Surface Topology-Regulated DNA Walking on Quasi-3D rGO Interfaces for Enhanced Electrochemical Kanamycin Sensing

Three-dimensional (3D) DNA tracks, typically assembled on nanoparticles, can improve DNA walker performance by creating locally confined environments that increase the probability of successive walker-track encounters. However, these architectures often require complex multistep fabrication and are difficult to integrate with planar electrode platforms. Here, we report a planar-electrode strategy in which the intrinsic wrinkled topology of reduced graphene oxide (rGO) provides a quasi-3D interfacial environment for DNA walker operation without nanoparticle assembly. A DNAzyme-driven, aptamer-locked DNA walker was operated on two comparative platforms: a conventional 2D DNA track on flat CVD graphene and a quasi-3D DNA track on wrinkled rGO. The corrugated rGO topology is proposed to reduce the effective three-dimensional distance between neighboring DNA tracks and increase the probability of repeated walker-track encounters. Consistent with this topology-induced local confinement, the rGO surface enabled a higher probe density than CVD graphene (6.52 × 1012 vs 3.39 × 1012 molecules cm-2), indicating a more closely packed interfacial track layer. This confined probe arrangement was associated with stronger apparent walker-track interaction (Kd = 0.69 vs 7.62 fM) and approximately 27% faster apparent signal-evolution kinetics on the rGO platform. As a result, the quasi-3D rGO sensor achieved an ultralow detection limit of 8.66 aM for kanamycin, substantially lower than that of the 2D CVD counterpart (0.52 fM), while maintaining reliable performance in spiked milk samples (recovery: 92-101%). These results suggest that electrode surface topology may serve as a practical design parameter for improving DNA walker-based electrochemical biosensing.