Labeless Analysis of Microarrays by Force Microscopy

通过力显微镜对微阵列进行无标记分析

• 批准号: 6790479
• 负责人: David Post Allison
• 金额: $ 22.05万
• 依托单位: MOLECULAR IMAGING CORPORATION
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-15 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6790479
• 关键词: Escherichia coli; atomic force microscopy; avidin; bacterial proteins; binding proteins; bioengineering /biomedical engineering; bioimaging /biomedical imaging; biomedical equipment development; biotechnology; biotin; computer program /software; microarray technology; molecular chaperones; nanotechnology; protein binding; protein protein interaction; recombinant proteins

DESCRIPTION (provided by applicant): The long-term objective of this project is to develop a robust screening technique for identifying biomolecular interactions. We intend to accomplish this using the sensitivity of atomic force microscopy (AFM) combined with the screening capabilities of the microarray format. A particular focus of this application is the screening for protein-protein interactions. Identifying protein-protein interactions is particularly challenging due to the heterogeneity among different proteins and the lack of generally applicable schemes for labeling and detection. These measurements would help in sorting out biochemical networks and in characterizing potential pharmaceutical targets. To realize this screening tool, the scanning module of a scanning probe microscope (SPM) will be combined with a programmable, translatable stage capable of micron to centimeter scale movements. This translation capability will allow assessment of conventional style microarrays with single molecule sensitivity and without the need for labels. A particular protein reagent (the probe) will be screened against an array of potential binding partners by tethering this probe to the microcantilever used in a conventional AFM. The array will then be screened for interaction forces. The first specific aim of the project is to integrate a multiple length scale translation stage with an AFM scanning head for screening microarrays. Appropriate software for positioning the probe tip and for sampling the array will be developed. The second aim will focus on optimizations of physical and chemical modifications that increase the likelihood of a protein-binding event. These optimizations will be performed using a standard system consisting of a biotin tethered microcantilever and avidin tethered to a surface. The third specific aim is to provide proof of principle of the intended instrument and application. A limited array of E. coli proteins will be produced using standard recombinant methods. One member of a well-characterized binding pair (e.g. GroEL and GroES) will be immobilized within the array while its complement will be tethered to the probe tip. The possibility of false negative and false positive binding events will be assessed.

描述（由申请人提供）：该项目的长期目标是开发一种强大的筛选技术来识别生物分子相互作用。我们打算利用原子力显微镜 (AFM) 的灵敏度与微阵列格式的筛选功能相结合来实现这一目标。该应用的一个特别重点是筛选蛋白质-蛋白质相互作用。由于不同蛋白质之间的异质性以及缺乏普遍适用的标记和检测方案，识别蛋白质-蛋白质相互作用特别具有挑战性。这些测量将有助于理清生化网络并表征潜在的药物靶标。为了实现这种筛选工具，扫描探针显微镜 (SPM) 的扫描模块将与能够进行微米到厘米级移动的可编程可平移台相结合。这种翻译能力将允许以单分子敏感性评估传统类型的微阵列，并且无需标签。通过将该探针连接到传统 AFM 中使用的微悬臂梁上，将针对一系列潜在的结合配偶体筛选特定的蛋白质试剂（探针）。然后将筛选阵列的相互作用力。该项目的第一个具体目标是将多长度平移台与 AFM 扫描头集成，用于筛选微阵列。将开发用于定位探针尖端和用于阵列采样的适当软件。第二个目标将侧重于物理和化学修饰的优化，以增加蛋白质结合事件的可能性。这些优化将使用由生物素系留的微悬臂梁和系留到表面的抗生物素蛋白组成的标准系统进行。第三个具体目标是提供预期文书和应用的原理证明。将使用标准重组方法生产有限的大肠杆菌蛋白质。充分表征的结合对（例如 GroEL 和 GroES）的一个成员将被固定在阵列内，而其互补体将被拴在探针尖端上。将评估假阴性和假阳性结合事件的可能性。