IDBR: High throughput single molecule AFM force-spectrometer

哥伦比亚大学获得了开发第一台高通量单分子AFM力谱仪的奖励。 原子力显微镜（AFM）的单分子力谱被广泛用于测量蛋白质在力作用下的动力学，在生物学中很常见。越来越多的生物学家正在利用从力谱测量中获得的详细信息。然而，目前的力谱仪面临着巨大的限制，因为它们的吞吐量非常低。该提案的目的是克服AFM光谱仪的局限性，并旨在构建一种新的仪器，其中吞吐量增加至少两个数量级。随着新仪器的构建，将开发分子共价连接的新型技术，这将允许对受力的单个蛋白质分子进行长期记录（数小时至数天）。 为了分析预期的大量数据，将采用基于扩展卡尔曼滤波器的新分析方法。通过增加至少100倍的AFM力谱的吞吐量，新的能力将被广泛的对蛋白质动力学，蛋白质力学和其他学科感兴趣的生物学家社区所获得。目前，越来越多的人认为蛋白质的机械扰动在体内是常见的。然而，即使是设想实验来发现影响蛋白质机械性质的小分子，如机械稳定性，也是极具挑战性的。这类化合物对于理解机械力在生理学和疾病中的作用非常有用，甚至可能找到治疗应用。能够在一个工作日内测试数百个小分子将允许识别这些小分子。这个跨学科的建议将用于培养本科生，研究生和博士后学生在仪器设计和单分子记录的艺术。本科生和研究生都将积极参与高通量力谱仪的设计和建造。特别是，本科生总是发现跨学科研究非常有吸引力，并利用暑期研究计划从事跨学科项目，如本提案中所述。一名研究生和一名博士后将全职从事该提案的不同方面。该提案包括为博士后研究员提供指导计划，这将有助于他向独立职位的过渡，增加他成为研究界成功成员的机会。传播结果对提案的成功至关重要。该提案的最终目标是通过与公司的伙伴关系，结合会议和研讨会上的演示，使新仪器可用。通过生物基础设施司生物研究仪器开发计划资助。

An award has been made to Columbia University to develop the first high throughput single molecule AFM force-spectrometer. Single-molecule force spectroscopy by atomic force microscopy (AFM) is widely used to measure the dynamic of proteins placed under force, of common occurrence in Biology. A growing number of biologists are taking advantage of the detailed information that emerges from force-spectroscopy measurements. However, current force-spectrometers face huge limitations since they are remarkably low-throughput. The aims of the proposal are designed to overcome the limitations of AFM spectrometers and aims to construct a new instrument where throughput is increased by at least two orders of magnitude. Together with the construction of the new instrument, novel technologies for the covalent attachment of the molecules will be developed which will allow for long-term recordings (hours to days) from a single protein molecule placed under force. In order to analyze the large volume of data anticipated, new methods of analysis based on Extended Kalman Filters will be implemented. By increasing at least by a factor of 100 the throughput of force-spectroscopy by AFM, new capabilities will be accessible to a wide community of Biologists interested in protein dynamics, protein mechanics and other disciplines. Currently, there is a growing consensus that mechanical perturbations of proteins are commonplace in vivo. However, it is extremely challenging even to conceive experiments to find small molecules that affect the mechanical properties of a protein, such as mechanical stability. Such compounds would be extraordinarily useful to understand the role of mechanical forces in physiology and disease, and they may even find therapeutic application. Being able to test hundreds of small molecules in a working day will allow the identification of such small molecules. This interdisciplinary proposal will be used to train undergraduate, graduate and postdoctoral students in the art of instrument design, and single molecule recordings. Both undergraduate and graduate students will be actively involved in the design and construction of the high-throughput force spectrometer. In particular, undergraduate students always find interdisciplinary research very attractive and make use of summer research programs to engage in interdisciplinary projects, such as the one described in this proposal. A graduate student and a postdoctoral fellow will work full time in different aspects of the proposal. The proposal includes a mentoring plan for the postdoctoral fellow that will ease his transition to an independent position, increasing his chances to become a successful member of the research community. The dissemination of the results is crucial to the success of the proposal. The ultimate goal of the proposal is to make the new instrumentation available through partnerships with companies, in combination with presentations in conferences and workshops.Funded through the Instrument Development for Biological Research program in the Division of Biological Infrastructure.