Electrochemical Detection of Alginate Penetration in Immobilized Layer-by-Layer Films by Unnatural Amino Acid Containing Antimicrobial Peptides

A simple electrochemical assay to monitor the alginate penetration ability of a series of antimicrobial peptides (AMP) containing unnatural amino acids is reported. Alginate, a component of P. aeruginosa biofilms, was used to modify pyrolytic graphite (PG) electrodes using well-established layer-by-layer (LbL) protocols, followed by exposure to AMP at varying concentrations for increasing lengths of time. Intact alginate layers inhibited surface binding and electron transfer of solution phase ferricyanide based on anionic repulsion. AMP exposure resulted in concentration-dependent alginate penetration, and an increase in electrochemical current at negative-shifted potentials. Controls showed that the AMP unnatural amino acid and cationic structure was important in facilitating alginate penetration. In an effort to validate this simple model as a potential anti-biofilm assay, biofilm dispersal and inhibition assays were performed in 96-well plates using Pseudomonas aeruginosa PAO1. The biological assays showed that the AMP were effective at dispersing PAO1 biofilm, providing a level of validation to the simple alginate electrochemical assay.