Microchip-based Cell Reactor Analysis System

描述（由申请人提供）：本区域更新申请的总体目标是开发可用于在分子水平上研究细胞间相互作用的微芯片方法。有许多例子表明，不同细胞类型的相互作用在正常生物功能或疾病发作中发挥作用。一个例子是神经元和经历炎症的神经胶质细胞之间的相互作用。已经提出，帕金森病中普遍存在的多巴胺能神经元的变性可能与小胶质细胞的慢性炎症有关，其中小胶质细胞产生一氧化氮和与神经元相互作用的其他活性氧物质。另一个例子是血管舒张过程，其中已经表明，当暴露于缺氧条件或变形时，红细胞通过ATP释放与内皮细胞相互作用，导致一氧化氮的产生和随后的平滑肌松弛。红细胞ATP释放的受损导致一氧化氮产生减少，并被认为在几种疾病中发挥作用。虽然已经有许多将细胞固定在芯片上并检测从细胞释放的特定分析物的例子，但是很少有将多种细胞类型整合在芯片上的报道，其中可以以可以监测许多神经递质/产物的方式研究细胞与细胞的通信。在这项资助中，我们建议继续开发微芯片方法，使细胞固定化与一般分析步骤（电泳）相结合，其中通过细胞层释放或运输的各种分析可以通过电化学分离并随后检测。重要的是，我们提出开发技术，使研究人员能够监测2层固定化细胞（PC 12细胞和小胶质细胞）之间或流动的细胞流（红细胞）和一层固定化细胞（内皮细胞）之间的细胞间通讯。此外，我们建议扩大这种微芯片的方法，使在体内研究，通过整合微透析采样，可用于研究不同细胞类型的相互作用，通过立体定向植入探针在感兴趣的区域，分段流，微芯片电泳和电化学检测。该基金的具体目标是：1）使用基于细胞固定化的细胞固定化和微芯片电泳与电化学检测来研究多种细胞类型之间的相互作用; 2）开发可用于研究红细胞与固定化内皮细胞之间的细胞间通讯的微芯片装置;以及3）将微透析采样和分段流与微芯片电泳和电化学检测集成用于体内采样。 公共卫生相关性：这个区域更新应用的总体目标是开发微芯片方法，可用于在分子水平上研究细胞与细胞的相互作用。所得到的技术将能够监测2层固定化细胞之间或流动细胞与固定化内皮之间的细胞与细胞通信。该方法还将通过将微透析采样与分段流、微芯片电泳和电化学检测相结合来实现体内研究。

DESCRIPTION (provided by applicant): The overall goal of this AREA renewal application is to develop microchip approaches that can be used to study cell-to-cell interactions at the molecular level. There are many examples in which the interaction of different cell types plays a role in normal biological function or in the onset of disease. One example is the interaction between neurons and glia cells that have undergone inflammation. It has been suggested that the degeneration of dopaminergic neurons that is prevalent in Parkinson's disease may be related to chronic inflammation of microglia, with the microglia producing nitric oxide and other reactive oxygen species that interact with the neurons. Another example is the vasodilatation process, where it has been shown that red blood cells, when exposed to hypoxic conditions or deformation, interact with endothelial cells via ATP release, leading to the production of nitric oxide and subsequent smooth muscle relaxation. Impairment of the ATP release from red blood cells leads to less nitric oxide production and has been postulated to play a role in several diseases. While there have been numerous examples of immobilizing cells on-chip and detecting a specific analyte released from the cells, there have been few reports of integrating multiple cell types on chip where cell-to-cell communication can be studied in a manner where numerous neurotransmitters/products can be monitored. In this grant we propose to continue the development of microchip approaches that enable the integration of cell immobilization with a general analysis step (electrophoresis), where a variety of analyses that are either released or transported through a layer of cells can be separated and subsequently detected via electrochemistry. Importantly, we propose to develop technology that will enable a researcher to monitor cell- to-cell communication between 2 layers of immobilized cells (PC 12 cells and microglia cells) or between a flowing stream of cells (red blood cells) and a layer of immobilized cells (endothelial cells). In addition, we propose to expand this microchip approach to enable in vivo studies by integrating microdialysis sampling, which can be used to study the interactions of different cell types by stereotaxically implanting a probe in the region of interest, with segmented flow, microchip electrophoresis, and electrochemical detection. The specific aims of the grant are to 1) Use of reservoir-based cell immobilization and microchip electrophoresis with electrochemical detection to study the interaction between multiple cell types; 2) Develop a microchip device that can be used to study cell-to-cell communication between red blood cells and an immobilized endothelium; and 3) Integration of microdialysis sampling and segmented flow with microchip electrophoresis and electrochemical detection for in vivo sampling. PUBLIC HEALTH RELEVANCE: The overall goal of this AREA renewal application is to develop microchip approaches that can be used to study cell-to-cell interactions at the molecular level. The resulting technology will enable monitoring of cell-to- cell communication between 2 layers of immobilized cells or between flowing cells and an immobilized endothelium. The approaches will also enable in vivo studies by integrating microdialysis sampling with segmented flow, microchip electrophoresis, and electrochemical detection.