An electrochemical aptasensor for sensitive kanamycin detection using an Nb.BbvCI-driven DNA walker-based signal amplification strategy

In this study, an electrochemical aptasensor dedicated to the sensitive detection of kanamycin (KAN) was constructed based on an Nb. Bbv CI-driven DNA walker-enabled signal amplification strategy. The sensor employed a gold electrode modified with polyethyleneimine-wrapped, nitrogen-doped multi-wall carbon nanotubes decorated with gold nanoparticles (N-MWCNTs-PEI-AuNPs). This composite was used to modify the gold electrode, providing an enhanced conductive interface and increasing the specific surface area for aptasensor construction. Meanwhile, a zirconium-based metal-organic framework decorated with gold nanoparticles (AuNPs@NH₂-UiO-66) was used both as an electrocatalyst and as a carrier for hairpin DNA (HP2), exhibiting strong catalytic activity toward the redox mediator thionine. In differential pulse voltammetry measurements, at the surface of the electrode, thionine undergoes a reversible, pH-dependent two-electron/two-proton transfer process at approximately −0.25 V on the electrode surface, generating a well-defined reduction current that serves as the analytical signal. Upon addition of KAN, the aptamer selectively binds to the analyte and dissociates from its complementary DNA (cDNA). The freed cDNA subsequently hybridizes with HP2 anchored on AuNPs@NH₂-UiO-66, thereby activating the Nb. Bbv CI-driven DNA walker, catalyzing the site-specific cleavage of HP2 and releasing multiple AuNPs@NH₂-UiO-66 labels. These released probes then hybridize with the captured hairpin DNA (HP1) fixed on the N-MWCNTs-PEI-AuNPs-modified gold electrode. The synergistic electrocatalytic effects of NH₂-UiO-66 and AuNPs significantly accelerate the electron transfer kinetics of thionine, leading to amplified voltammetric current responses that correlate proportionally with KAN concentration. The aptasensor demonstrated highly sensitive performance, including a linear detection range of 10 pM to 5 μM and a detection limit of 1.65 pM and a quantitation limit of 5.46 pM. The aptasensor also showed satisfactory selectivity and reproducibility and was successfully applied for KAN determination in real milk samples. This approach provides a robust signal amplification strategy and offers a promising tool for the rapid and sensitive detection of KAN in real-world samples.