Microchip devices to assay quantal exocytosis

描述(申请人提供)：我们研究的长期目标是开发用于高通量测量神经元和神经内分泌细胞的量子胞吐的微型设备。膜片钳电生理学和碳纤维电化学法代表了高时间分辨率和高信息含量胞吐分析的最新技术，但速度慢，劳动强度大。细胞群体分泌物的生化分析具有有限的时间分辨率，不能分辨单个量子融合事件。我们将利用微芯片技术开发能够在一天内检测数千个细胞的量子胞吐的设备，以大大加快基础神经科学研究的步伐。这种方法还将首次实现对影响神经递质胞吐的候选药物进行快速和高信息含量的筛选。例如，L多巴通过增加多巴胺释放的量子含量来治疗帕金森氏症。这种方法将是跨学科的，将汇集具有生物医学、电气和机械工程、材料科学、物理、电化学、生理学和生物物理学专业知识的研究人员。 具体目标是：1)开发将单个电池自动对准微制造设备上的电化学微电极的方法。2)发展在微设备上刺激细胞胞吐的方法，包括快速微流控溶液交换、笼状钙的光解和电刺激动作电位。3)将新的电化学电极材料集成到微器件中，以提高灵敏度和性能。4)开发电子仪器，以允许同时记录多个通道的电化学或电生理数据。 该项目的五年目标是让实际设备投放市场，为胞吐研究社区提供服务，这些设备至少比目前的碳纤维方法快一个数量级。我们对每个目标的第一年里程碑是：1)将一个或多个细胞放置在微芯片上的预定位置。2)在微芯片上交换100ms的胞外液。3)表征类金刚石碳微电极的电化学性质。4)开发廉价的模块化电路，使用现成的组件进行基本的电化学测量，这些组件可以轻松扩展到大约12个同时使用的通道。

DESCRIPTION (provided by applicant): The long-term objective of our research is to develop microdevices for high-throughput measurement of quantal exocytosis from neurons and neuroendocrine cells. Patch-clamp electrophysiological and carbon-fiber electrochemical approaches represent the state-of-the-art for high time resolution and high information content assays of exocytosis but are slow and labor-intensive. Biochemical assays of secretion from cell populations have limited time resolution and can not resolve individual quantal fusion events. We will use microchip technology to develop devices that can assay quantal exocytosis from thousands of cells in a day in order to greatly accelerate the pace of basic neuroscience research. This approach will also enable, for the first time, rapid and high information content screening of drug candidates that affect exocytosis of neurotransmitter. For example, L-DOPA used to treat Parkinson's disease acts by increasing the quantal content of dopamine release. The approach will be interdisciplinary and will bring together investigators with expertise is biomedical, electrical and mechanical engineering, materials science, physics, electrochemistry, physiology and biophysics. The specific aims are: 1) Develop approaches to automatically target individual cells to electrochemical microelectrodes on microfabricated devices. 2) Develop approaches to stimulate exocytosis from cells on microdevices including rapid microfluidic solution exchange, photolysis of caged Ca and electrical stimulation of action potentials. 3) Integrate new electrochemical electrode materials into microdevices to increase sensitivity and performance. 4) Develop electronic instrumentation to allow simultaneous recording of many channels of electrochemical or electrophysiological data. The five-year goal of the project is to have actual devices on the market to serve the exocytosis research community that are at least an order of magnitude faster than current carbon-fiber approaches. Our first-year milestones for each aim are: 1) Position one or more cells at predetermined sites on a microchip. 2) Exchange extracellular solution in < 100 ms on a microchip. 3) Characterize the electrochemical properties of a diamond-like carbon microelectrode. 4) Develop inexpensive modular circuitry for basic electrochemical measurements using off-the-shelf components that can be easily scaled for approximately 12 simultaneous channels.