HIGH-THROUGHPUT MICROARRAY-BASED MICRODIALYSIS (FAST-MD) WITH PARALLEL MEASUREMEN

描述(申请人提供)：微透析是一种研究大脑和身体其他器官细胞外微环境中化合物(分析物)的通用方法。尽管它在基础、临床和转化性研究中具有巨大的重要性，但目前的方法有两大局限性。其中之一是测量的分析物的低时间分辨率，这受到扩散速度和所需样本量的限制。第二个缺点是缺乏与对应于某种化合物浓度的电过程的直接耦合。因此，非常需要一种仪器或方法，能够接近实时地测量分析物，以便与电活动的变化并行地进行分析和关联。NeuroInDx提出了一种新的方法，利用荧光产生的酶反应和基于微阵列的样本收集和检测，几乎实时地测量大脑分析物。与现有的微透析方法不同，样品将使用机械臂连续沉积在多赖氨酸涂层玻璃片的表面，从而创建微阵列。样品经过前处理后，将用微阵列扫描仪进行分析。采集的与特定分析物浓度成正比的信号强度数据将使用改进的DataPac 2K2软件(Run Technologies，Inc.)与原始电生理文件整合。因此，可以建立脑电活动与分析物浓度之间的关联。与现有的只能测量一种化合物的生物传感器不同，快速分子动力学方法将允许同时测量几种化合物。NeuroInDx的长期目标是开发这种用于临床和基础研究的Fast-MD设备并将其商业化。第一阶段项目的主要目标是基于对大脑中主要的兴奋性神经递质谷氨酸和大脑代谢的主要相关物质葡萄糖的测量，论证快速-MD方法用于脑神经递质分析的可行性。建议的Fast-MD系统可用于分析大脑、肌肉、肺、肝脏和身体其他部位细胞外空间中不同化合物的浓度。这些实验可以在重症监护期间的人身上进行，也可以在自由活动和麻醉的动物身上进行，并在脑电、局部场电位和单个神经元的水平上记录电活动的宽带平行。与公共健康相关：该方法利用微流控连续收集脑脊液，使用产生荧光信号的酶反应检测分析物，以微阵列格式自动沉积显影样本，以及并行监测脑电活动。第一阶段项目的重点是开发Fast-MD原型、其关键组件、工艺优化以及谷氨酸和葡萄糖的酶检测。

DESCRIPTION (provided by applicant): Microdialysis is a versatile method for investigation of chemical compounds (analytes) in the extracellular microenvironment of a brain and other organs of the body. In spite of its tremendous importance in basic, clinical and translational research, current approach has two major limitations. One of them is a low temporal resolution of measured analytes that is limited by the rate of diffusion and required sample volumes. Second drawback is the lack of direct coupling with the electrical processes that correspond to the concentration of a certain chemical compound. Therefore, there is a great need for an instrument or approach that would allow measuring the analytes close to real time permitting their analysis and correlation in parallel with the changes in electrical activity. NeuroInDx proposes a novel method of nearly real time brain analyte measurements utilizing fluorescence generating enzymatic reactions and microarray-based sample collection and detection. In contrast to the existing microdialysis approach, samples will be continuously deposited using a robotic arm on the surface of a polylysine coated glass slide that creates a microarray. After preprocessing, samples will be analyzed with a microarray scanner. Acquired signal intensity data that is proportional to the specific analyte's concentration will be integrated with the original electrophysiological file using modified DataPac 2K2 software (RUN Technologies, Inc). Therefore, the correlation between brain electrical activity and the analyte's concentration can be established. In contrast to the existing biosensors, which can measure only one compound, Fast MD approach will allow to measure several compounds simultaneously. The long-term goal of NeuroInDx is to develop and commercialize this Fast-MD device for clinical and basic research use. The main goal of the Phase I project is to demonstrate the feasibility of the Fast-MD approach for brain neurotransmitter analysis on the basis of the measurement of glutamate, which is a major excitatory neurotransmitter in the brain, and glucose, a major correlate of brain metabolism. The proposed Fast-MD system can be used for the analysis of concentrations of different chemical compounds in the extracellular space of the brain, muscle, lung, liver and other parts of the body. The experiments can be performed in the human during intensive care monitoring, as well as in freely moving and anesthetized animals with parallel wide frequency band recording of electrical activity at the level of EEG, local field potentials and single neurons. PUBLIC HEALTH RELEVANCE: The approach utilizes continuous collection of cerebro-spinal fluid using microfluidics, analyte detection using enzymatic reaction that generates a fluorescent signal, robotic deposition of the developed samples in a microarray format, and parallel monitoring of brain electrical activity. Phase I project is focused on the development of the Fast-MD prototype, its key components, process optimization and enzymatic detection of glutamate and glucose.