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    New Approaches to Stabilize Black Lipid Membranes - Towards Ion Channel Functionalized Detectors for Capillary Separations

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    Author
    Bright, Leonard Kofi
    Issue Date
    2015
    Keywords
    Capillary electrophoresis
    Detector
    Ion channel
    Microchip electrophoresis
    Sensor patform
    Chemistry
    Black lipid membrane (BLM)
    Advisor
    Apsinwall, Craig A.
    
    Metadata
    Show full item record
    Publisher
    The University of Arizona.
    Rights
    Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
    Embargo
    Release after 4-Aug-2016
    Abstract
    Capillary electrophoresis (CE) is an excellent analytical separation method with promising features such as small sample volumes (µL to pL), fast analysis times (s), high selectivity and efficiency, and excellent compatibility with biological samples. However, the inability of conventional CE detectors to sense biologically active compounds that are optically and electrochemically inactive limits their use for biosensing and drug screening. We have developed a highly stable electrophysiological detection platform consisting of ion channel (IC) reconstituted in synthetic bilayer membrane also known as black lipid membranes (BLM) suspended across a functionalized microaperture to be coupled to a high resolution capillary separation channel. Low energy surface modifiers were used to drastically improve the electrical, mechanical, and temporal stability of BLMs. Glass microapertures modified using tridecafluoro 1, 1, 2, 2-tetrahydrodimethylchlorosilane facilitated the rapid formation of highly stable BLMs due to the amphiphobic property (H₂O/oil repellency). Furthermore, a combination of chemically modified aperture surfaces and chemical cross-linking within the lipid membrane were used to dramatically improve BLM stability. Partial cross-linking within the bilayer maintained fluidity which allowed reconstitution of ion channel proteins while maintaining the stability of BLM-IC platform. The stable BLM-IC across glass pipette aperture was coupled to microchip electrophoresis and was shown to withstand field strength (>250 V/cm) from separation channel. Additionally, planar microapertures fabricated in SU8 were used for the formation of stable BLM-IC platform towards the construction of an integrated device. The chemical properties of the SU8 supported the formation and cross-linking within polymerizable lipid or lipid bilayer doped with polymerizable methacrylate monomers. Additionally, we expressed ion channel coupled receptor fusion protein in HEK 293 cells towards the development of ion channel sensors for wide range of ligand detection in BLM sensor platforms. The pharmacology of IC functionalized with muscarinic acetyl choline (M2-K) receptor using cell based assay by patch clamp electrophysiology showed activation by acetylcholine and inhibition by atropine. Thus this platform holds a great promise as the next-generation integrated analysis system for rapid screening of biologically active compounds (eg. glucagon) in complex matrix such as whole blood and urine for the diagnosis and management of chronic disease such as diabetes.
    Type
    text
    Electronic Dissertation
    Degree Name
    Ph.D.
    Degree Level
    doctoral
    Degree Program
    Graduate College
    Chemistry
    Degree Grantor
    University of Arizona
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