IDBR: Nanodroplet reactor arrays and imaging system for biomolecular structure and kinetics

IDBR：纳米液滴反应器阵列和成像系统的生物分子结构和动力学技术说明。本项目的目的是开发一种能够在油中的水性纳米液滴阵列中进行高通量单分子灵敏荧光测量的仪器。 该仪器将使单分子轨迹的观察更准确，更高通量，更方便。它还将促进新型测量，以解决分子生物学和生物物理学中一些最基本但仍未得到解答的问题，计划开展以下活动：（1）建造一个集成射流的显微镜;（2）设计、制作原型和开发一个廉价的无源微流控装置，与单分子敏感成像兼容，用于制作纳米液滴阵列;（3）开发一个用于制造纳米液滴阵列的微流控装置;（4）开发一个用于制造纳米液滴阵列的微流控装置。（3）通过对核酸结构和结构动力学的研究，演示和验证综合仪器;（4）通过出版、合作和教育广泛传播;（5）对各级学生进行仪器、微流体和单分子敏感技术方面的培训;以及（6）通过东北研究生教育和教授联盟（NEAGEP）的意义进行外联和招聘。单荧光团和单粒子敏感测量现在通常用于更好地理解生物分子的结构转换和生物分子之间的相互作用。 这些测量所揭示的前所未有的细节正在促进对驱动生命系统的生物分子机制的新理解。 单分子观测的一个要求是，所研究的分子必须留在显微镜的检测区域内。 对于体外测量，这通常是通过将分子化学地束缚到异质玻璃表面或将它们限制在凝胶的孔中来实现的。 在这两种情况下，异质表面的存在经常干扰或灭活生物分子，并改变用于研究它们的染料的光学性质。这些问题构成了对体外单分子敏感测量的重大限制，被广泛承认但很少得到解决。液滴阵列为单分子测量和成像提供了表面附着或凝胶限制的令人信服的替代方案。 油中的纳米级水滴为受限分子提供了均匀的环境。大多数生物分子在这些液滴中功能良好。液滴的功能是纳米级的？试管？阵列中的每个液滴提供独特的环境，使得可以在许多不同的条件下同时观察分子。 因此，这项工作的智力价值是三方面的。(1)将开发一种新的体外单分子敏感观察方法，以简化和改善测量。 (2)在研究核酸的灵活性和结构转换中使用这种仪器将导致对这些分子如何起作用的物理理解的改进。(3)更简单、更快速的单分子测量平台将带来更多的研究;这些研究的结果将影响材料科学和药物发现等多个领域。除了上文（3）中提到的广泛的知识影响外，教育和外联活动也将产生教育和社会影响。作为这项工作的一部分，各级学生和许多背景将介绍跨学科研究和单分子敏感技术。计划通过新的实验室单位为本科生和研究生提供培训机会。该仪器将直接用于研究生培训。为了帮助打破与妇女和少数民族成员参与STEM（科学，技术，工程和数学）学科有关的社会障碍，将通过NEAGEP的外联活动促进从尽可能广泛的申请者中招募和留住最优秀的学生。

IDBR: Nanodroplet reactor arrays and imaging system for biomolecular structure and kineticsTechnical description. This objective of this project is to develop an instrument capable of high-throughput single-molecule sensitive fluorescence measurement in arrays of aqueous nanodroplets in oil. This instrument will make the observation of single-molecule trajectories more accurate, higher-throughput, and more convenient. It will also facilitate new types of measurements to address some of the most basic but still unanswered questions in molecular biology and biophysics.The following activities are planned: (1) Construction of a microscope with integrated fluidics; (2) The design, prototyping and development of an inexpensive, passive microfluidic device, compatible with single-molecule sensitive imaging, for making nanodroplet arrays; (3) Demonstration and validation of the integrated instrument through a study of nucleic acid structure and structural kinetics; (4) Widespread dissemination through publication, collaboration, and education; (5) Training of students at all levels on the instrument and in microfluidics and single-molecule sensitive techniques; and (6) Outreach and recruitment through the Northeast Alliance for Graduate Education and the Professoriate (NEAGEP)Significance. Single-fluorophore and single-particle sensitive measurements are now commonly used to better understand structural transformations of, and interactions between, biological molecules. The unprecedented detail revealed by these measurements is facilitating new understandings of the biomolecular mechanisms that drive living systems. One requirement of single-molecule observations is that a molecule under study must be made to stay in the detection region of a microscope. For in-vitro measurements, this is most often accomplished by chemically tethering the molecules to a heterogeneous glass surface, or confining them in the pores of a gel. In either case, the presence of heterogeneous surfaces frequently perturbs or inactivates biomolecules and modifies the optical properties of the dyes that are used to study them. These problems, which constitute a significant limitation to in-vitro single-molecule-sensitive measurement, are widely acknowledged but rarely addressed. Droplet arrays provide a compelling alternative to surface attachment or gel confinement for single-molecule measurement and imaging. Nanoscopic aqueous droplets in oil provide a homogeneous environment for the confined molecules. Most biomolecules function well inside these droplets. The droplets function as nanoscopic ?test-tubes,? with each droplet in an array providing a unique environment, so that molecules can be observed under many different conditions simultaneously. The intellectual merit of this work is therefore threefold. (1) A new approach for single-molecule sensitive observations in vitro will be developed that will simplify and improve measurements. (2) Use of this instrument in studies of the flexibility and structural transformations of nucleic acids will result in an improved physical understanding of how these molecules function. (3) Many additional studies will be engendered by a simpler, faster platform for single-molecule measurement; the outcome of these studies will impact fields as diverse as materials science and drug discovery.Broader Impact. In addition to the broad intellectual impact noted in (3) above, educational and societal impacts will result from education and outreach activities. As part of this work, students at all levels and with many backgrounds will be introduced to interdisciplinary research and single-molecule sensitive techniques. Training opportunities through new laboratory units are planned for students at the undergraduate and graduate level. The instrument will be used directly in graduate-level training. To help break societal barriers associated with the participation of women and minority members in the STEM (science, technology, engineering and mathematics) disciplines, recruitment and retention of the best students from the broadest possible pool of applicants will be facilitated by outreach activities through NEAGEP.