The proposed studies describe an innovative approach for detecting protein association to spatially localized surface-immobilized small molecule (ligand) and protein microarrays via chiral imaging with second harmonic generation (SHG). The use of microarray-based protein assays has the potential to dramatically increase the analysis of global protein populations within living organisms. The practical implementation of these technologies will have a dramatic impact in proteomics, pharmacological screening of drug candidates, and the investigation of protein-protein interactions. Most conventional microarray-based strategies for detecting protein-ligand, and protein-protein association or for the screening of protein populations are based on fluorescence methods to visualize the interaction of interest. Such methodologies require the fluorescent labeling of a large population of proteins with fluorophores. The inability to accurately perform this operation in a controlled manner for the complete protein output of a cell or organism is a major drawback to currently proposed techniques for high throughput protein screening. This proposal will address the hypothesis that the intrinsic chirality of proteins can be used to """"""""image"""""""" their association on patterned microarrays. This hypothesis will initially be tested by exploring the ability to monitor protein association to ligands presented on the outer leaflet of planar supported lipid bilayers, utilizing the proteins' native chirality as a spectroscopic probe. Then, SHG chiral imaging for protein-ligand based microarray assay will be examined using photolithographic and micro-contact printing methods to produce ordered arrays of ligands for protein adsorption studies. Finally, the ability of chiral SHG imaging to measure protein-protein interactions on patterned protein microarrays will be assessed. In order to fully characterize the spatial arrangement of interfacial protein species in these studies, SHG microscopy will be used in combination with fluorescence microscopy to provide a precise evaluation of the proposed chiral SHG imaging methodology. The proposed studies represent a novel experimental approach that should provide answers to a growing number of questions concerning detection for high throughput proteome screening. ? ?
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