Bivariate Cooperator-Catalyzed Hairpin Assembly for Amplifying Conformation-Guided Label-Free Ratiometric Fluorescence Biosensing

The traditional cycling hairpin assembly amplifier usually suffers from high nonspecific background leakage and insufficient conversion. To address these challenges, developing a bivariate cooperator-catalyzed hairpin assembly ( bc CHA) strategy might be intriguing and significant for specifically and efficiently interpreting and amplifying two interrelated targeting biomarkers, e.g., adenosine triphosphate (ATP) and miRNA-21 (miR-21). The proof-of-concept example begins with the design of a functional birecognizable hairpin ( br H) including the modular segment with two kissing extremities in the quasi-hairpin ATP-aptamer binding complex, and the complementary element of miR-21. In a typical bivariate-responsive route, the simultaneous distinguishing hybridization at br H makes its stem-loop structure rapidly disassemble. This further triggers the progressive bc CHA operation forward via repetitive strand migration and self-catalysis recycling under a thermodynamically favorable Gibbs free energy change. In the resulting duplex products, the exposed toeholds are merged to unfold the bitemplating hairpin hosting dual-emissive Ag nanoclusters as signaling reporters, thereby illuminating their ratiometric fluorescence for sensitive bivariate assay at the picomolar level. Following the bicooperator authentication mechanism with built-in correction, our label-free ratiometric approach is achievable in simplifying design, lowering false-positive background, and improving selectivity and accuracy. This bc CHA-based strategy would offer new insights into establishing multiplex biomimetic platforms for applicable biosensing, cell imaging, logic computation, or precise diagnostics.