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可靠的筛选技术来识别生物分子相互作用。我们打算使用原子力显微镜（AFM）的灵敏度结合微阵列格式的筛选能力来实现这一目标。该应用的一个特殊重点是筛选蛋白质 - 蛋白质相互作用。由于不同蛋白质之间的异质性以及缺乏通常适用的标记和检测方案，鉴定蛋白质蛋白相互作用尤其具有挑战性。这些测量将有助于整理生化网络并表征潜在的药物靶标。为了实现此筛选工具，将扫描探针显微镜（SPM）的扫描模块与可编程的，可翻译的阶段结合使用，能够以微米为单位到厘米的运动。这种翻译能力将允许评估具有单分子灵敏度的常规样式微阵列，而无需标记。特定的蛋白质试剂（探针）将通过将此探针链接到常规AFM中使用的微型验证剂来筛选一系列潜在的结合伙伴。然后将筛选阵列以获得相互作用力。该项目的第一个具体目的是将多个长度的翻译阶段与AFM扫描头进行筛选微阵列。将开发用于定位探针尖端和采样数组的合适软件。第二个目的将集中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束缚的微型抗体和抗生物素系在一起的标准系统进行。第三个具体目的是提供预期工具和应用原理的证明。使用标准重组方法生产有限的大肠杆菌蛋白。特征良好的结合对（例如凹槽和凹槽）的一个成员将被固定在阵列中，而其补体将束缚在探针尖端。将评估假阴性和假阳性结合事件的可能性。