9402762 Wood This Small Business Innovation Research Phase II project will develop ways to increase the amount of information available in luminescence assays by concurrently using different colors of luminescence. Current luminescent bioassays rely on measurement of light intensity only to transmit information, thus providing only one information channel. Greater information could be gained through a multiplexing strategy using different colors for channel separation. The only bioluminescent system capable of emitting a range of wavelength, without requiring intermolecular energy transfer, are the beetle luciferases. Previous research by the company shows that individual amino acid substitutions in these enzymes control the color of light emitted. From empirical and theoretical evidence, luminescent colors ranging from green to red should be possible. The objectives of this research is to create a red-emitting enzyme by engineering specific substitutions into a green-emitting enzyme. When completed, the two enzymes should differ by only 3-10 amino acids, and thus their physical and enzymological properties should by nearly equivalent. The development process will use data and methods gained from Phase I to search for the optimal combination of amino acid substitutions. Newly created isozymes will be analyzed for spectral, physical, and enzymotogical characteristics. After development, both enzymes will be used together to demonstrate the feasibility of luminescence multiplexing in quantifying cellular physiology. The technology will advance quantitative measurements in complex biological systems for many commercial and basic research applications. The multiplexing system has novel and important applications in several markets, including "real time" detection of toxins in environmental samples, new drug discovery and food industry quality assurance. Advantages of this technology versus current "rapid" diagnostics include sensitivity, specificity, and the potential to develop tests for defined groups of contaminants in a single biosensor kit.
