A pseudo-circular DNA walker-mediated electrochemical biosensor for sensitive detection of the apolipoprotein E4 gene

In this work, we report a pseudo-circular DNA walker (PDW) integrated with G-quadruplex (G4) and triplex DNA to construct an electrochemical biosensor for ultrasensitive detection of apolipoprotein E4 (T-e4). PDW contains double embedded DNAzyme sequences, leveraging the intrinsic advantages of enhanced structural rigidity for stable spatial conformation, continuous autonomous rolling capability, and efficient cyclic cleavage of hairpin probes due to its closed-loop topology. After binding to the target, the catalytic domain of the PDW is activated, rolling and cutting the hairpin probe in the presence of Mg²⁺. The triplex helix hairpin orbital (THO) serves as a rigid scaffold to immobilize hairpin DNA probes, enhancing interfacial stability and signal probe loading efficiency. The cleavage releases G-rich DNA fragments, which subsequently self-assemble into hemin/G-quadruplex (HG4) nanostructures. This design achieves label-free detection with exceptional sensitivity, exhibiting a linear range of 0.05-10 pM and a detection limit of 27 fM. This strategy opens up a new way for the sensitive detection of neurodegenerative disease biomarkers and shows broad application prospects in the fields of early disease screening and clinical monitoring.