Proximity-localized catalytic hairpin assembly (plCHA) is intriguing for rapid and sensitive assay of an HIV-specific DNA segment ( T *). Using template-integrated green Ag nanoclusters (igAgNCs) as emitters, herein, we report the first design of a T *-activated plCHA circuit that is confined in a three-way-junction architecture (3WJA) for the fluorescence sensing of T *. To this end, the T *-recognizable complement is programmed in a stem-loop hairpin (H1), and two split template sequences of igAgNCs are separately overhung contiguous to the paired stems of H1 and another hairpin (H2). The hybridization among H1, H2, and two single-stranded linkers (L1 and L2) allows the stable construction of 3WJA. Upon presenting the input T *, the 3WJA-localized plCHA is operated through toehold-mediated strand displacements of H1 and H2 reactants, and T * is rationally displaced and repeatably recycled, analogous to a specific catalyst, inducing more hairpin assembly events. Resultantly, the hybridized products enable the collective combination of two splits in the parent scaffold for hosting igAgNCs, outputting T *-dependent fluorescence response. Because of 3WJA structural confinement, the spatial proximity of two reactive hairpins yielded high local concentrations to manipulate the plCHA operation, achieving rapider reaction kinetics via T *-catalyzed recycling than typical catalytic hairpin assembly (CHA). This simple assay strategy would open the arena to develop various plCHA-based circuits capable of modulating the fluorescence emission of igAgNCs for applicable biosensing and bioanalysis.
Exploring the ratiometric fluorescence biosensing of DNA-templated biemissive silver nanoclusters (AgNCs) is significant in bioanalysis, yet the design of a stimuli-responsive DNA device is a challenge. Herein, using the anti-digoxin antibody (anti-Dig) with two identical binding sites as a model, a tweezerlike DNA architecture is assembled to populate fluorescent greenand red-AgNCs (g-AgNCs and r-AgNCs), aiming to produce a ratio signal via specific recognition of anti-Dig with two haptens (DigH). To this end, four DNA probes are programmed, including a reporter strand (RS) dually ended with a g-/r-AgNC template sequence, an enhancer strand (ES) tethering two same G-rich tails (G18), a capture strand (CS) labeled with DigH at two ends, and a help strand (HS). Initially, both g-AgNCs and r-AgNCs wrapped in the intact RS are nonfluorescent, whereas the base pairing between RS, ES, CS, and HS resulted in the construction of DNA mechanical tweezers with two symmetric arms hinged by a rigid "fulcrum", in which g-AgNCs are lighted up due to G18 proximity ("green-on"), and r-AgNCs away from G18 are still dark ("red-off"). When two DigHs in proximity recognize and bind anti-Dig, the conformation switch of these tweezers resultantly occurs, taking g-AgNCs away from G18 for "green-off" and bringing r-AgNCs close to G18 for "red-on". As such, the ratiometric fluorescence of r-AgNCs versus g-AgNCs is generated in response to anti-Dig, achieving reliable quantization with a limit of detection at the picomolar level. Based on the fast stimulated switch of unique DNA tweezers, our ratiometric strategy of dual-emitting AgNCs would provide a new avenue for a variety of bioassays.
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 (λem = 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.
Ratiometric assays of label-free dual-signaling reporters with enzyme-free amplification are intriguing yet challenging. Herein, yellowand red-silver nanocluster (yH-AgNC and rH-AgNC) acting as bicolor ratiometric emitters are guided to site-specifically cluster in two template signaling hairpins (yH and rH), respectively, and originally, both of them are almost non-fluorescent. The predesigned complement tethered in yH is recognizable to a DNA trigger (T OC ) related to SARS-CoV-2. With the help of an enhancer strand (G 15 E) tethering G-rich bases (G 15 ) and a linker strand (LS), a switchable DNA construct is assembled via their complementary hybridizing with yH and rH, in which the harbored yH-AgNC close to G 15 is lighted-up. Upon introducing T OC , its affinity ligating with yH is further implemented to unfold rH and induce the DNA construct switching into closed conformation, causing T OC -repeatable recycling amplification through competitive strand displacement. Consequently, the harbored rH-AgNC is also placed adjacent to G 15 for turning on its red fluorescence, while the yH-AgNC is retainable. As demonstrated, the intensity ratio dependent on varying T OC is reliable with high sensitivity down to 0.27 pM. By lighting-up dualcluster emitters using one G 15 enhancer, it would be promising to exploit a simpler ratiometric biosensing format for bioassays or clinical theranostics.
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