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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 抽象的： 目前，正在应用微流体技术来开发用于时间分辨率的低温电子显微镜（TRCEM）应用的微弹器。 TRCEM需要在毫秒或更短的时间尺度上快速且均匀的大量大分子进行均匀的预混合。工作也只是开始，将该技术应用于标本沉积问题上的问题和快速冻结。我们对该赠款的其余部分的目标是设计和测试基于微流体的设备，该设备允许TRCEM实验以毫秒的时间分辨率进行定期进行，并且仅需要研究人员通常可以使用的少量生物分子。 Nano设备设计和制造是通过P41 Grant的分包合同与Rensselaer理工学院的Toh Ming Lu博士和Zonghuan Lu博士合作进行的。 微型物。 微分解是实现快速混合目标的承诺技术。去年，我们描述了我们在各种设计的混合器结构及其初始测试中的最初进展。对于设备原型制作和流体混合实验，将聚二甲基硅氧烷（PDMS）材料与快速成型和铸造复制技术一起使用，以准备设备。 在使用荧光分子和光学显微镜进行密集的计算和实验测试之后，我们达到了与TRCEM的动力学和体积需求兼容的混合器的基本设计。最终配置包含两个T形预定器，它们将馈入一个单个通道，其中包含2-6个柱子的阵列，这些柱子在横截面中具有蝴蝶形状。这些设备的总体积通常少于一个微柱，以200-360微升/秒的流速进行最佳性能，并允许以不到一毫秒的速度进行完整的混合。这些结果已在两个会议摘要中提出，并且已经提交了全部手稿供出版。 Micromixer/Microsprayer合并成一个“单片”设备。在将优化的微米物设计与气动微喷雾剂设计相结合（见上文）方面取得了重大进展。五个设备已从硅制造并粘合到玻璃盖上。下面显示了其中一种设备的照片，即最广泛表征的设备（左图显示了从顶部和底部查看的整个设备的照片；右图显示通过扫描设备的搅拌器和喷雾器区域的电子显微镜获得的放大视图）。 如当前实施，两个解决方案入口连接到将反应物传递到设备的双注射器泵。该设备具有简单的喷嘴，该喷嘴由两个空气通道横向周围的单个液体通道组成。已经制造了另外两种喷雾器喷嘴，其中液态通道分叉成两个亚渠道。已经确定了两个喷雾器设计的液滴尺寸分布，但它们没有显示较大的差异。所有经过测试的设备产生的液滴大小的分布相当大，平均液滴直径比我们认为是最佳的几倍（直径约为20微米）（基于AIM 1中描述的宏刺激器的结果）。然而，当使用含有合适的亲水性碳膜的网格时，即使大型微弹头也会散布以形成足够薄的膜以允许在冷冻后进行成像。已经收集了针对完整细菌核糖体和从显微照片确定的初步3D重建（23€分辨率）的冷冻EM数据。令人鼓舞的是，我们没有发现整体混合器/喷雾器会对核糖体造成任何结构性损害。同样，当酶（苹果酸脱氢酶）通过设备时，我们发现活性损失没有损失。 在撰写本报告时，我们正在进行实验，以表征核糖体亚基的关联以形成完整的核糖体。这里的目的是在核糖体组装中寻找中间体，这些中间体有望根据生物化学实验的结果（例如Hennelly等人（2006）J。Mol。Biol。346：1243）。在接下来的几个月中，我们期望完成这些关于核糖体亚基组装的最初研究，并在TRCEM中建立微发行版的实用性。此外，尤其是微喷雾器组件的设备设计将继续优化设备的设计。