NEW TECHNOLOGIES FOR TIME-RESOLVED INVESTIGATIONS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 摘要： 目前，微流体技术正被应用于开发用于时间分辨低温电子显微镜（TRCEM）应用的微混合器。 TRCEM需要在毫秒或更短的时间内快速均匀地预混合微量的大分子。这项技术将应用于EM网格上的样品沉积和快速冷冻问题的工作也刚刚开始。 我们的目标是设计和测试一种基于微流体的设备，使TRCEM实验能够以毫秒级的时间分辨率常规进行，并且只需要研究人员经常使用的少量生物分子。 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博士通过P41赠款合作进行的。 微型混合器。 微加工技术是实现快速混合的一种很有前途的技术。 去年，我们介绍了我们在制造各种设计的混频器及其初步测试方面的初步进展。 对于装置原型和流体混合实验，聚二甲基硅氧烷（PDMS）材料与快速模制和铸造复制技术一起用于制备装置。 经过密集的计算和实验测试，使用荧光分子和光学显微镜，我们已经达到了一个基本的设计，是与TRCEM的动力学和体积要求相兼容的混合器。 最终的配置包括两个T形预混器，其进料到单个通道中，该通道包含横截面为蝴蝶形的2-6个柱的阵列。 该装置通常具有小于1微升的总体积，在200-360微升/秒的流速下最佳地执行，并且允许在小于1毫秒内完全混合。 这些结果已在两份会议摘要中呈现，并已提交完整手稿以供出版。 微型混合器/微型喷雾器组合成一个“单片”装置。 在将优化的微混合器设计与气动微喷雾器设计（见上文）相结合方面已经取得了实质性进展。 五个器件已经由硅制成并粘合到玻璃盖上。 其中一个装置的照片（已被最广泛表征的装置）如下所示（左图显示从顶部和底部观察的整个装置的照片;右图显示通过扫描电子显微镜获得的装置混合器和喷雾器区域的放大视图）。 如目前实施的，两个溶液入口连接到将反应物递送到装置的双注射器泵。 该装置具有简单的喷嘴，该喷嘴由被两个空气通道横向包围的单个液体通道组成。 已经制造了两种其它类型的喷雾器喷嘴，其中液体通道分叉成两个子通道。 已经确定了两种喷雾器设计的液滴尺寸分布，但它们没有显示出很大的差异。 所有测试的装置都产生相当大的液滴尺寸分布，并且平均液滴直径比我们认为最佳的（基于目标1中描述的宏观喷雾器的结果）大几倍（直径约20微米）。 然而，当使用含有适当亲水性碳膜的网格时，即使是大的微滴也会扩散以形成足够薄的膜，以允许在冷冻后成像。 收集了完整细菌核糖体的Cryo-EM数据，并从显微照片中确定了初步的3D重建（23“分辨率）。 令人鼓舞的是，我们没有发现任何证据表明整体混合器/喷雾器对核糖体造成任何结构损伤。 此外，我们发现，当一种酶，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，我们正在进行实验以表征核糖体亚基的缔合以形成完整的核糖体;这里的目标是基于生物化学实验的结果来寻找预期发生的核糖体组装中的中间体（例如Hennelly等人（2006）J. Mol. Chem. 346：1243）。 在接下来的几个月里，我们预计将完成这些初步的研究核糖体亚基组装，并建立在TRCEM的微型设备的效用。 此外，将继续努力优化装置的设计，特别是微型喷雾器组件。

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.