Electrophoresis of Filamentous Viruses Through Solid State Nanopores

丝状病毒通过固态纳米孔的电泳

• 批准号: 1505878
• 负责人: Jay Tang
• 金额: $ 33万
• 依托单位: Brown University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1505878&HistoricalAwards=false
• 关键词: Electrophoresis; Filamentous; Viruses; Through; Solid

Non-technical: This award by the Biomaterials program in the Division of Materials Research to Brown University is to study how filamentous viruses pass through extremely tiny pores. The pores are drilled through a thin layer of silicon nitride, which is a solid-state material and why they are referred to as solid-state nanopores. The viruses being used for this study belong to a class of bacterial phage and are isolated from infected E. coli. The viruses are skinny filaments of about one micrometer in length and only a few nanometers in diameter, and each one is identical in size because it consists of a single DNA chain coated by about three thousand copies of a small coat protein. Because the coat protein carries a few charges, the virus filament is charged along its length. When a solution containing the virus fills one side of a device containing a single nanopore, viruses can be driven through the nanopore, one by one, by applying a voltage across the device. When a virus passes through the nanopore, it blocks a characteristic amount of current for a brief but measurable duration. The goal of this project is to use the nanopore as a tool to study in microscopic detail how stiff charged molecules move through nanopores. This study essentially consists of comprehensive sets of measurements under carefully controlled conditions and with a number of different parameters. How the amount of current blocked and the speed of translocation change with parameters such as the amount of salt added to the solution, the size of the nanopore, and the voltage applied can tell us a great deal about the physical processes at play, all the way down to molecular scale. In spite of the conceptual simplicity of the study, the knowledge acquired can have profound technological implications. It may, for instance, lead to tiny devices for efficiently detecting and sorting various virus strains. The program will also provide multidisciplinary training of graduate and undergraduate students. The PIs and the graduate students have also planned, through the Brown Science Prep program, to deliver guest lectures, perform demonstrations, and host lab tours, all in order to broaden the impact of laboratory research on the surrounding area K-12 students and the general public. Technical: In this project, filamentous viruses will be used to study the fundamental physics of driven electrophoresis through nanoscale pores. The physical properties of filamentous viruses, including their charge density, their stiffness, and their length, are both precisely known and easily tunable. That control enables us to systematically test theoretical models of electrophoresis inside a nanopore at the single-molecule level. The planned study includes measurements of the capture process, the amount of current blocked, the translocation speed, and the spread of individual translocation times. With this study, the effects of filament stiffness on translocation speed under a range of driving voltages will be studied. In addition, factors that account for the dispersion in translocation speed will also be determined. The experimental results will be compared with theoretical calculations based on the coupled Navier-Stokes and Poisson Boltzmann equations, and also compared with computer simulations driven by Langevin dynamics. By addressing every facet of nanopore translocations, including the capture process, the electrokinetic pulling force, and the viscous drag that opposes it, the planned experiments will provide quantitative tests of the established theoretical models. Thus, students working on the program will acquire comprehensive training in the cutting-edge, nano-technology field. The planned outreach efforts, coordinated by the Brown Science Prep program, focus on connecting physics knowledge with device technology, via guest lectures, lab tours, and demos both live and through online videos.

非技术性：布朗大学材料研究部的生物材料项目的这个奖项是为了研究丝状病毒如何通过极其微小的孔隙。这些孔是通过氮化硅薄层钻出来的，氮化硅是一种固态材料，也是为什么它们被称为固态纳米孔的原因。用于本研究的病毒属于一类细菌噬菌体，从感染的大肠杆菌中分离得到。杆菌这些病毒是细长的丝状体，长度约为1微米，直径仅为几纳米，每一种病毒的大小都是相同的，因为它由一条DNA链组成，外面包裹着大约3000个小的外壳蛋白。由于外壳蛋白携带少量电荷，病毒丝沿着其长度方向带电荷。当含有病毒的溶液填充含有单个纳米孔的装置的一侧时，通过在装置上施加电压，可以一个接一个地驱动病毒通过纳米孔。当病毒通过纳米孔时，它会在短暂但可测量的时间内阻断特征量的电流。该项目的目标是使用纳米孔作为工具，在微观细节上研究刚性带电分子如何通过纳米孔移动。这项研究基本上包括在精心控制的条件下，用一些不同的参数进行的全面的测量。阻断的电流量和移位的速度如何随着添加到溶液中的盐的量、纳米孔的大小和施加的电压等参数而变化，可以告诉我们很多关于物理过程的信息，一直到分子尺度。尽管这项研究的概念简单，但所获得的知识可能具有深远的技术影响。例如，它可能会导致微型设备用于有效检测和分类各种病毒株。该计划还将为研究生和本科生提供多学科培训。PI和研究生还计划通过布朗科学准备计划，提供客座讲座，进行演示，并举办实验室图尔斯之旅，所有这些都是为了扩大实验室研究对周边地区K-12学生和公众的影响。技术：在这个项目中，丝状病毒将用于研究通过纳米级孔驱动电泳的基本物理学。丝状病毒的物理性质，包括它们的电荷密度、刚度和长度，都是精确已知的，而且很容易调节。这种控制使我们能够在单分子水平上系统地测试纳米孔内电泳的理论模型。计划中的研究包括测量捕获过程、阻断的电流量、易位速度和个体易位时间的分布。通过本研究，将研究在一定范围的驱动电压下，灯丝刚度对移位速度的影响。此外，还将确定导致易位速度分散的因素。实验结果将与基于耦合Navier-Stokes和Poisson Boltzmann方程的理论计算进行比较，并与Langevin动力学驱动的计算机模拟进行比较。通过解决纳米孔易位的每个方面，包括捕获过程，电动拉力和与之相反的粘性阻力，计划的实验将提供对已建立的理论模型的定量测试。因此，参与该项目的学生将获得尖端纳米技术领域的全面培训。由布朗科学预备计划协调的计划外展工作，重点是通过客座讲座，实验室图尔斯参观和现场演示以及在线视频将物理知识与设备技术联系起来。

项目成果

Osmotically Driven and Detected DNA Translocations

• DOI: 10.1038/s41598-019-51049-4
• 发表时间: 2019-10
• 期刊: Scientific Reports
• 影响因子: 4.6
• 作者: A. McMullen;George Araujo;M. Winter;D. Stein