Bioassays and Molecular Characterization by Microchip CE

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DESCRIPTION (provided by applicant): The goal of this research is to develop plastic microchip analytical technologies that yield high-throughput approaches for the trace determination and/or physical properties of biological and small molecule therapeutics that are increasingly important to pharmaceutical and medical research. In support of this long-term goal are two specific aims. The first aim is to develop appropriate assays from three categories that would benefit significantly from the advantages of the microchip platform. The research focus will include efforts to significantly improve the determination of trace amounts of biological compounds with respect to speed and selectivity as compared with the standard immunoassay methodology. Aptamer-based assays of proteins will be used as illustrative chemical systems, and capillary electrophoresis on a multilane plastic chip with detection by laser induced fluorescence (LIF) will be the analytical technique. Analysis time will be shortened from hours to minutes by rapid, high-resolution separation of protein-photoaptamer complexes without the need for prior sample preparation. Additionally, a relatively high-throughput non-equilibrium separation-based approach that allows the accurate determination of Kd and IC-50 values from a wide range of ligand-binding systems will be developed. Non-equilibrium affinity capillary electrophoresis performed on a multilane plastic microchip with LIF, indirect LIF or flow-assisted mass spectrometry detection will be used with several model ligand-binding systems. As the final illustration, a universal, low sample consumption, high throughput and low cost separation-based platform to determine two of the key physiochemical properties of small molecules (MW 200-1,000), pKa and log Pow will be developed. Multilane plastic microchips coupled with either indirect laser-induced fluorescence or mass spectrometric detection can provide a rapid means of assessing these key parameters. The second aim will be to develop appropriate plastic microchip platforms from which these assays can be performed. In particular, these platforms must be compatible with the assays developed in the first Specific Aim. Efforts will be directed at developing multilane plastic chips having appropriate properties for use with LIF or indirect LIF as well as the appropriate laser system for LIF-associated methods. Efforts will also focus on appropriate chip designs and plastic substrates for multi-analyte analyses with mass spectrometric detection methods. Upon completion, appropriate microchip-sets will be created for assays of biological and small molecule therapeutics.