Guanine-Lighting-Up Fluorescence Biosensing of Silver Nanoclusters Populated in Functional DNA Constructs by a...

Dark or weak-emissive DNA-harbored silver nanoclusters (AgNCs) can be remarkably lighted up when approaching to guanine bases. The resultant bright AgNCs acting as a fluorescent reporter are fascinating in biosensing. To explore the applicable guanine-enhanced emission of AgNCs for biosensing microRNA-155 (miR-155) as a model, here we designed a unique stem-loop hairpin beacon (HB) encoding with an miR-155-recognizable sequence and a AgNCs-nucleable template, as well as a hairpin helper tethering a partially locked guanine-rich (15-nt) tail (G₁₅H), while two identical cytosine-rich segments were inserted in HB and G₁₅H to merge for folding/unfolding of i-motif at changed pHs. Initially, the intact clusters populated in HB (HB/AgNCs) were almost nonfluorescent in a buffer (pH 7.0). Then, miR-155 was introduced to trigger a repeated hairpin assembly of HB and G₁₅H by competitive strand displacement reactions at decreased pH 5.0 within 10 min, consequently generating numerous duplex DNA constructs (DDCs). With the resultant template of pH-responsive i-motifs incorporated in DDCs, their folding at pH 5.0 brought the proximity of unlocked G₁₅ overhang to the clusters in a crowded environment, remarkably lighting up the red-emitting fluorescence of HB/AgNCs (λ_em = 628 ± 5 nm) for amplified signal readout. About 3.5-fold enhancement of quantum yield was achievable using different variants of i-motif length and G₁₅ position. Simply by adding OH^-, the configuration fluctuation of i-motifs was implemented for switchable fluorescence biosensing to variable miR-155. Based on a one-step amplification and signaling scheme, this subtle strategy was rapid, low-cost, and specific for miR-155 with high sensitivity down to 67 pM.