Engineered Proteins for Continuous Metabolic Monitoring

描述（由申请人提供）：糖尿病是一种代谢性疾病，其特征是葡萄糖的吸收和利用缺陷。减少糖尿病并发症的一个关键步骤是频繁或连续的血糖监测。虽然在许多情况下，单独监测葡萄糖就足够了，但我们相信，同时监测几种相互作用的代谢物将提供更完整的诊断信息，并将这种设备的适用性扩展到其他领域，如重症监护、急救护理、太空医学甚至生物处理。在这里，我们提出了一种用于监测葡萄糖，乳酸盐和谷氨酰胺的设备，有可能在未来的原型中扩大分析物的数量。该装置的新奇之处在于使用了一系列荧光标记的结合蛋白（而不是酶），这些蛋白在与其特异性配体结合时发生构象变化。蛋白质用两种不同的染料标记，一种对配体结合有反应但衰变寿命短，另一种是非反应性染料，但衰变寿命比另一种长数百倍。双标记的蛋白质允许寿命辅助比率（LAR）传感。LAR传感还允许设计一种小型化、低成本和用户友好的设备，但其精度可与研究级仪器相媲美。这些蛋白质以阵列形式固定在一个表面上，该表面是为了最大限度地传输信号而模制的。连续监测是用最初设计用于神经化学研究的市售微透析探针完成的。微泵驱动灌注流体通过微透析探针到达传感器阵列。该项目的一个主要任务是确定适合生物传感器的微摩尔灵敏度和待询问的组织或样品的复杂性的微透析参数。生物传感器阵列配备有光学器件和驱动电子器件，以及用于可视化和数据存储的微控制器。将使用标准HPLC方法和脉冲电化学检测器进行验证研究。

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.