MRI: Development of a Microscope with Simultaneous Electrical and Optical Measurement of Single Molecules

用于观察单分子的光学技术获得了2014年诺贝尔化学奖，因为它们正在彻底改变生命科学的研究。 在世界各地的实验室中，使用荧光来“看到”正在发生化学变化的单个分子，有助于揭示蛋白质等复杂分子的真正工作方式，或者这些蛋白质何时因基因突变或药物而失效。 最近，加州大学欧文分校的研究人员发明了一种新的全电子方法来“倾听”单个分子。 这种电子技术并不观察来自蛋白质的荧光，而是对蛋白质表面电荷的移动很敏感。 这个MRI项目的目标是开发一种新型的显微镜，可以将这种新的电子监听技术联合收割机结合到更传统的单分子荧光显微镜平台中。 具体来说，该项目将拆开荧光显微镜，修改显微镜以与电子技术兼容，并建立一个单一的集成仪器，可以在单个蛋白质工作时同时观察和倾听它们。 例如，一些蛋白质需要一部分弯曲，然后另一部分才能变得具有化学活性。 当药物结合并阻止特定运动时，其他蛋白质可以被禁用。 能够监测同一种蛋白质的两个不同部分将是一种令人兴奋的新能力，有助于理解对健康生命至关重要的生化过程中的因果关系。 该项目开发的仪器将增加位于加州大学欧文分校的两个主要研究中心的能力，并作为未来商业化工作的原理证明。 此外，该仪器还将为本科生、研究生和博士后研究人员提供持续的跨学科指导和培训，他们中的任何一个都可以为显微镜行业的类似开发和商业化努力做出贡献。单分子荧光技术正在揭示蛋白质、肽和生物聚合物之间复杂的结构-功能关系。 对机械组织的直接观察和单分子动力学的实时记录有助于揭示复杂的生化过程如何工作以及它们如何受到突变和辅助因子的影响。 为了补充基于光的技术，加州大学欧文分校的研究人员最近利用纳米级场效应晶体管的灵敏度，创造了一种新的单分子技术，该技术对蛋白质表面上电荷的移动敏感。 这些运动产生的电子信号代表了一种独立于传统单分子荧光的物理机制，这表明两个自由度的组合可以用于探测单个分子。 这个MRI项目的目标是展示一种验证这一前提的显微镜。 通过将纳米晶体管电子技术与全内反射荧光成像相结合，该项目旨在实现利用两种同时监测模式的新型单分子科学。 例如，同步监测可以区分复杂过程之间的因果关系，这些复杂过程涉及能量转移、底物结合、键断裂和结构域运动，每个过程发生在一个蛋白质内的不同位点。

Optical techniques for watching single molecules received the 2014 Nobel Prize in Chemistry because of the way they are revolutionizing research in the life sciences. In laboratories around the world, the use of fluorescence to "see" individual molecules undergoing chemical changes is helping to reveal how complex molecules like proteins really work or when these proteins become disabled by genetic mutations or pharmaceutical drugs. Recently, researchers at UC Irvine have invented a new, all-electronic method for "listening" to single molecules. Instead of looking at fluorescent light coming from a protein, the electronic technique is sensitive to the movement of charges on a protein's surface. The goal of this MRI project is to develop a new type of microscope that can combine this new, electronic listening technique into the more conventional platform of single-molecule fluorescence microscopy. Specifically, the project will take apart a fluorescence microscope, modify the microscope to be compatible with the electronic technique, and build up a single, integrated instrument that can simultaneously watch and listen to single proteins as they do their work. For example, some proteins require one portion to bend before another portion can become chemically active. Other proteins can be disabled when a drug binds and blocks a particular motion. Being able to monitor two different portions of the same protein will be an exciting new capability for understanding cause and effect in the biochemical processes that are fundamental to healthy lives. The instrument developed in this project will add to the capabilities of two major research centers located at UC Irvine and serve as a proof-of-principle for future commercialization efforts. In addition, the instrument will contribute to the ongoing, interdisciplinary mentoring and training of undergraduates, graduates, and postdoctoral researchers, any of whom could contribute to similar development and commercialization efforts in the microscope industry.Single-molecule fluorescence techniques are exposing the complex structure-function relationships among proteins, peptides, and biopolymers. Direct observation of mechanical organization and real-time recordings of single-molecule kinetics are helping reveal how complex biochemical processes work and how they are affected by mutations and co-factors. To complement light-based techniques, researchers at UC Irvine have recently exploited the sensitivity of nanometer-scale field effect transistors to create a new single-molecule technology that is sensitive to the movement of charges on a protein surface. The electronic signals generated by these movements represent a physical mechanism that is independent of conventional single-molecule fluorescence, suggesting that a promising combination of two degrees of freedom can be used to probe individual molecules. The goal of this MRI project is to demonstrate a microscope that validates this premise. By combining the nano-transistor electronic technique with total internal reflection fluorescence imaging, the project aims to enable new types of single-molecule science that take advantage of two modes of simultaneous monitoring. For example, synchronized monitoring may distinguish between cause and effect among complex processes involving energy transfer, substrate binding, bond cleavage, and domain motions, each occurring at different sites within one protein.