Abstract
The life-threatening diarrheal disease caused by Shiga toxin-producing Escherichia coli (STEC) necessitates rapid, sensitive, portable, and low-cost diagnostic tools to strengthen on-field food safety monitoring, improve disease prognosis, and guide timely therapeutic interventions. In this study, an impedimetric aptasensor was developed against Shiga like toxin-1 produced by STEC using a 71-mer ssDNA aptamer (sT3), enriched from an initial library (∼1014 sequences) through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique, and an interdigitated chain-type microelectrode fabricated by depositing Ti and Au layers on a glass substrate via electron-beam deposition, photolithography, and wet-chemical etching. The aptamer exhibited a strong binding affinity with a dissociation constant (K d) of 273 ± 0.03 nM, as determined by isothermal titration calorimetry. The resulting aptasensor achieved a detection limit of 2.88 pM, a linear dynamic range of 10-450 pM, and a sensitivity of 107.02 Ω pM-1, underscoring its strong analytical performance. It retained 97.6% of its initial response after 25 days with five intermittent operations and showed high reproducibility, with only 3.9% standard deviation across five independently fabricated sensors. These stable performances were attributed to the strong aptamer-surface coupling enacted by covalent immobilization through a self-assembled monolayer strategy. The aptasensor further demonstrated high selectivity and practical applicability by reliably detecting the toxin protein in spiked milk samples. Overall, the developed aptasensor offer a great prospect for its practical application as a diagnostic device for rapid and reliable detection of Shiga toxin-induced gastroenteritis at point-of-care settings.