DEVELOPMENT AND CHARACTERIZATION OF STABILIZED PHOSPHOLIPID COATINGS FOR OPEN TUBULAR AND PACKED CAPILLARY SEPARATIONS

Persistent Link:
http://hdl.handle.net/10150/204067
Title:
DEVELOPMENT AND CHARACTERIZATION OF STABILIZED PHOSPHOLIPID COATINGS FOR OPEN TUBULAR AND PACKED CAPILLARY SEPARATIONS
Author:
Adem, Seid Muhie
Issue Date:
2010
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:
Embargo: Release after 4/14/2012
Abstract:
Phosphorylcholine (PC) based phospholipid bilayers have been explored as coating materials for various substrates due to their inherent resistance to non-specific protein adsorption. Phospholipids have been used for coatings in capillary electrophoresis (CE) to suppress electroosmotic flow (EOF) and to obtain better separation of proteins. Here, a series of investigations geared towards developing highly stable phospholipid based biomimetic stationary phases for chromatographic separations was performed.Fluid phospholipid bilayers lack the desired chemical and physical stability to serve as long-term coatings. In this work, highly stable phospholipid coatings generated via crosslinking polymerization of bis-SorbPC monomers were investigated. Reproducible EOF and migration times for model proteins were obtained for coated capillaries that were kept at room temperature for up to two months. Furthermore, the effects of surfactants, pH and capillary inner diameter (i.d.) on the stability of the lipid coating were investigated.In an alternate approach, stabilized phospholipid coatings for capillary electrophoresis were investigated via formation of hybrid monolayers. The capillary surface was chemically modified with a cyano group followed by deposition of phospholipid monomers. In this approach, marked enhancements in coating stability were attained with commercially available reagents. The hybrid coating was utilized for protein separations and gave efficiencies comparable to non-stabilized lipid coated capillaries.Fused silica capillaries were modified with phospholipid bilayers that were chemically tuned to introduce specific affinity binding agents, while minimizing nonspecific protein adsorption to the capillary wall. The wall of fused silica was functionalized with DOGS-NTA-Ni2+ lipid to present binding sites inside the capillary for 6xHis-tagged proteins. Fluorescence microscopy and changes in electrophoretic mobility were used to follow the interaction of the model proteins with the functionalized silica surface.The structural similarity of lipid vesicles to cell membranes made them attractive in developing stationary phases for both liquid chromatography and capillary electrophoresis to study interactions between analytes and phospholipid membranes. Stabilized PLB coated silica microspheres were prepared via polymerization of lipid monomers and displayed enhanced stability to extended storage and organic solvent. These highly stable microspheres, while minimizing nonspecific protein adsorption, were also functionalized with DOGS-NTA-Ni2+ and effectively bind 6xHis-EGFP proteins.
Type:
text; Electronic Dissertation
Keywords:
Capillary electrophoresis; Packed capillary columns; Phospholpid Bilayers; Stabilized phospholpid coatings; Surface functionalization
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry
Degree Grantor:
University of Arizona
Advisor:
Aspinwall, Craig A.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleDEVELOPMENT AND CHARACTERIZATION OF STABILIZED PHOSPHOLIPID COATINGS FOR OPEN TUBULAR AND PACKED CAPILLARY SEPARATIONSen_US
dc.creatorAdem, Seid Muhieen_US
dc.contributor.authorAdem, Seid Muhieen_US
dc.date.issued2010-
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.releaseEmbargo: Release after 4/14/2012en_US
dc.description.abstractPhosphorylcholine (PC) based phospholipid bilayers have been explored as coating materials for various substrates due to their inherent resistance to non-specific protein adsorption. Phospholipids have been used for coatings in capillary electrophoresis (CE) to suppress electroosmotic flow (EOF) and to obtain better separation of proteins. Here, a series of investigations geared towards developing highly stable phospholipid based biomimetic stationary phases for chromatographic separations was performed.Fluid phospholipid bilayers lack the desired chemical and physical stability to serve as long-term coatings. In this work, highly stable phospholipid coatings generated via crosslinking polymerization of bis-SorbPC monomers were investigated. Reproducible EOF and migration times for model proteins were obtained for coated capillaries that were kept at room temperature for up to two months. Furthermore, the effects of surfactants, pH and capillary inner diameter (i.d.) on the stability of the lipid coating were investigated.In an alternate approach, stabilized phospholipid coatings for capillary electrophoresis were investigated via formation of hybrid monolayers. The capillary surface was chemically modified with a cyano group followed by deposition of phospholipid monomers. In this approach, marked enhancements in coating stability were attained with commercially available reagents. The hybrid coating was utilized for protein separations and gave efficiencies comparable to non-stabilized lipid coated capillaries.Fused silica capillaries were modified with phospholipid bilayers that were chemically tuned to introduce specific affinity binding agents, while minimizing nonspecific protein adsorption to the capillary wall. The wall of fused silica was functionalized with DOGS-NTA-Ni2+ lipid to present binding sites inside the capillary for 6xHis-tagged proteins. Fluorescence microscopy and changes in electrophoretic mobility were used to follow the interaction of the model proteins with the functionalized silica surface.The structural similarity of lipid vesicles to cell membranes made them attractive in developing stationary phases for both liquid chromatography and capillary electrophoresis to study interactions between analytes and phospholipid membranes. Stabilized PLB coated silica microspheres were prepared via polymerization of lipid monomers and displayed enhanced stability to extended storage and organic solvent. These highly stable microspheres, while minimizing nonspecific protein adsorption, were also functionalized with DOGS-NTA-Ni2+ and effectively bind 6xHis-EGFP proteins.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectCapillary electrophoresisen_US
dc.subjectPacked capillary columnsen_US
dc.subjectPhospholpid Bilayersen_US
dc.subjectStabilized phospholpid coatingsen_US
dc.subjectSurface functionalizationen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorAspinwall, Craig A.en_US
dc.contributor.committeememberSaavedra, S. S.en_US
dc.contributor.committeememberWysocki, Vicki H.en_US
dc.contributor.committeememberSanov, Andreien_US
dc.identifier.proquest10851-
dc.identifier.oclc659753749-
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