Engineered Proteins for Continuous Metabolic Monitoring

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DESCRIPTION (provided by applicant): Diabetes is a metabolic disease characterized by defective absorption and utilization of glucose. A crucial step in minimizing diabetes complications is frequent or continuous glucose monitoring. Although monitoring glucose alone is sufficient in many instances, we believe that monitoring several interacting metabolites at the same time will provide more complete diagnostic information as well as broaden the applicability of such a device to other areas, such as critical care, emergency care, space medicine and even bioprocessing. Here we propose a device for monitoring glucose, lactate and glutamine, with the possibility of expanding the number of analytes in future prototypes. The novelty of this device is in the use of a series of fluorescentlylabeled binding proteins (not enzymes) that undergo conformational changes when bound to their specific ligands. The proteins are labeled with two distinct dyes, one that is responsive to ligand binding but with a short decay lifetime, and the other is a non-responsive dye but with a decay lifetime that is hundreds of times longer than the other. The dual labeled proteins allow for lifetime-assisted ratiometric (LAR) sensing. LAR sensing also permits the design of a device that is miniaturized, low-cost and user-friendly but comparable in accuracy to research-grade instrumentation. The proteins are immobilized in an array format on a surface molded for maximum signal transmission. Continuous monitoring is accomplished with a commercially available microdialysis probe originally designed for neurochemical studies. A micropump drives the perfusion fluid through the microdialysis probe to the sensor array. A major undertaking in this project is the determination of microdialysis parameters that are suited for the micromolar sensitivity of the biosensors and the complexity of tissues or samples to be interrogated. The biosensor array is equipped with optics and the driving electronics, as well as a microcontroller for visualization and data storage. Validation studies will be carried out using standard HPLC methods with a pulsed electrochemical detector.