Responsive hairpin assembly to actuate and cascade π-π stacking G-quadruplexes for amplified electrochemical...

To explore isothermal, enzyme-free amplification strategy for sensitive detection of Ustilaginoidea (U.) virens, herein we developed a responsive hairpin assembly (RHA) to facilitate π-π stacking G-quadruplexes (G4) for electrochemical biosensing a short-stranded DNA fragment of U. virens-specific biomarker as targeting trigger (tT). The as-designed RHA was executed by programming a recognizable hairpin (rH), a helping hairpin (hH), and two guanine-rich hairpins (gH1 and gH2) with the same G-rich sequence capable of folding into K⁺-stabilized G4 structure when losing hairpin conformation. Upon inputting tT, the specific recognition initiated progressive RHA operation via cross-unfolding and contiguous hybridization among four hairpins, thereby guiding repeatedly displacement of tT for self-catalytic cycling and complementary chain extension between gH1 and gH2. In the resulting long linear product, free G-rich motifs rationally adopted parallel G-tetrad secondary structures through Hoogsteen hydrogen bonds and Mg²⁺-aided π-π stacking. Thus, numerous electroactive ferrocene (Fc) at 5'-end of gH1 was cascaded closely on the modified electrode surface to generate significantly amplified current signal. Benefited from integration of catalytic recycling and compact G4 stacking, this RHA-based electrochemical method featured fast reaction kinetics, efficient transduction yield, high specificity and high sensitivity, and would suggest a new paradigm for potentially applicable biosensing, bioanalysis or even agriculture areas.