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.

Nanoscopic spatial reorganization of selectin ligands, CD44 and PSGL-1, during the initial step of hematopoietic stem/progenitor cell (HSPC) homing, tethering and rolling of migrating cells over E-selectins, has been recently reported. However, the exact spatial distribution of these ligands and their spatial reorganization during the cell rolling on E-selectins are still an open question. The spatiotemporal characterization at the nanoscale level requires high resolution imaging methods. In this study, I quantitatively characterize nanoscopic spatiotemporal behavior of the selectin ligands on the migrating cells to understanding the molecular mechanism of the cell rolling at the nanoscale level by means of a microfluidics-based 3D super-resolution fluorescence microscopy technique. The obtained results suggest that PSGL-1 on the cell shows significant change in the axial distribution on the cell during the cell rolling on E-selectin whereas the spatial distribution of CD44 along the axial direction is not affected significantly by the cell rolling. These findings indicate that each selectin ligand has a distinct contribution to the initial step of the HSPC homing because of their distinct spatial localizations on the cells that regulate at least partly the accessibility of these ligands to the surface E-selectin.