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ENHANCING NANOPORE BASED BIOSENSENING TECHNOLOGY USING PORE FORMING PROTEINS
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Abstract
Pore forming proteins (PFPs) are membrane channels that are essential for various biological processes. For example, some PFPs act as gatekeepers of the cell, controlling the traffic of ions and macromolecules flowing into and out of cells; while others are involved in causing cell death (Reiner et al., 2012). Our fundamental understanding of PFPs determines our ability to employ these proteins for use in biomedical research and nanopore technology. Given their nanoscale dimensions, reproducibility and functionality these PFPs are widely used in the growing field of nanopore technology, particularly nanopore sensing (Reiner et al., 2012; Feng et al., 2015). These biological nanopores are powerful tools enabling the production of a real-time, sensitive and selective technology. Ultimately, we aim to enhance our current understanding of PFPs to improve the currently available nanopore sensing technology. In this work, we report a nontraditional biological nanopore, Outer membrane protein G (OmpG) and its ability to detect protein. The principle of detection for this biosensor is harnessed via the dynamic movement of loop 6. Through chemically modifying the pore we attached a ligand to search or fish for our target protein of interest. In addition to successful protein detection, we were also able to discriminate between homologous within an antibody mixture. Moreover, the sensitivity of our sensor discloses a unique fingerprint for each protein target detected. This result led us to further investigate the loop network of OmpG which is vital to our detection system. The OmpG pore is pH-sensitive, pH influences the conformation of the pore. We hypothesized that the loop network of OmpG can be influenced by charge, allowing us to manipulate the pore conformation independent of pH. We report a pH-insensitive pore that allows us to tailor OmpG for sensing an array of protein analytes. Also included in this work, we investigate protein and peptide detection and translocation by traditional nanopores to build on our research interest.
Type
Dissertation
Date
2019-02