NEW TECHNOLOGIES FOR TIME-RESOLVED INVESTIGATIONS

用于时间分辨调查的新技术

• 批准号: 7954591
• 负责人: TERENCE C WAGENKNECHT
• 金额: $ 1.12万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954591
• 关键词: Air; Biochemistry; Biological; Butterflies; Caliber; Carbon; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; Data; Deposition; Device Designs; Devices; Enzymes; Film; Freezing; Funding; Glass; Goals; Grant; Image; Imagery; Institutes; Institution; Investigation; Kinetics; Left; Liquid substance; Malate Dehydrogenase; Manuscripts; Microfabrication; Microfluidics; Molds; Publications; Pump; Reporting; Research; Research Personnel; Resolution; Resources; Ribosomes; Scanning Electron Microscopy; Shapes; Silicon; Solutions; Source; Specimen; Syringes; Techniques; Technology; Testing; Time; United States National Institutes of Health; Work; Writing; abstracting; base; design; feeding; light microscopy; macromolecule; meeting abstracts; millisecond; nanodevice; new technology; polydimethylsiloxane; reconstruction; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Currently, microfluidics technology is being applied to develop micromixers for time resolved cryo-electron microscopy (TRCEM) application. TRCEM requires fast and homogeneous premixing of tiny amounts of macromolecules in the time scale of milliseconds or less. Work is also just beginning in which this technology will be applied to the problems of specimen deposition onto EM grids and rapid freezing. Our goal for the remainder of this grant is to design and test a microfluidics-based device that allows TRCEM experiments to be conducted routinely with millisecond time resolution and requires only the small amounts of biomolecules that are often available to researchers. The nano device design and fabrication is conducted in collaboration with Dr. Toh Ming Lu and Dr. Zonghuan Lu at Rensselaer Polytechnic Institute via subcontract from the P41 grant. Micromixers. Microfabrication is a promising technique to achieve the objective of rapid mixing. Last year we described our initial progress in the fabrication of mixers of various designs and their initial testing. For device prototyping and fluid mixing experiments, polydimethylsiloxane (PDMS) material was used with a fast molding and casting replication technique to prepare devices. After intensive computational and experimental testing using fluorescent molecules and light microscopy we have arrived at a basic design for mixers that is compatible with the kinetic and volume requirements of TRCEM. The final configurations comprise two T-shaped premixers that feed into a single channel containing an array of 2-6 pillars that have a butterfly shape in cross-section. The devices typically have a total volume of less than one microliter, perform optimally at flow rates of 200-360 microliters/sec, and allow complete mixing in less than one millisecond. These results have been presented in two meeting abstracts and a full manuscript has been submitted for publication. Micromixer/microsprayer combined into one "monolithic" device. Substantial progress has been achieved in integrating the opitimized micromixer designs with pneumatic micro-sprayer designs (see above). Five devices have been fabricated from silicon and bonded to a glass cover. Photographs of one of the devices, the one that has been most extensively characterized, are shown below (left panels show photographs of the entire device as viewed from the top and bottom; right panels show magnified views obtained by scanning electron microscopy of the mixer and sprayer regions of the device). As currently implemented, the two solution inlets are connected to a dual syringe pump that delivers reactants to the device. The device has a simple spray nozzle consisting of a single liquid channel surrounded laterally by two air channels. Two other types of sprayer nozzles have been fabricated in which the liquid channel bifurcates into two subchannels. Droplet size distributions have been determined for two of the sprayer designs, but they do not show large differences. All of the devices tested produce rather large distributions of droplet sizes and the average droplet diameter is severalfold larger (~ 20 microns in diameter) than we believe to be optimal (based upon results with the macrosprayer described in aim 1). Nevertheless, when grids containing suitably hydrophilic carbon films are used, even the large microdroplets spread to form a film that is sufficiently thin to allow imaging following freezing. Cryo-EM data has been collected for intact bacterial ribosomes and a preliminary 3D reconstruction (23 ¿ resolution) determined from the micrographs. Encouragingly, we find no evidence that the monolithic mixer/sprayer causes any structural damage to the ribosomes. Also, we find no loss in activity when an enzyme, malate dehydrogenase, is passed through the devices. At the time of writing this report, we are engaged in experiments to characterize the association of ribosomal subunits to form intact ribosomes; the objective here is to search for intermediates in ribosomal assembly that are expected to occur based upon results of biochemistry experiments (e.g. Hennelly et al. (2006) J. Mol. Biol. 346:1243). In the coming months we expect to complete these initial studies on ribosomal subunit assembly and to establish the utility of the microdevices in TRCEM. Also, efforts will continue to optimize the design of the devices, in particular, the microsprayer component.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 抽象的： 目前，微流体技术正在应用于开发用于时间分辨冷冻电子显微镜（TRCEM）应用的微混合器，该应用需要在毫秒或更短的时间尺度内快速均匀地预混合微量大分子。技术将应用于解决样品沉积到电磁网格上和快速冷冻的问题，我们的剩余资金目标是设计和测试基于微流体的设备，该设备允许。 TRCEM 实验通常以毫秒时间分辨率进行，仅需要研究人员通常可以获得的少量生物分子。纳米器件的设计和制造是与伦斯勒理工学院的 Toh Ming Lu 博士和 Zonghuan Lu 博士合作进行的。通过 P41 拨款的分包。 微混合器。 微加工是实现快速混合目标的一项有前途的技术，去年我们描述了我们在制造各种设计的混合器及其初步测试方面的初步进展，在设备原型设计和流体混合实验中，使用了聚二甲基硅氧烷（PDMS）材料。快速成型和铸造复制技术来制备设备。 经过使用荧光分子和光学显微镜的密集实验测试，我们得出了与 TRCEM 的动力学和体积要求兼容的混合器的基本设计，最终配置包括两个 T 形预混合器，它们馈入包含阵列的单个通道。该装置由 2-6 个横截面呈蝶形的柱子组成，总体积通常小于 1 微升，在 200-360 微升/秒的流速下性能最佳，并允许完全流动。这些结果已在两份会议摘要中呈现，完整的手稿已提交出版。 微混合器/微喷雾器组合成一个“整体”装置，在将优化的微混合器设计与气动微喷雾器设计集成方面取得了实质性进展（见上文），其中一个装置的照片是由硅制成的。具体来说，这些器件，即最具特征性的器件，如下所示（左图显示从顶部和底部观察的整个器件的照片；右图显示通过扫描电子获得的放大视图）设备的混合器和喷雾器区域的显微镜检查）。 目前，两个溶液入口连接到一个双注射泵，该泵将反应物输送到该装置，该装置具有一个简单的喷嘴，该喷嘴由横向被两个空气通道包围的单个液体通道组成。已确定两种喷雾器设计的液体通道分叉成两个子通道，但它们并没有表现出很大的差异。并且平均液滴直径比我们认为的最佳直径大几倍（直径约 20 微米）（基于目标 1 中描述的大型喷雾器的结果），然而，当使用包含适当亲水性碳膜的网格时，即使是大的微液滴。扩散形成足够薄的薄膜，以便在冷冻后进行成像，并收集完整的细菌核糖体和初步的 3D 重建（23 ¿令人鼓舞的是，我们没有发现任何证据表明整体式混合器/喷雾器会对核糖体造成任何结构损伤。此外，我们发现当酶（苹果酸脱氢酶）通过该装置时，活性没有损失。 在撰写本报告时，我们正在进行实验来表征核糖体亚基与形成完整核糖体的关联；这里的目标是寻找基于生物化学实验结果预计会发生的核糖体组装中间体（例如Hennelly）等人 (2006) J. Mol. 346:1243），我们预计将在接下来的几个月内完成这些关于核糖体亚基组装的初步研究。此外，还将继续努力优化设备的设计，特别是微型喷雾器组件。