Structure-Guided Computational Design and Electrochemical Validation of a DNA Aptamer for Impedimetric Detection of...

The extensive application potential of nucleic acid aptamers in biosensing and therapeutic domains necessitates the development of cost-effective, time-efficient, and technically simpler synthesis protocols compared with conventional SELEX methods. In this study, an in silico-based de novo approach has been proposed to design an aptamer targeting the B subunits of Shiga-like toxin 2 (Stx2-B) protein, intended for use as a recognition element in biosensors for detecting Stx2. A single-stranded DNA library comprising 5000 sequences was generated and ranked based on their Gibbs free energy (ΔG) of the 2D structures. The top 100 sequences with the lowest ΔG values were modeled in 3D and individually docked with the Stx2 protein. Sequences exhibiting the highest docking scores were further analyzed through molecular dynamics simulations. Among the analyzed candidates, sequence A330 demonstrated the highest stability, rigidity, and compactness in complex with Stx2B, as indicated by root-mean-square deviation, root-mean-square fluctuation, radius of gyration, and hydrogen-bond analysis over 100 ns simulation trajectories. The strong binding affinity of A330 for the target (K_d = 459 ± 7 nM) was confirmed through circular dichroism and isothermal titration calorimetry analyses. This aptamer was subsequently employed as a recognition element in the fabrication of an electrochemical impedimetric aptasensor for Stx2 detection, utilizing a microscale interdigitated wave-type electrode. The developed sensor exhibited high sensitivity, a limit of detection of 4.63 pM, and a linear detection range of 10-400 pM (R² = 0.99). The aptasensor's practical applicability was validated in milk samples, achieving a detection recovery of >93.06%. Overall, the proposed aptasensor shows strong potential for the sensitive and reliable monitoring of Shiga toxin-producing Escherichia coli contamination in real samples.