Potentiometric Detection of Listeria monocytogenes via a Short Antimicrobial Peptide Pair-Based Sandwich Assay

Peptide-based sandwich assays are promising tools in molecular detection, but may be restricted by the availability of "pairs" of affinity peptides. Herein, a new potentiometric sandwich assay for bacteria based on peptide pairs derived from an antimicrobial peptide (AMP) ligand is demonstrated. As a model, the original AMP with a well-defined structure for  Listeria monocytogenes (LM) can be split into two fragments to serve as the peptide pairs for the sandwich assay. The recognition and binding of the short peptide pairs to the target can be verified by circular dichroism, flow cytometry, fluorometry, and optical microscopy. The potentiometric magnetic bead-based sandwich assay is designed by using horseradish peroxidase as a label. The enzyme can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine with H₂O₂ to induce a potential change on a polymeric membrane ion-selective electrode. Under optimal conditions, the concentration of LM can be determined potentiometrically in a linear range of 1.0 × 10² to 1.0 × 10⁶ CFU mL^-1 with a detection limit of 10 CFU mL^-1 (3σ). The proposed sensing strategy expands the applications of peptides in the field of bioassays.