Biolayer interferometry-SELEX for Shiga toxin antigenic-peptide aptamers & detection via chitosan-WSe₂ aptasensor

We report biolayer interferometry based in-vitro selection technique (BLI-SELEX) for fishing out specific aptamers against E. coli Shiga toxin subtypes viz., stx1 & stx2 via epitopic peptides. BLI-SELEX is a one-step technique for rapidly generating aptamers against protein biomarkers in a microtiter plate format, obliterating the need for multiple enrichment rounds to harvest high-affinity aptamers as in conventional SELEX. Two unique aptamers selected against stx1 & stx2 with picomolar K_d (~47 pM & ~29 pM, respectively) were successfully used to fabricate voltammetric diagnostic assay via immobilization onto chitosan exfoliated 2D tungsten diselenide (WSe₂) nanosheet platform. These aptamers modified nanosensors showed high sensitivity of ~ 5.0 μA ng^-1 mL, a dynamic response range from 50 pg mL^-1 to 100 ng mL^-1, with a detection limit of 44.5 pg mL^-1 & 41.3 pg mL^-1 for stx subtypes, respectively and showed low cross-reactivity in spiked urine, serum and milk samples. The synergistic effect of selective aptamers & high sensitivity imparted by 2D transition metal dichalcogenide (TMD) highlights the superior potential of a fabricated nanosensor for bacterial toxin detection.