| Description |
This work describes the use of second harmonic generation (SHG) and ultraviolet-visible sum-frequency generation (UV-Vis SFG) to directly detect ligand-protein recognitions and drug-membrane associations. First, SHG spectroscopy was employed to detect protein-ligand binding for several model systems: avidin, streptavidin, neutrAvidinTM and anti-biotin antibody binding to biotin. The equilibrium binding affinities of these model systems were measured and compared with those values reported in literature to validate the ability of SHG to detect protein-ligand interactions without any chemical modification. Furthermore, the energetics of the protein-ligand binding and the protein nonspecific adsorption were evaluated to provide useful information about these protein-ligand pairs which are commonly used in many bioanalytical applications. Next, UV-Vis SFG spectroscopy was developed and utilized to detect drug-lipid membrane association for four drugs: ibuprofen, azithromycin, tetracaine, and tolnaftate. Drug association was measured on planar supported lipid bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine. The drugs' equilibrium association constants were obtained and correlated with the drugs' partition coefficients in the membrane-water system. Furthermore, the drug surface excess in the lipid membrane was quantitatively determined using the combination of the UV-Vis SFG and the bulk partition coefficient. It was shown that UV-Vis SFG is a powerful and novel technique to directly measure the association of drugs to a lipid membrane without chemical modification. Finally, SHG imaging was employed in the label-free detection of the interactions between tetracaine and a multicomponent planar supported lipid bilayer array (MLBA). The MLBAs allowed the effects of lipid phase and cholesterol content on tetracaine binding to be examined simultaneously. Additionally, tetracaine binding at different charge states and the effect of the charged lipids were investigated. The maximum surface excess of tetracaine in the lipid bilayers was also determined. This demonstrates that SHG imaging is a sensitive technique which can directly image and quantitatively measure the association of tetracaine in a high-throughput manner. This work has demonstrated that SHG and UV-Vis SFG are valuable alternatives in detection of biomolecular interactions at a lipid bilayer surface. The use of SHG or UV-Vis SFG imaging in combination with the MLBAs offer potential applications in high-throughput screenings of proteins and small molecules. |
