Cross-linked Phospholipid Coatings in Micron-Sized Channels for Spatially Discrete Bioassays and Protein Separations

Persistent Link:
http://hdl.handle.net/10150/193940
Title:
Cross-linked Phospholipid Coatings in Micron-Sized Channels for Spatially Discrete Bioassays and Protein Separations
Author:
Mansfield, Elisabeth
Issue Date:
2007
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.
Abstract:
Supported phospholipid bilayers (SPBs) are attractive surface coatings since they are resistant to non-specific protein adsorption, self-assemble, support the function of many integral and membrane bound proteins, and can be functionalized with a variety of available headgroup chemistries. Phospholipid capillary coatings have been reported for applications in capillary electrophoresis (CE), although these coatings suffer from inherent stability problems, which arise from the fragility of the interactions between the bilayer and supportive substrate. Patterned immobilized domains can be functionally diversified, enabling for the screening of multiple analytes in a condensed region. The application of polymerized phospholipids was investigated for applications in CE and preparation of low volume biosensing arrays. A polymerized phospholipid, bis-SorbPC, was used to stabilize the phospholipid bilayer through cross-linking on the surface of fused silica capillaries and compared to other phospholipid capillary coatings for stability and separation quality. Polymerized bis-SorbPC coatings have significantly longer stability against degradation and can be stored for > one year with improved separation efficiency of proteins over bare capillary separations.Three-dimensional fluidic channels were modified with polymerized phospholipid features, which can be chemical "tuned" to introduce specific affinity binding, such as biotin-avidin or antibody-antigen binding, while minimizing nonspecific protein binding to the area of interest. Patterning of bis-SorbPC channels was achieved and the void regions between poly(bis-SorbPC) regions can be filled with other phospholipids to monitor the non-covalent binding (e.g. biotin-avidin, NTA-Ni2+-6xHis-protein) and covalent binding of proteins to DOPE-PEG-pNP and maleimide functionalized lipids, while minimizing non-specific adsorption and protein denaturation. Analytes can be immobilized on the surface via immunobinding events for detection and immunoassay applications. SPBs were used to support membrane proteins within patterned regions of the channel. Functionality can be added to the poly(bis-SorbPC) regions with copolymerization allowing regions of distinct functionality to reside adjacently in the channel. Through multiple polymerization steps, channels with multiple functionalities are demonstrated, with the possibility for higher-throughput multi-functional biosensing channels in the future.
Type:
text; Electronic Dissertation
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Chemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Aspinwall, Craig A.
Committee Chair:
Aspinwall, Craig A.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleCross-linked Phospholipid Coatings in Micron-Sized Channels for Spatially Discrete Bioassays and Protein Separationsen_US
dc.creatorMansfield, Elisabethen_US
dc.contributor.authorMansfield, Elisabethen_US
dc.date.issued2007en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © 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.en_US
dc.description.abstractSupported phospholipid bilayers (SPBs) are attractive surface coatings since they are resistant to non-specific protein adsorption, self-assemble, support the function of many integral and membrane bound proteins, and can be functionalized with a variety of available headgroup chemistries. Phospholipid capillary coatings have been reported for applications in capillary electrophoresis (CE), although these coatings suffer from inherent stability problems, which arise from the fragility of the interactions between the bilayer and supportive substrate. Patterned immobilized domains can be functionally diversified, enabling for the screening of multiple analytes in a condensed region. The application of polymerized phospholipids was investigated for applications in CE and preparation of low volume biosensing arrays. A polymerized phospholipid, bis-SorbPC, was used to stabilize the phospholipid bilayer through cross-linking on the surface of fused silica capillaries and compared to other phospholipid capillary coatings for stability and separation quality. Polymerized bis-SorbPC coatings have significantly longer stability against degradation and can be stored for > one year with improved separation efficiency of proteins over bare capillary separations.Three-dimensional fluidic channels were modified with polymerized phospholipid features, which can be chemical "tuned" to introduce specific affinity binding, such as biotin-avidin or antibody-antigen binding, while minimizing nonspecific protein binding to the area of interest. Patterning of bis-SorbPC channels was achieved and the void regions between poly(bis-SorbPC) regions can be filled with other phospholipids to monitor the non-covalent binding (e.g. biotin-avidin, NTA-Ni2+-6xHis-protein) and covalent binding of proteins to DOPE-PEG-pNP and maleimide functionalized lipids, while minimizing non-specific adsorption and protein denaturation. Analytes can be immobilized on the surface via immunobinding events for detection and immunoassay applications. SPBs were used to support membrane proteins within patterned regions of the channel. Functionality can be added to the poly(bis-SorbPC) regions with copolymerization allowing regions of distinct functionality to reside adjacently in the channel. Through multiple polymerization steps, channels with multiple functionalities are demonstrated, with the possibility for higher-throughput multi-functional biosensing channels in the future.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorAspinwall, Craig A.en_US
dc.contributor.chairAspinwall, Craig A.en_US
dc.contributor.committeememberArmstrong, Neal R.en_US
dc.contributor.committeememberGhosh, Indraneelen_US
dc.contributor.committeememberMiranda, Katrinaen_US
dc.contributor.committeememberSaavedra, S. Scotten_US
dc.identifier.proquest2403en_US
dc.identifier.oclc659748300en_US
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