Enzyme Assay Chips for Apoptosis Discovery

描述(由申请人提供)：而基因组和表型分析 随着DNA芯片和阵列的广泛使用，达到了巨大的规模 蛋白质组学的世界仍然受到蛋白质复杂性和 他们的分析难度很大。我们寻求创造一种负担得起的、轻松的 低成本“酶分析芯片”形式的可实现的蛋白质组学工具 允许在每平方厘米400个反应的水平上评估酶活性或 每张幻灯片最多有4000个反应。每个反应都将针对检测进行量身定做 在存在个性化激活剂的情况下特定酶的活性， 辅因子，以及在每个感测时检测的目标酶的抑制物 元素。一项新的微流控技术已经获得专利，并已为此进行了测试 申请。具体目标1(设计驱动)将定义运营/质量 高密度芯片中酶活性快速检测的控制条件 使用不到100,000个细胞。这一目标还将利用纯化的酶来 对酶活性敏感表面进行了测试和标定。具体目标2 (假说驱动)将寻求创造一种“细胞凋亡”芯片来绘制细胞凋亡图 细胞中的通路受到几种化学试剂的影响。我们假设 几种凋亡诱导剂的亚临界水平将相互作用，导致 芯片读数有明显变化。具体目标3(设计/假设 Driven)寻求创建去卷积软件来预测隐藏变量 (许多酶的浓度)传递到传感器表面。我们 假设酶感应的复杂模式可以被解卷为 每100次酶测定，样品中超过10种酶活性 在存在酶串扰的情况下。第一阶段的活动将导致 为商业化芯片建立芯片化学以帮助细胞凋亡， 癌症，药物毒性研究，以及二次药物测试和 工业生物反应器监测。这将使第二阶段活动能够 将专注于开发自动化且经济实惠的工作台 用于利用的技术。 建议的商业应用： 一种酶分析芯片技术将被验证，可以使用放置在传感芯片上的极小的细胞或生物样本来分析许多细胞内的蛋白酶。这项技术可用于研究、医疗和工业，并可用于监测细胞内酶状态和识别潜在的药物靶点。

DESCRIPTION (provided by applicant): While genomic and phenotypic analysis has reached a massive scale with the widespread use of DNA chips and arrays, the world of proteomics remains hindered by the complexity of proteins and the difficulty of their analysis. We seek to create an affordable and easily implementable proteomic tool in the form of low cost "enzyme assay chips" that allow evaluation of enzyme activities at the level of 400 reactions per cm2 or up to 4,000 reactions per slide. Each reaction will be tailored for detection of a specific enzyme activity in the presence of individualized activators, cofactors, and inhibitors of the target enzyme of assay at each sensing element. A novel microfluidics technology has been patented and tested for this application. Specific Aim 1 (design driven) will define the operational/quality control conditions for rapid sensing of enzyme activities on high-density chips using less than 100,000 cells. This aim will also utilize purified enzymes to test and calibrate the enzyme activity-sensing surface. Specific Aim 2 (hypothesis driven) will seek to create an "apoptosis" chip to map apoptotic pathways in cells subjected to several chemical agents. We hypothesize that sub-critical levels of several apoptotic inducers will interact to cause a marked change in the readout of the chip. Specific Aim 3 (design/hypothesis driven) seeks to create de-convolution software to predict hidden variables (concentrations of numerous enzymes) delivered to the sensor surface. We hypothesize that complex patterns of enzyme sensing can be de-convoluted for over 10 enzyme activities in a sample for every 100 enzyme determinations even in the presence of enzyme cross-talk. Phase I activities will lead toward establishing chip chemistries for a commercialized chip to aid in apoptosis, cancer, drug toxicity research, as well as, secondary drug testing and industrial bioreactor monitoring. This will enable Phase II activities that will focus on the development of an automated, yet affordable bench top technology for the utilization. PROPOSED COMMERCIAL APPLICATION: An enzyme assay chip technology will be validated for the assay of numerous intracellular proteases using extremely small cellular or biological samples placed on a sensing chip. This technology can have research, medical, and industrial uses with respoect to the monitoring of intracellular enzyme states and identifying potential drug targets.