Ratiometric Fluorescence Biosensor of Allosteric Ag Nanoclusters by G-Quadruplex-Aided Displacement Amplification

Conventional displacement reactions often suffer from drawbacks such as high background signal and low reaction rates. To overcome this limitation, we propose a G-quadruplex (G4) folding-aided displacement amplification (G4DA) strategy to illuminate the ratiometric fluorescence of allosteric Ag nanoclusters as bicolor signaling reporters ( a Ag 554 and a Ag 610 ) for the rapid and sensitive detection of a specific targeting trigger ( t T). The recognizable element is totally blocked in the stem of a modular hairpin for minimizing nonspecific background responses, while it can be exclusively unlocked by a short-stranded key effector via disturbing the sticky toehold. Preferably, red a Ag 610 emitters are developed only in two proximal template splits through directional complementary hybridization. Upon the effector invasion and affinity binding, the strand-exchange events are executed to drive progressive G4DA operation, which is kinetically sped up by the rapid intramolecular folding of rigid G4 structures with more stable geometry, thereby displacing t T for repetitive recycling amplification. During this process, the disassembly of a duplex complex switches red a Ag 610 into green a Ag 554 to produce reversely changed fluorescence for conformation-dependent ratiometric signaling. Without enzyme participation and tedious chemical modification, this G4DA-based approach is achievable with simplified operation, reaction dynamics, productive yield, and assay sensitivity, further suggesting a new methodological paradigm for potential biosensor, bioanalysis, and therapeutic applications.