The goal of this research was to develop a novel biosensor for detecting and eliminating pathogenic E. coli. Traditionally, identifying pathogenic E. coli and distinguishing it from harmless environmental strains includes serotyping and DNA sequencing, which can take days or weeks. Our biosensor platform makes use of a material that is part of the immune system from single- multi- cellular organisms that target viruses, fungi, and bacteria called antimicrobial peptides (AMPs). Using the quartz crystal microbalance with dissipation monitoring (QCM-D), we characterized non-specific binding between CP1 to silicon nitride and gold, and covalent binding of cysteine-terminated CP1 (CP1-cys) to gold. QCM-D monitors frequency and dissipative changes resulting from adsorbed mass, and peptide film thickness and density can be calculated using Voigt Viscoelastic modeling. Viability of the E. coli was monitored using a live/dead kit consisting of nucleic acid stains Syto 9 and Propidium Iodide. Successfully immobilizing peptide to a substrate is particularly important if CP1 would be applied on a food processing surface. By immobilizing CP1 to silicon nitride, we measured the binding forces between bacteria and peptides with the atomic force microscope (AFM), and explored important bacterial features such as LPS composition and length that influence binding affinity with CP1. The structure of the LPS is comprised of 3 sections: lipid A, core group, and O-antigen. We are mostly interested in the initial binding between AMP and LPS since our goal is to develop a novel biosensor that can detect pathogenic bacteria within seconds of exposure. Considering the short exposure period, the AMP would only be exposed to the O-antigen and outer core groups, which are repeating sugar chains that are essential for bacterial pathogenicity and adhesion to substrates. Although geared for use as a novel biosensor, results of this study can also be applied to the use of AMPs for replacing or enhancing the activity of antibiotics. Our work suggests that CP1 may not be serotype-specific, but targets the O-antigen before interfering with phospholipid groups of the bacterial membrane. Other factors that assist in pathogenicity, such as LPS length, may also be important for the consideration of CP1 potency.
| Identifer | oai:union.ndltd.org:wpi.edu/oai:digitalcommons.wpi.edu:etd-theses-1114 |
| Date | 20 January 2009 |
| Creators | Strauss, Joshua |
| Contributors | , , , , , |
| Publisher | Digital WPI |
| Source Sets | Worcester Polytechnic Institute |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses (All Theses, All Years) |
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