Spelling suggestions: "subject:"supported lipid bilayer""
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Micro/nano-patterning of supported lipid bilayers: biophysical studies and membrane-associated species separationShi, Jinjun 15 May 2009 (has links)
Micro/nano-patterning of supported lipid bilayers (SLBs) has shown considerable potential for addressing fundamental biophysical questions about cell membrane behavior and the creation of a new generation of biosensors. Herein are presented several novel lithographic methods for the size-controlled patterning of SLBs from the microscale to the nanoscale. Using these methods, chemically distinct types of phospholipid bilayers and/or Escherichia Coli (E. Coli) membranes can be spatially addressed on a single microchip. These arrays can, in turn, be employed in the studies of multivalent ligand-receptor interactions, enzyme kinetics, SLBs size limitation, and membrane-associated species separation. The investigations performed in the Laboratory for Biological Surface Science include the following projects. Chapters II and III describe the creation of lab-on-a-chip based platforms by patterning SLBs in microfluidic devices, which were employed in high throughput binding assays for multivalent ligand-receptor interactions between cholera toxin B subunits (CTB) and ganglioside GM1. The studies on the effect of ligand density for multivalent CTB-GM1 interactions revealed that the CTB-GM1 binding weakened with increasing GM1 density. Such a result can be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibits the binding of CTB. Chapter IV characterizes the enzymatic activity of phosphatase tethered to SLBs in a microfluidic device. Higher turnover rate and catalytic efficiency were observed at low enzyme surface densities, ascribing to the low steric crowding hindrance and high enzyme fluidity, as well as the resulting improvement of substrate accessibility and affinity of enzyme catalytic sites. Chapter V presents sub-100 nm patterning of supported biomembranes by atomic force microscopy (AFM) based nanoshaving lithography. Stable SLBs formed by this method have a lower size limit of ~ 55 nm in width. This size limit stems from a balance between a favorable bilayer adhesion energy and an unfavorable bilayer edge energy. Finally, chapter VI demonstrates the electrophoretic separation of membrane-associated fluorophores in polymer-cushioned lipid bilayers. This electrophoretic method was applied to the separation of membrane proteins in E. Coli ghost membranes.
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Micro/nano-patterning of supported lipid bilayers: biophysical studies and membrane-associated species separationShi, Jinjun 15 May 2009 (has links)
Micro/nano-patterning of supported lipid bilayers (SLBs) has shown considerable potential for addressing fundamental biophysical questions about cell membrane behavior and the creation of a new generation of biosensors. Herein are presented several novel lithographic methods for the size-controlled patterning of SLBs from the microscale to the nanoscale. Using these methods, chemically distinct types of phospholipid bilayers and/or Escherichia Coli (E. Coli) membranes can be spatially addressed on a single microchip. These arrays can, in turn, be employed in the studies of multivalent ligand-receptor interactions, enzyme kinetics, SLBs size limitation, and membrane-associated species separation. The investigations performed in the Laboratory for Biological Surface Science include the following projects. Chapters II and III describe the creation of lab-on-a-chip based platforms by patterning SLBs in microfluidic devices, which were employed in high throughput binding assays for multivalent ligand-receptor interactions between cholera toxin B subunits (CTB) and ganglioside GM1. The studies on the effect of ligand density for multivalent CTB-GM1 interactions revealed that the CTB-GM1 binding weakened with increasing GM1 density. Such a result can be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibits the binding of CTB. Chapter IV characterizes the enzymatic activity of phosphatase tethered to SLBs in a microfluidic device. Higher turnover rate and catalytic efficiency were observed at low enzyme surface densities, ascribing to the low steric crowding hindrance and high enzyme fluidity, as well as the resulting improvement of substrate accessibility and affinity of enzyme catalytic sites. Chapter V presents sub-100 nm patterning of supported biomembranes by atomic force microscopy (AFM) based nanoshaving lithography. Stable SLBs formed by this method have a lower size limit of ~ 55 nm in width. This size limit stems from a balance between a favorable bilayer adhesion energy and an unfavorable bilayer edge energy. Finally, chapter VI demonstrates the electrophoretic separation of membrane-associated fluorophores in polymer-cushioned lipid bilayers. This electrophoretic method was applied to the separation of membrane proteins in E. Coli ghost membranes.
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Kinetics of an Inverse Temperature Transition Process and Its Application on Supported Lipid BilayerChang, Chin-Yuan 2010 August 1900 (has links)
This dissertation focuses on the study of inverse temperature transition
processes of the poly(N-isopropylacrylamide) (PNIPAM) and the elastin-like
polypeptides (ELPs). A novel temperature jump microfluidic system is introduced and
this system shows the ability to measure the kinetics of the PNIPAM and the ELPs
collapse without a heat transfer problem. The conformational change of the ELPs during
the phase transition process is utilized as a nanoscale protein filter to modulate ligandreceptor
binding events on supported lipid bilayers (SLBs).
This research study is divided into three main parts. The first part is the
development of the temperature jump microfluidics. The kinetics of PNIPAM collapse is
used as a model system to show the capability of this new device to measure millisecond
time scale phase transition processes. The effects of salts on the kinetics of PNIPAM
collapse are also shown in this part. To our knowledge, this is the first study which
shows the effects of salts on PNIPAM collapse kinetics.
The second part of this research is the application of the novel temperature jump
microfluidics. The hydrophobic collapse of ELPs composed of identical sequence but
different chain length is investigated. By controlling the molecular weight of the ELPs, the thermodynamic contributions from intermolecular hydrophobic interactions, and
intramolecular hydrophobic interactions could be calculated individually for this unique
system.
The third part is the application of the phase transition property of ELPs. The
ELPs are conjugated on the surface of the SLBs as a nanoscale protein filter. The
conformation of the ELPs can be modulated by ionic strength of the buffer solution or
ambient temperature. The ELPs conjugated SLBs platform showed the ability to block
IgG binding to biotin conjugated on the SLBs when the ELPs were in the extended coil
state and open the access for protein to bind to biotin in compact globule conformation.
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Geometry-Dependent Nonequilibrium Steady-State Diffusion and Adsorption of Lipid Vesicles in Micropillar ArraysLiu, Fangjie, Abel, Steven M., Collins, Liam, Srijanto, Bernadeta R., Standaert, Robert, Katsaras, John, Collier, Charles Patrick 09 May 2019 (has links)
Micro- and nanofabricated sample environments are useful tools for characterizing diffusion in confined aqueous environments. The steady-state diffusion and adsorption of unilamellar lipid vesicles in arrays of hydrophilic micropillars is investigated. Gradients in the coverage of fluorescently labeled, pillar-supported lipid films, formed from vesicle fusion, are determined from 3D z-stack images using confocal microscopy. The gradients are the result of preferential adsorption of vesicles near the tops of the pillars, which progressively deplete them from solution as they diffuse toward the base of the array. However, the increased propensity for vesicle adsorption near the pillar tops compared to the confined spaces between pillars results in the formation of confluent supported lipid bilayers at the pillar tops that resist the adsorption of additional vesicles while leaving the pillar surfaces below available for binding. This results in a reduction in the numbers of depleted vesicles compared to what one would anticipate based on diffusive fluxes. The resulting inhomogeneous spatial profiles of lipid structures on the pillars are the result of the system being maintained in a dissipative, nonequilibrium steady state during incubation of the pillar arrays in the vesicle solution, which is ultimately quenched by rinsing away the unbound, freely diffusing vesicles.
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FORMATION, DYNAMICS AND CHARACTERIZATION OF SUPPORTED LIPID BILAYERS ON SiO2 NANOPARTICLESAhmed, Selver January 2012 (has links)
This work is devoted to understanding the formation of supported lipid bilayers (SLBs) on curved surfaces as a function of lipid properties such as headgroup charge/charge density and alkyl chain length, and nanoparticle properties such as size and surface characteristics. In particular, the formation of SLBs on curved surfaces was studied by varying the size of the underlying substrate SiO2 nanoparticles with size range from 5-100 nm. Curvature-dependent shift in the phase transition behavior of these supported lipid bilayers was observed for the first time. We found that the phase transition temperature, Tm of the SLBs first decreased with decreasing the size of the underlying support, reached a minimum, and then increased when the size of the particles became comparable with the dimensions of the lipid bilayer thickness; the Tm was above that of the multilamellar vesicles (MLVs) of the same lipids. The increase in Tm indicated a stiffening of the supported bilayer, which was confirmed by Raman spectroscopic data. Moreover, Raman data showed better lipid packing and increased lateral order and trans conformation for the SLBs with increasing the curvature of the underlying support and decrease of the gauche kinks for the terminal methyl groups at the center of the bilayer. These results were consistent with a model in which the high free volume and increased outer headgroup spacing of lipids on highly curved surfaces induced interdigitation in the supported lipids. These results also support the symmetric lipid exchange studies of the SLBs as a function of the curvature, which was found to be slower on surfaces with higher curvature. Further, the effect of surface properties on the formation of SLBs was studied by changing the silanol density on the surface of SiO2 via thermal/chemical treatment and monitoring fusion of zwitterionic lipids onto silica (SiO2) nanoparticles. Our findings showed that the formation of SLBs was faster on the surfaces with lower silanol density and concomitantly less bound water compared to surfaces with higher silanol density and more bound water. Since the two SiO2 nanoparticles were similar in other respects, in particular their size and charge (ionization), as determined by zeta potential measurements, differences in electrostatic interactions between the neutral DMPC and SiO2 could not account for the difference. Therefore the slower rate of SLB formation of DMPC onto SiO2 nanoparticles with higher silanol densities and more bound water was attributed to greater hydration repulsion of the more hydrated nanoparticles. Lastly, we have investigated the effect and modulation of the surface charge of vesicles on the formation of SLBs by using different ratios of zwitterionic and cationic DMPC/DMTAP lipids. Through these studies we discovered a procedure by which assemblies of supported lipid bilayer nanoparticles, composed of DMPC/DMTAP (50/50) lipids on SiO2, can be collected and released from bilayer sacks as a function of the phase transition of these lipids. The lipids in these sacks and SLBs could be exchanged by lipids with lower Tm via lipid transfer. / Chemistry
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Studying the Interactions of Biomacromolecular Assemblies with Surfaces Using the Microcantilever Sensor and Quartz Crystal MicrobalanceJanuary 2011 (has links)
This thesis uses surface sensitive tools to characterize the effect of a solid surface on immobilized biomacromolecules. This includes understanding how the surface can change the affinity of these macromolecules to small molecules compared to bulk studies. Two classes of immobilized biomacromolecules, the supported lipid bilayer (SLB) and the Lac repressor protein (LacI), are characterized using microcantilever sensors and quartz crystal microbalance with dissipation (QCM-D). The first part of this thesis reports the use of microcantilever beams, an ultrasensitive sensor for measuring the surface free energy changes on a substrate induced by molecular adsorptions, to probe the interaction between a solid surface and a phospholipid bilayer. This sensing method integrates two well-developed techniques: solid-supported lipid bilayers (SLBs) and the microcantilever (MC) sensors. Studying the adsorption free energy of lipid bilayers on a solid surface allows better characterizing of the formation and stability of SLBs. Microcantilever converts the Gibbs free energy change taking place on its surface into a mechanical deformation. As molecules physisorb or chemisorb onto the surface of the microcantilevers, the microcantilevers bend, either due to induced compressive or tensile stresses, which result from the surface free energy change. By monitoring the deflection values of the microcantilevers, the real-time surface free energy change during the SLB formation can be detected. This thesis has led to the development of a novel biosensor--lipid membrane coated microcantilevers--to detect the adsorption, insertion, aggregation and solubilizing effect of membrane-active substances, such as surfactants and peptides, on the phospholipid membranes. To better characterize the surface free energy, SLBs doped with charged lipids or cholesterol are shown to alter the surface free energy. We can predict this change in surface free energy using a thermodynamic model. Application of this membrane-coated cantilever is put into use for detecting how amphiphilic molecules interact with SLBs, as well as for probing the abrupt conformational change of SLBs during a temperature induced phase-transition. This study systematically demonstrates various usage aspects of microcantilever to characterize the SLBs, and how this technique may advance the biophysical knowledge of the lipid membrane, one of the essential building blocks of life. The second part of this thesis reports the use of both microcantilever sensors and QCM-D to measure the adsorption free energy and mass of a model protein, the Lac repressor (LacI), and compare how a modified T334C mutant that includes a cysteine group to orient the protein on the gold surface through a covalent sulfur bonds retains its binding capabilities over that of wild type LacI. The main challenge of this work is to unravel how the adsorption of biomacromolecules at the solid/liquid interface leads to surface free energy changes and ultimately changes the stress of the underlying solid surface (the cantilever). The uses of microcantilever sensors and QCM to probe the interactions that take place on SLBs and surface-bound proteins have the advantage of being a sensitive, real-time, and label-free technique.
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Supported Lipid Bilayer Electrophoresis: A New Paradigm in Membrane Biophysics and SeparationsPace, Hudson 1982- 14 March 2013 (has links)
The motivation of this work was to produce novel analytical techniques capable of probing the physical properties of the cell surface. Many researchers have used supported lipid bilayers (SLBs) as models to study the structure and function of the cell membrane. The complexity of these models is consistently increasing in order to better understand the myriad of physiologically relevant processes regulated by this surface. In order to aid researchers in studying such phenomenon, the following contributions were made.
To manipulate components within the cell membrane, an electrophoretic flow cell was designed which can be used as a probe to study the effect of electrical fields on charged membrane components and for the separation of these components. This devise allows for the strict control of pH and ionic strength as species are observed in real-time using fluorescence microscopy. Additionally, advancements have been made to the production of patterned heterogeneous SLBs for use in separations and to probe the interactions of membrane components. The methodology to couple SLB separations and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) imaging was devised. This technology allows for the label-free mapping of the SLB surface post electrophoresis in order to observe naturally occurring species unperturbed by the addition of extrinsic tags. The final contribution, and perhaps the greatest, is the development of a procedure to create highly mobile SLBs from native membranes. These surfaces have vast potential in that they are no longer simple models of the cell surface, they are in fact the actual cell surface made planar. This advancement will be of great use to biophysicists and biochemists interested in using surface specific analytical methods to better understand physiological processes. These highly mobile native membrane surfaces have been coupled with the SLB electrophoresis technology to separate discrete bands of lipids and proteins, a proof of principle that will hopefully be further developed into a standard method for membrane proteomic studies.
Collectively the tools and methodologies described herein show great potential in allowing researchers to further add to mankind’s understanding of the cellular membrane.
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Molecular Recognition at the MembraneGong, Yun 15 January 2010 (has links)
No description available.
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Development of Advanced Optics and High Resolution Instrumentation for Mass Spectrometry Based ProteomicsSherrod, Stacy D. 14 January 2010 (has links)
Imaging mass spectrometry (MS) analysis allows scientists the ability to obtain
spatial and chemical information of analytes on a wide variety of surfaces. The ability to
image biological analytes is an important tool in many areas of life science research,
including: the ability to map pharmaceutical drugs in targeted tissue, to spatially
determine the expression profile of specific proteins in healthy vs. diseased tissue states,
and to rapidly interrogate biomolecular microarrays. However, there are several avenues
for improving the imaging MS experiment for biological samples. Three significant
directions this work addresses include: (1) reducing chemical noise and increasing
analyte identification by developing sample preparation methodologies, (2) improving
the analytical figures of merit (i.e., spatial resolution, analysis time) by implementing a
spatially dynamic optical system, and (3) increasing both mass spectral resolution and
ion detection sensitivity by modifying a commercial time-of-flight (TOF) MS.
Firstly, sample methodology schemes presented in these studies consist of
obtaining both ?top-down? and ?bottom-up? information. In that, both intact mass and
peptide mass fingerprinting data can be obtained to increase protein identification. This sample methodology was optimized on protein microarrays in preparation for bio tissue
analysis. Other work consists of optimizing novel sample preparation strategies for
hydrated solid-supported lipid bilayer studies. Sample methods incorporating
nanomaterials for laser desorption/ionization illustrate the ability to perform selective
ionization of specific analytes. Specifically, our results suggest that silver nanoparticles
facilitate the selective ionization of olefin containing species (e.g., steroids, vitamins).
Secondly, an advanced optical design incorporating a spatially dynamic optical
scheme allows for laser beam expansion, homogenization, collimation, shaping, and
imaging. This spatially dynamic optical system allows user defined beam shapes,
decreases analysis times associated with mechanical movement of the sample stage, and
is capable of increasing the MS limits of detection by simultaneously irradiating multiple
spots.
Lastly, new data acquisition strategies (multiple anode detection schemes) were
incorporated into a commercial time-of-flight mass spectrometer to increase both
sensitivity and resolution in a matrix assisted laser desorption/ionization mass
spectrometer. The utility of this technique can be applied to many different samples,
where high mass spectral resolution allows for increased mass measurement accuracy.
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Příprava modelových membrán pro studium jejich interakcí s biopolymery pomocí fluorescenční korelační spektroskopie / Preparation of model membranes to study their interactions with biopolymers using fluorescence correlation spectroscopyAdamcová, Zuzana January 2015 (has links)
This diploma thesis is focused on preparation and characterization of supported lipid bilayers as simplified models of cell membranes. The bilayers were prepared from source system of lecithin liposomes in phosphate buffer using the vesicle fusion method on a cover glass sufrace hydrophilized by plasma. Three fluorescent probes – Nile red, Oregon Green DHPE and DiO – were utilized to characterize diffusion within the bilayer using fluorescence correlation spectroscopy. For this purpose Z-scan FCS, which is a method developed specially for planar samples, was used. After the process of preparation and characterization of supported lipid bilayer was optimalized, interaction between this artificial membrane and solution of hyaluronic acid in phosphate buffer was studied. It was found out, that addition of this biopolymer causes slowing the diffusion of the fluorescent probe within the bilayer.
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