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

描述（由申请人提供）：微透析是一种用于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、临床和转化研究中具有巨大的重要性，但目前的方法有两个主要的局限性。其中之一是被测分析物的低时间分辨率，受扩散速率和所需样品量的限制。第二个缺点是缺乏与与某种化合物浓度相对应的电过程的直接耦合。因此，非常需要一种仪器或方法，可以接近实时地测量被分析物，使其分析和相关性与电活动的变化并行。NeuroInDx提出了一种利用荧光产生酶促反应和基于微阵列的样品收集和检测的近实时脑分析物测量的新方法。与现有的微透析方法相反，样品将使用机械臂连续沉积在聚赖氨酸涂覆的玻璃载玻片表面，从而产生微阵列。预处理后，样品将用微阵列扫描仪进行分析。获取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）与原始电生理文件集成。因此，可以建立脑电活动与分析物浓度之间的相关性。与现有的只能测量一种化合物的生物传感器相比，快速MD方法将允许同时测量几种化合物。NeuroInDx的长期目标是开发和商业化这种用于临床和基础研究的快速md设备。第一阶段项目的主要目标是在谷氨酸（大脑中主要的兴奋性神经递质）和葡萄糖（大脑代谢的主要相关物质）测量的基础上，证明Fast-MD方法用于脑神经递质分析的可行性。提出的Fast-MD系统可用于分析大脑、肌肉、肺、肝脏和身体其他部位的细胞外空间中不同化合物的浓度。该实验可在重症监护监护期间进行，也可在自由活动和麻醉动物中进行，并在脑电图、局部场电位和单个神经元水平上进行宽频带平行记录电活动。公共卫生相关性：该方法利用微流体连续收集脑脊液，利用产生荧光信号的酶促反应检测分析物，以微阵列形式将开发的样品进行机器人沉积，并并行监测脑电活动。一期项目重点是Fast-MD原型的研制、关键部件的研制、工艺优化以及谷氨酸和葡萄糖的酶促检测。

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.