Development of an Electrokinetic Trap for Single Biomolecules in Solution

描述(由申请人提供)：仪器设备的进步往往会带来概念上的突破。例如，基于激光的光学镊子导致了对生物分子拉伸和结合所涉及的物理力的实验。这一探索性计划的长期目标是提供一种捕获水溶液中单个生物分子的新技术--反布朗电泳法(Abel Trap)。这种设备将允许捕获比通常用光学镊子捕获的最小物体小50-100倍的物体，一直延伸到溶液中的单一蛋白质和核酸。 第一个目标是开发和扩展Abel捕集概念，使其能够捕获缓冲液中的单个微小、模糊的生物分子，例如单个GFP、单个量子点生物标记和仅用一个荧光团标记的伴侣蛋白GroEL的单个分子。这将涉及通过使用硬件反馈以及微流控设计和表面处理的优化来减少延迟。第二个目标是开发分析程序，允许提取作为时间函数的单个被捕获生物分子的迁移率和扩散系数。 阿贝尔陷阱将使在自由溶液中对单个生物分子的扩展研究成为可能--这是以前不存在的能力。阿贝尔陷阱直接提供有关被捕获物体的运输性质的信息，例如蛋白质-蛋白质相互作用事件的发生。在未来的工作中，这一能力将导致对单体逐一添加时聚集形成的研究，最终阿贝尔陷阱应该有助于揭示导致这些异常引起的疾病中的普恩或其他蛋白质聚集和/或错误折叠的初始事件。 该项目还将在单生物分子研究中引入一个新概念：反馈可以用来导致单个分子(或小分子集合)违反第二定律，同时在宏观尺度上保持第二定律。项目描述详细说明了实现主要目标所需的物理、化学和生物物理实验；但一旦仪器可用于生物医学界，其他人将想到目前无法想象的实验。正是出于这个原因，这项研究代表着生物医学应用的新的和强大的潜在进展。

DESCRIPTION (provided by applicant): Advances in instrumentation often lead to conceptual breakthroughs. For instance, laser-based optical tweezers led to experiments on the physical forces involved in stretching and binding of biomolecules. The long-term goal of this exploratory program is to provide a new technology for trapping individual biomolecules in aqueous solution, the Anti-Brownian ELectrophoretic trap (ABEL trap). This device will allow trapping of objects 50-100 times smaller than the smallest objects typically trapped with optical tweezers, extending all the way to single proteins and nucleic acids in solution. The first aim will develop and extend the ABEL trapping concept to enable trapping of individual small, dim biomolecules in buffer, such as a single GFP, a single quantum dot biolabel, and a single molecule of the chaperonin GroEL labeled with only one fluorophore. This will involve reduction of latency by using hardware feedback and optimization of the microfluidic design and surface treatment. A second aim will develop analysis procedures that will allow extraction of the mobility and diffusion coefficient of the single trapped biomolecule as a function of time. The ABEL trap will enable extended study of single biomolecules in free solution-a capability which has not previously existed. The ABEL trap directly provides information on the transport properties of the trapped object, such as the occurrence of protein-protein interaction events. In future work, this capability will lead to study of aggregate formation as monomers are added one by one, and eventually the ABEL trap should shed light on the initial events that lead to aggregation and/or misfolding of prions or other proteins in diseases that arise from these abnormalities. This project will also introduce a new concept into single-biomolecule research: that feedback can be used to cause a single molecule (or small collection of molecules) to violate the Second Law, while preserving the Second Law on the macroscale. The Project Description details the physical, chemical, and biophysical experiments that are required to achieve the main aims; but once the instrumentation is availble to the biomedical community, others will think of experiments that cannot be imagined at the present. It is for this reason that this research represents a new and powerful potential advance for biomedical applications.