Electrochemical Detection of Anti-Biofilm Activity Using Unnatural Amino Acid-Containing Antimicrobial Peptides

Abstract

Bacterial infections are a significant health problem that can be detrimental to the human population. It is estimated that bacterial infections are responsible for billions of dollars’ worth of damages in the health care field alone, and numerous deaths annually. Bacterial infections can become so detrimental because they produce a structure called biofilm, which facilitates antibiotic resistance and is a major cause of chronic infections. In order to combat this threat, new anti-biofilm and antibiotic therapies are being developed and their efficiency must be tested. A series of antimicrobial peptides (AMP) containing unnatural Tic-Oic amino acids have been developed for this purpose. Traditional methods such as biological assays are the standard by which antibiotics are judged, but they have their drawbacks, such as the lengthy test times and the costs associated with it and the reagents. Electrochemical biosensors can remedy some of those drawbacks by offering speed and cost benefits. Electrochemical biosensors consisting of Layer-by-Layer (LbL) modified electrodes were constructed. These sensors were fabricated to test the anti-biofilm activity of the aforementioned unnatural amino acid-containing antimicrobial peptides against a model of Pseudomonas aeruginosa or against the bacteria itself. P. aeruginosa is a common biofilm producing bacteria. First, we employed alginate as one of the layers in our sensor as P. aeruginosa is known to produce this as its major biofilm component. We show that the penetration of the alginate layer by the AMP can be detected electrochemically utilizing a solution-phase redox active molecule that produces an increasing signal upon electrochemical reduction when the film becomes compromised. Biological assays are presented that provide some validation for the sensor, but elucidated a particular AMP as compared to the electrochemical alginate sensor. Based on this slight disagreement between our electrochemical model and the biological assay, we employed sensors that featured directly immobilized P. aeruginosa PAO1 on the electrode surface. These bacteria are electroactive, which negated the need for an external redox active molecule, and allowed the monitoring of anti-biofilm activity via a signal decrease over time. The P. aeruginosa sensor showed more agreement with the biological assay, highlighting the same AMP as active toward biofilm degredation at low (<1 mircoM) concentrations. Overall, these electrochemical biosensors utilizing models of and actual P. aeruginosa have opened up new avenues to test for effectiveness of potential anti-biofilm agents toward biofilm forming bacteria.