NK-lysin antimicrobial peptide-functionalized nanoporous alumina membranes as biosensors for label-free bacterial endotoxin detection

Authors: JIANG, NIANYU - Iowa State University; SHROTRIYA, PRANAV - Iowa State University; Dassanayake, Rohana

Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2022
Publication Date: 12/25/2022
Citation: Jiang, N., Shrotriya, P., Dassanayake, R.P. 2022. NK-lysin antimicrobial peptide-functionalized nanoporous alumina membranes as biosensors for label-free bacterial endotoxin detection. Biochemical and Biophysical Research Communications. 636:18-23. https://doi.org/10.1016/j.bbrc.2022.10.097.
DOI: https://doi.org/10.1016/j.bbrc.2022.10.097

Interpretive Summary: Small antimicrobial proteins (NK-lysin) produced by cattle white blood cells show strong antimicrobial activity. The antimicrobial activity of small proteins is due to the selective and specific binding to bacterial membranes followed by pore formation in the membranes. We explored whether this selective binding property of small antimicrobial proteins with bacterial membranes can be used to develop a biosensor system to label-free detection of bacterial products. The small antimicrobial protein functionalized biosensor was able to quickly bind nanogram amounts of bacterial products (lipopolysaccharides) in the samples. Further studies are needed to assess whether this novel detection system can be used to rapid detection of bacterial products (or bacteria) from blood samples.

Technical Abstract: We report an NK-lysin peptide-functionalized nanoporous anodized aluminum oxide (NAAO) based biosensor to detect bacterial endotoxin. Bovine NK-lysin-derived peptides show antimicrobial activity against bacterial pathogens, and bactericidal activity is primarily due to the membranolysis activity. Antimicrobial activity of NK-lysin NK2A was confirmed against a Gram-negative Mannheimia haemolytica and a Gram-positive Staphylococcus aureus. Electron microscopic examination showed the localization of NK2A conjugated silver nanoparticles, but not unconjugated silver nanoparticles used as control, to the bacterial outer membrane and cell wall. NK2A functionalized NAAO membranes were used in a previously developed four-electrode electrochemical configuration to detect the presence of Gram-negative bacterial lipopolysaccharides (LPS) and Gram-positive bacterial lipoteichoic acid (LTA) molecules. NK2A-functionalized NAAO biosensor could detect LPS with a detection limit of 10 ng/mL within an appreciable signal/noise ratio. Biosensors functionalized with a scrambled amino acid version of NK2A (Sc-NK2A) that lacks antimicrobial activity could not detect the presence of LPS. However, both NK2A and Sc-NK2A functionalized biosensors showed sensing signals with Gram-positive bacterial lipoteichoic acids. These results suggest that the specific binding of NK2A-LPS on the NAAO membrane surface is responsible for the observed biosensor signals. These findings suggest that NK2A-functionalized biosensors can be used for rapid and sensitive label-free LPS detection.