Bio-Engineering Consortium - Abstract
High throughput (HT) screening is integral to drug discovery. While flow cytometry is known for its ability to measure cell responses, its power in the homogeneous analysis of ligand binding or molecular assembly and its potential for high throughput are not well-recognized. The possibility of displaying virtually any molecule in a format compatible with particle-based analysis as well as the novel approach of plug-flow flow cytometry for sampling times ~1 sec could make flow cytometry a powerful alternative for the real-time analysis of molecular interactions. Thus, we propose four projects that bring together expertise in bioengineering and biomaterials, receptors and cell biology, and flow cytometry instrumentation. The first two projects concern biomaterials. In the first project, we propose to express the proteins relevant to signal transduction and termination (seven trans-membrane receptors - 7TMR, receptor tails, G protein sub-units, arrestins, and receptor kinases) in forms appropriate for flow cytometry. These proteins will have epitope tags suitable for homogeneous attachment to beads as well as fluorescent groups suitable for detection by conventional flow cytometry. In the second project, we will employ biomaterial display and detection strategies compatible with flow cytometric analysis. Beads will be used as platforms to display the molecules, to analyze molecular assemblies, to examine enzymatic activities, and to examine inhibition by combinatorial drug libraries. Projects 3 and 4 will involve instrumentation development, fluidics, micro-machines, and automation. In the third project, we will develop fluid handling approaches for cells and beads. We will target throughput rates of 1 sample per second, or near the industrial standard of 100,000 samples per day, using commercial fluid handling components for the types of assays described in Projects 1 and 2. In the fourth project, we will develop and implement micro-fluidic sample handling approaches compatible with flow cytometry using novel elastomer-based micromachine technology. We have set a goal of 10 samples per second or 864,000 samples per day, exceeding the industrial throughput standard by nearly an order of magnitude. By integrating bioengineering, biomaterial, molecular, cellular and flow cytometric expertise, we expect to develop test platforms for high throughput analysis of molecular interactions with commercial potential in drug development. These resulting technological advances will also allow us at the same time to define mechanistic details of cell activation through 7TMR mediated pathways.