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acase@tulane.edu / This work embraces the fabrication of functional materials through the interactions at interfaces and covers two topics. First, we investigate the assembly of hydrophobically modified polypeptoids (HMPs) with lipid bilayers and the structural transition of lipid bilayers induced by HMPs. Polypeptoids are a class of pseudo-peptidic polymers. Properties such as biocompatibility, biodegradability, enzymatic resistance, and good processibility make polypeptoid a promising material for biotechnological applications. With decyl groups as hydrophobic side chains, HMPs interact with lipid bilayers by inserting the hydrophobes into the lipid bilayers through hydrophobic interaction. The hydrophobe insertion results in the disruption of lipid bilayers and the formation of lipid bilayer fragments containing HMPs and HMP-lipid complexes depending on HMP-to-lipid ratio. These polymer-lipid “rafts” can attach to the bare lipid bilayers forming close-spaced multilamellar structures, with HMPs connecting across the adjacent lipid bilayers. Also, HMP-lipid rafts can significantly increase the solubility of sorafenib in aqueous solution through hydrophobic interaction. The ability to assemble to lipid bilayers and to encapsulate hydrophobic drugs demonstrate the promise of HMP-lipid rafts in the application of drug delivery. The second topic is the rapid fabrication of hollow and yolk-shell functional materials through an aerosol assisted synthesis. Materials with hollow and yolk-shell structures have potentials in many applications, such as catalysis and energy storage. But the fabrication of such materials often suffers time-consuming and tedious procedures, which limits the practical application of these materials. We first demonstrated the rapid fabrication of hollow and yolk-shell Fe2O3 microspheres for enhanced photo-Fenton reactions. The placement of iron salts on the external surface of a carbonaceous microsphere generated from an aerosol droplet allows the formation of a Fe2O3 capsule by calcination, where colloid particles can be encapsulated. The Fe2O3 microspheres show significant enhancement in the photo-Fenton reaction. We then illustrated the fabrication of hollow and cage-like mesoporous Fe2O3/SiO2 microspheres. Through salt bridge effect, iron salts and cationic surfactants form colloidal aggregates which are locked within a rapidly formed SiO2 shell during the aerosol process. Sucrose as a pore generating agent leads to the formation of mesopores in the shell, which was proven favored features for CO2 capture. / 1 / Yueheng Zhang

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Cell membranes are integral for many biological processes. In addition to containing and protecting cellular contents and maintaining the chemical integrity of the cell, these interfaces host a variety of ligand-receptor interactions. These ligand-receptor interactions are important for cell signaling and transport and the ability to monitor them is key to understanding these processes. In addition, therapeutics and drug discovery is also aided by membrane-specific study, as the majority of drugs target receptors associated with the cell surface. The cell membrane can be effectively mimicked by the use of supported lipid bilayers, which provide a robust platform exhibiting the lateral fluidity and composition associated with cell membranes. The ability to study both ligand-receptor interactions as well as small molecule-membrane interactions on these model membranes is aided by the fact that these assays can be multiplexed and are amenable to use with low sample volumes with high throughput. Our laboratory has recently developed a strategy for fluorescent microscopy studies of ligand-receptor interactions on supported lipid bilayers without the use of fluorescently-labeled analytes. This technique involves the incorporation of pH-sensitive fluorophores into the composition of the supported lipid bilayer as embedded reporter dyes. It was determined that this assay can operate as either a “turn-on” or a “turn-off” sensor depending on the analyte to be detected. It was additionally found that modulating the ionic strength of the operating buffer allows for tuning the operating pH and sensitivity of the assay. This label-free technique can be utilized to monitor small peptide interactions with bilayers containing specific phospholipids. Basic amino acid sequences which are associated with transporting contents across membranes or anti-microbial activity can be monitored binding to negatively charged bilayers without the use of labels. Not only is this a sensitive technique for detecting small peptides, but thermodynamic data can be extracted as well. In a final set of experiments, the interaction of proteins with phosphatidylserine (PS) in supported lipid bilayers is observed by utilizing PS-Cu2+-induced quenching of fluorophores. Disruption of this metal-phospholipid, specifically by Ca2+-dependent protein kinases, results in a turn-on fluorescent assay, which can be used to monitor the binding of the protein to PS and the effects of other metal interference.

The research presented within this dissertation explores the photocatalytic deposition of metal carried out within sealed microfluidic channels. Micro scale patterning of metals inside sealed microchannels is investigated as well as nanoscale control over the surface morphology of the nanoparticles making up the patterns. This is achieved by controlling solution conditions during deposition. Finally, the nanoparticle patterns are used in fabricating a sensor device, which demonstrates the ability to address multiple patches within a sealed channel with different surface chemistries. Also presented here is the construction of the first epifluorescence/total internal reflection macroscope. Its ability to carry out high numerical aperture imaging of large arrays of solid supported phospholipid bilayers is explored. For this, three experiments are carried out. First, imaging of a 63 element array where every other box contains a different bilayer is preformed, demonstrating the ability to address large scale arrays by hand. Next, a protein binding experiment is preformed using two different arrays of increasing ligand density on the same chip. Finally, a two-dimensional array of mixed fluorescent dyes contained within solid supported lipid bilayers is imaged illustrating the ability of the instrument to acquire fluorescent resonance energy transfer data. Additionally, the design and fabrication of an improved array chip and addressing method is presented. Using this new array chip and addressing method in conjunction with the epifluorescence/total internal reflection macroscope should provide an efficient platform for high throughput screening of important biological processes which occur at the surfaces of cell membranes.

Ligand-receptor interactions are ubiquitous on cell membranes. Indeed, many important physiological functions primarily involve such interactions. These include cell signaling, pathogen binding, trafficking of lymphocytes, and the immune response.1-4 Therefore, studying ligand-receptor interactions at appropriate model membrane is of importance for both proper understanding of biological functions and applications to biosensors and bioseparations. Supported lipid bilayers are composed of the same lipid molecules found in the plasma cell membranes of living cells and possess the same two-dimensional fluidity as cell membranes, making them capable of mimicking the cell surface. Moreover, supported lipid bilayer-based in vitro assays are appealing because they require only very small sample volumes and they are suitable for multiplexing and high-throughput screening. Recently, our laboratory has combined supported lipid bilayer-coated microfluidic platforms with total internal reflection fluorescence microscopy to obtain equilibrium dissociation constant data for protein-ligand interactions. Using this method, it was found that equilibrium dissociation constants of antibody-ligand interactions at lipid membrane interfaces can be strongly affected by ligand lipophilicity and linker length/structure. These results are described in Chapter III. Monitoring protein-ligand interactions is routinely performed by fluorescently labeling the proteins of interest. Protein labeling can, however, interfere with detection measurements and be highly inconvenient to employ. To solve these problems, a simple and highly sensitive technique for detection of protein-ligand binding at biointerfaces has been developed. The method is based upon modulation of the interfacial pH when the protein binds. This change is detected by pH-sensitive fluorescent dye molecules embedded into the biointerface. The dye fluoresces strongly in the protonated state but becomes inactive upon deprotonation. These results are demonstrated in Chapter IV. Finally, the study of supported lipid bilayer-based electrophoresis is described in Chapter V. Bilayer electrophoresis is an attractive alternative to gel electrophoresis for the separation of membrane components such as lipids and membrane proteins because it is run in native-like environments and avoids exposing the analytes of interest to harsh chemicals. In this study, lipid rafts of varying size were used as separation matrices to separate two similar lipids with different alkyl chains. Lipid rafts of varying size were formed by a process controlled by varying treatment of the solid substrate. Depending on which method was employed, the results showed that lipid raft size could be modulated over five orders of magnitude. Moreover, it was found that the electrophoretic separation of the two lipid components depended on the size of rafts in the bilayer matrix.

The presented thesis is a continuation of the bachelor work, in which the effects of monovalent ions on neutral model lipid membranes were characterized. Herein physical properties of physiologically relevant anionic membranes in the presence of monovalent cations and oxidized lipids were studied. Hydration and mobility of the lipid bilayer at glycerol level were investigated using fluorescent solvent relaxation technique. In the first part of this work the interactions of cations (Na+ , K+ , Cs+ ) with negatively charged POPC/POPS lipid mixture, which is a good model of inner leaflet of cellular membrane, were studied. The presence of cations resulted in dehydration and substantial hinderence of mobility of hydrated lipids at the glycerol level probed by Laurdan. Clear specificity of those effects, which correlated with Hofmeister series have been observed. In the second part of the work truncated oxidized phospholipids, oxPLs (PazePC, PoxnoPC, PGPC, POVPC), which are known to be important in pathophysiology of numerous diseases, were investigated. 10 mol% of each oxPL was incorporated into neutral and anionic lipid bilayers, the hydration and mobility of which were measured in water or in KCl solution. The results reveal complex interactions between oxPLs, negatively charged lipids, and K+ . In...

In this thesis we study the interactions of ions and cationic peptides with a negatively charged lipid bilayer in an ionic solution where the electrostatic interactions are screened. <br /><br /> We first examine the problem of charge renormalization and inversion of a highly charged bilayer with low dielectric constant. To be specific, we consider an asymmetrically charged lipid bilayer, in which only one layer is negatively charged. In particular, we study how dielectric discontinuities and charge correlations among lipid charges and condensed counterions influence the effective charge of the surface. When counterions are monovalent, e. g. , Na⁺, our mean-field approach implies that dielectric discontinuities can enhance counterion condensation. A simple scaling picture shows how the effects of dielectric discontinuities and surface-charge distributions are intertwined: Dielectric discontinuities diminish condensation if the backbone charge is uniformly smeared out while counterions are localized in space; they can, however, enhance condensation when the backbone charge is discrete. In the presence of asymmetric salts such as CaCl₂, we find that the correlation effect, treated at the Gaussian level, is more pronounced when the surface has a lower dielectric constant, inverting the sign of the charge at a smaller value of Ca²⁺ concentration. <br /><br /> In the last chapter we study binding of cationic peptides onto a lipid-bilayer membrane. The peptide not only interacts electrostatically with anionic lipids, rearranging their spatial distributions, but it can also insert hydrophobically into the membrane, expanding the area of its binding layer (i. e. , the outer layer). We examine how peptide charges and peptide insertion (thus area expansion) are intertwined. Our results show that, depending on the bilayer's surface charge density and peptide hydrophobicity, there is an optimal peptide charge yielding the maximum peptide penetration. Our results shed light on the physics behind the activity and selective toxicity of antimicrobial peptides, i. e. , they selectively rupture bacterial membranes while leaving host cells intact.

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