Single Molecule Detection of Binding Kinetics

结合动力学的单分子检测

• 批准号: 0852581
• 负责人: Andrea Armani
• 金额: $ 119.82万
• 依托单位: University of Southern California
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2014-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0852581&HistoricalAwards=false
• 关键词: Single; Molecule; Detection; Binding; Kinetics

The ability to understand and to manipulate protein-protein interactions will contribute to improvements in biological imaging and methods for drug discovery and delivery. One crucial component of protein interactions is the binding site. The strength of the interaction determines the utility of the two proteins, which could be an antibody-antigen or an enzyme-substrate pair. Different methods have been developed to characterize this interaction, yet these techniques require either high protein pair concentrations or imaging of the interaction. High concentrations often cause blurring in the final data. Therefore, to accurately determine the interaction between two proteins, it is important to perform both types of measurements.The primary focus of this project is to develop and to demonstrate an optical sensor capable of characterizing antibody-antigen binding at the single molecule level without relying on images. This instrument is based on an optical sensor that relies on resonance, somewhat like an acoustic tuning fork. The optical sensor is capable of single molecule detection, because as molecules bind to the sensor, the sensor?s resonant frequency changes, resulting in a fast response time (100ns) and improved sensitivity. This technique will enable new types of measurements that will have immediate impact on the fields of cell biology and biochemistry, with longer-term benefits to systems biology. Training future scientists and engineers at all age levels is an integral component of this project. In addition to the involvement of a graduate student and a post-doctorate scholar, several undergraduate researchers whose stipends are provided through USC?s Undergraduate Research Fund will be involved in different aspects of this project. They will have the opportunity to learn state-of-the-art instrumentation and fabrication methods in USC?s Class 10 cleanroom and electron microscopy center. Results will be presented at professional conferences and the equipment will be demonstrated to middle school and high school visitors to the laboratory.

理解和操纵蛋白质-蛋白质相互作用的能力将有助于改进生物成像以及药物发现和递送方法。蛋白质相互作用的关键组成部分之一是结合位点。相互作用的强度决定了两种蛋白质的效用，这两种蛋白质可以是抗体-抗原或酶-底物对。已经开发了不同的方法来表征这种相互作用，但这些技术需要高蛋白质对浓度或相互作用的成像。高浓度通常会导致最终数据模糊。因此，为了准确确定两种蛋白质之间的相互作用，执行两种类型的测量非常重要。该项目的主要重点是开发和演示一种能够在单分子水平上表征抗体-抗原结合的光学传感器，而无需依赖图像。该仪器基于依赖共振的光学传感器，有点像声学音叉。光学传感器能够进行单分子检测，因为当分子与传感器结合时，传感器的谐振频率会发生变化，从而实现快速响应时间（100ns）并提高灵敏度。这项技术将实现新型测量，对细胞生物学和生物化学领域产生直接影响，并为系统生物学带来长期利益。培训各个年龄段的未来科学家和工程师是该项目不可或缺的组成部分。除了一名研究生和一名博士后学者的参与外，几名由南加州大学本科生研究基金提供津贴的本科生研究人员也将参与该项目的不同方面。他们将有机会在南加州大学的 10 级洁净室和电子显微镜中心学习最先进的仪器和制造方法。结果将在专业会议上公布，设备将向实验室的初中和高中参观者展示。