Single Molecule Detection of Binding Kinetics

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传递的方法。蛋白质相互作用的一个重要组成部分是结合部位。相互作用的强度决定了这两种蛋白质的效用，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，然而这些技术要么需要高蛋白质对浓度，要么需要相互作用的成像。高浓度往往会导致最终数据的模糊。因此，为了准确地确定两种蛋白质之间的相互作用，进行这两种类型的测量是很重要的。本项目的主要重点是开发和展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，而不依赖于图像。该仪器基于光学传感器，依靠共振，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S共振频率会发生变化，从而导致快速的响应时间(100 Ns)和提高的灵敏度。这项技术将使新类型的测量能够对细胞生物学和生物化学领域产生立竿见影的影响，并对系统生物学产生更长期的好处。培训各个年龄段的未来科学家和工程师是这一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，几名通过南加州大学S本科生研究基金提供津贴的本科生研究人员将参与这个项目的不同方面。他们将有机会在南加州大学S 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，设备将向来到实验室的初中和高中参观者展示。

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.