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

描述（由申请人提供）：微透析是一种研究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时间分辨率低，这受到扩散速率和所需样品体积的限制。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，其允许接近真实的时间测量分析物，从而允许它们的分析和与电活动的变化并行的相关性。NeuroInDx提出了一种利用荧光生成酶反应和基于微阵列的样品收集和检测的近真实的时间脑分析物测量的新方法。与现有的微透析方法相比，样品将使用机械臂连续沉积在多聚赖氨酸涂覆的载玻片表面上，从而产生微阵列。预处理后，将用微阵列扫描仪分析样品。使用改良的DataPac 2K 2软件（RUN Technologies，Inc）将与特定分析物浓度成比例的采集信号强度数据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该Fast-MD器械，用于临床和基础研究。第一阶段项目的主要目标是证明Fast-MD方法用于脑神经递质分析的可行性，该方法基于谷氨酸盐（脑中主要的兴奋性神经递质）和葡萄糖（脑代谢的主要相关物）的测量。所提出的Fast-MD系统可用于分析脑、肌肉、肺、肝和身体其他部位的细胞外空间中不同化合物的浓度。该实验可以在重症监护监测期间在人类中进行，以及在自由移动和麻醉的动物中进行，其中在EEG、局部场电位和单个神经元的水平上并行宽频带记录电活动。公共卫生相关性：该方法利用微流体连续收集脊髓液，使用产生荧光信号的酶反应进行分析物检测，以微阵列格式自动沉积开发的样品，并并行监测脑电活动。第一阶段项目的重点是Fast-MD原型的开发，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.