Responsive reconstruction-driven autocatalysis DNA circuit for efficient and amplifiable electrochemical biosensing

To overcome low strand utilization and slow kinetics in traditional entropy-driven DNA circuits, herein we propose a novel responsive reconstruction-driven Autocatalysis DNA Circuit (rrADC) for constructing highly sensitive electrochemical biosensor. Using 17β-estradiol as a targeted ligand, the specific ligand-aptamer binding triggers the release of an initiator strand (I) via magnetic separation. To operate rrADC process, two single strands (s and s‾) each encoding with one split of I, together with a linker strand are designed to assemble a three-stranded duplex substrate. Upon invading by the as-interpreted I, strand migration reactions are activated to displace s, and further liberate s‾ and I by a fuel strand, till perfect complementary hybridization. The released s and s‾ instantly pair to form a double-stranded duplex via conformational ordering process, in which an analogue of I (I*) in the merged tails is reconstructed as endogenous trigger and cooperates with I to execute repeated interferences for catalyzing rrADC forward for cycling amplification. In the resulting complexes, the labeled electroactive ferrocene generates dose-dependent electrochemical current signal in the modified electrode surface. The distinct structural ordering of the whole system sustains continuous, efficient rrADC operation with minimized steric hindrance, maximized strand utilization and accelerated reaction kinetics. By reusing otherwise discarded intermediate strands, the biosensor achieves an ultralow E2 detection limit of 16.6 fM. This strategy offers a new paradigm for simplified, enzyme-free, efficient electrochemical biosensing to detect trace environmental pollutants and biomolecules.