A Dual-Capture and Dual-Output 3D DNA Walker System Integrated with Ligases Enables Ultrasensitive Detection of Single-Nucleotide Polymorphisms

DNA walkers, as structurally and functionally programmable signal amplification tools, exhibit great potential for application in the field of biosensing. Traditional DNA walkers often rely on enzymes for operation, posing compatibility challenges, while the handful of existing enzyme-free DNA walkers demonstrate limited performance. To address this, we innovatively developed an efficient enzyme-free 3D DNA walker with dual capture and dual output capabilities. Coupled with ligase chain reaction (LCR), this system facilitates highly sensitive and specific detection of single nucleotide polymorphisms (SNPs). Specifically, LCR precisely identifies single-base mutations, effectively transmitting biological information. The 3D DNA walker system is based on entropy-driven circuit cycling reaction technology. In this system, LCR products serve as the driving strands for the DNA walker, independently binding to track strands and walking legs immobilized on gold nanoparticles, forming a unique dual signal capture mechanism. Each track strand carries two signal chains, significantly enhancing signal amplification efficiency. Benefiting from this novel enzyme-free 3D DNA walker strategy, our biosensing system exhibits exceptional sensitivity to mutant targets (MT), detecting MT at concentrations as low as 30.3 aM and distinguishing heterozygous samples with a 0.01% mutation frequency. Furthermore, this system has been successfully applied to genotyping and mutation abundance assessment of genomes from fresh soybean leaves, demonstrating its vast potential for practical applications. In summary, this research pioneers a novel enzyme-free 3D DNA walker with dual capture and dual output capabilities, and develops an ultrasensitive genotyping tool. This provides strong technical support for the advancement of genetic research.