs of cAgNC-clustering and emission-converting segments are tethered in aT (template-a1-a1-template) and aC (converter-a2-a2-converter), respectively. Two tandem red cAgNC emitters are preferably developed in aT. Upon presenting ATP, the affinity recognition by a specific aptamer (a1/a2) entity enables cooperative binding events for triggering rapid complementary hybridization of aT and aC. The other proximal a1/a2 pair is bulged and merged to speed up the second ATP affinity binding. The structurally stable aT·2ATP·aC complex is formed to orient two ended converters and clusters together, thereby modulating its red fluorescence into green through electronic interactions between Ag0 and particular nucleobases. The resulting signal ratio of dual-emissive cAgNC exhibits a linear dependence on ATP, achieving sensitive and accurate quantification via inherent built-in correction. In contrast to the monovalent mechanism, the bbHA-based strategy features superior assay performances such as higher target responsiveness, faster reaction kinetics, and more efficient transduction. Thus, this label-free method would offer a new paradigm to construct simplified, specific, robust, and cost-effective sensing platforms for applicable assay of various small-molecule analytes.