Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering , National Institutes of Health , Bethesda , Maryland 20892 , United States.
Departments of Biophysics and Microbiology , UT Southwestern Medical Center , Dallas , Texas 75390 , United States.
Anal Chem. 2019 May 7;91(9):5866-5873. doi: 10.1021/acs.analchem.9b00202. Epub 2019 Apr 15.
The centerpiece of the sample cell assembly in analytical ultracentrifugation holds the sample solution between windows, sealed against high vacuum, and is shaped such that macromolecular migration in centrifugal fields exceeding 200 000g can proceed undisturbed by walls or convection while concentration profiles are imaged with optical detection systems aligned perpendicular to the plane of rotation. We have recently shown that 3D printing using various materials allows inexpensive and rapid manufacturing of centerpieces. In the present work, we expand this endeavor to examine the accuracy of the measured sedimentation process, as well as short-term durability of the centerpieces. We find that 3D-printed centerpieces can be used many times and can provide data equivalent in quality to commonly used commercial epoxy resin centerpieces. Furthermore, 3D printing enables novel designs adapted to particular experimental objectives because they offer unique opportunities, for example, to create well-defined curved surfaces, narrow channels, and embossed features. We present examples of centerpiece designs exploiting these capabilities for improved AUC experiments. This includes narrow sector centerpieces that substantially reduce the required sample volume while maintaining the standard optical path length; thin centerpieces with integrated window holders to provide very short optical pathlengths that reduce optical aberrations at high macromolecular concentrations; long-column centerpieces that increase the observable distance of macromolecular migration for higher-precision sedimentation coefficients; and three-sector centerpieces that allow doubling the number of samples in a single run while reducing the sample volumes. We find each of these designs allows unimpeded macromolecular sedimentation and can provide high-quality sedimentation data.
在分析超速离心的样品池组件中，中心部件将样品溶液夹在两个窗口之间，窗口与高真空密封，并采用这样的形状，使在超过 200,000g 的离心场中大分子的迁移能够在不受壁或对流干扰的情况下进行，同时浓度分布可以通过与旋转平面垂直的光学检测系统进行成像。我们最近表明，使用各种材料的 3D 打印可以实现中心部件的低成本和快速制造。在本工作中，我们扩展了这一努力，以检查测量的沉降过程的准确性，以及中心部件的短期耐用性。我们发现，3D 打印的中心部件可以多次使用，并且可以提供与常用商业环氧树脂中心部件质量相当的数据。此外，3D 打印可以实现适应特定实验目标的新型设计，因为它们提供了独特的机会，例如，创建明确定义的曲面、狭窄的通道和压花特征。我们展示了利用这些功能改进 AUC 实验的中心部件设计示例。这包括大幅减少所需样品量的窄扇形中心部件，同时保持标准的光程长度；带有集成窗口支架的薄型中心部件，可提供非常短的光程长度，从而减少高浓度大分子时的光学像差；长柱型中心部件，可增加大分子迁移的可观察距离，以获得更高精度的沉降系数；以及允许在单个运行中增加两倍样品数量同时减少样品体积的三扇形中心部件。我们发现，这些设计中的每一种都允许大分子不受阻碍地沉降，并可以提供高质量的沉降数据。
