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Title: TIR-Raman spectroscopy of model supported lipid bilayers
Author: Churchwell, John Hampilton
ISNI:       0000 0004 5366 3510
Awarding Body: Durham University
Current Institution: Durham University
Date of Award: 2014
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Abstract:
In this thesis the technique of total internal reflection (TIR) Raman spectroscopy was applied to study the properties and interactions of supported lipid bilayers (SLBs) at the silica-water interface, both kinetically and at equilibrium. First, the formation kinetics of SLB systems from lipid aggregate suspensions was investigated. The lipid systems comprised POPC, POPE, egg-SM and a 1:1:1 mixture of POPE, egg-SM and cholesterol, all in tris buffer with and without added NaCl and CaCl2. Vesicle/aggregate suspensions were prepared by bath sonication and their size distributions were quantified with nanoparticle tracking analysis (NTA). The additional group I and group II salts altered the size distribution of the lipid vesicle/aggregate suspensions produced and played large role in the kinetics observed. For POPC, by changing the buffer conditions the adsorption of extraneous vesicles on the SLB could be tuned. For POPE, a previously unknown formation pathway was observed, whereby larger aggregates spread following adsorption to the interface. For the mixed system, the final ratio of components was found to be the same as that in the initial suspension. Second, the physical transformations of SLBs composed of DMPC, egg-SM and POPE were examined and the role of NaCl and CaCl2 upon these phase transitions was investigated. Raman spectra were obtained as a function of temperature and quantified using order parameter analysis. The resulting data were interpreted using the Zimm and Bragg model, which yielded the cooperativity of each phase transition. Cooperativity was controlled by the interfacial energy between regions of Lβ/Pβ and Lα phase. The presence or absence of the above salts altered the number of molecules within the cooperative unit for each of the species listed and controlled the interfacial energy. The most striking result was that of the POPE main phase transition with added CaCl2, for which cooperativity was massively reduced yielding a structural transition over a broad temper- ature range; AFM images confirmed the nature of this transition, showing domain like structures over a matching broad temperature range. Third, the interaction of SDS with SLBs composed of POPE, POPC, egg-SM and the 1:1:1 mixture of POPE, egg-SM and cholesterol was explored. Partitioning isotherms were constructed from equilibrium data and interpreted with a non-ideal partitioning model previously applied to vesicular systems. Accounting for the theoretical build-up of surface charge was found to be unnecessary probably owing to counterion binding. Kinetic data of the partitioning process for the different SLBs were obtained and qualitatively interpreted. For POPC at low dSDS concentrations dSDS translocation or flip-flop from the distal to proximal bilayer leaflets did not occur. At higher concentrations a period of rapid uptake lasting for approximately 100 s was followed by a slower increase lasting on the order of 10 minutes thus indicating that translocation was occurring. Upon subsequent rinsing, there was an initial rapid decrease in dSDS followed by a slower protracted decrease indicating that reverse flip-flop was occurring. The most intriguing result was that of the overall lipid signal upon rinsing, often it was observed to recover to levels equal to those prior to dSDS addition. These data suggest the formation of blebs or tubules as a result of dSDS induced spontaneous curvature; kinetic data from the CH region provided further evidence. Comparable data was obtained for POPE and egg-SM which showed very similar dSDS partitioning and rinsing kinetics, although the equilibrium behaviour differed in the strength of the dSDS lipid interaction. Less dSDS partitioned into the 1:1:1 mixture of POPE, egg-SM and cholesterol than any of the other species studied indicating its detergent resistance. Partial removal of this SLB from the interface left the contour of the CH region unchanged.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.614407  DOI: Not available
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