A radial calibration window for analytical ultracentrifugation.
A radial calibration window for analytical ultracentrifugation.
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DOI：
10.1371/journal.pone.0201529
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Zhao H
中科院分区：
文献类型：
--
作者：
LeBrun T;Schuck P;Wei R;Yoon JS;Dong X;Morgan NY;Fagan J;Zhao H
分析超离心（AUC）是一种基于第一性原理的方法，用于通过在高离心场存在下监测其径向浓度分布随时间的演变来研究溶液中的大分子和颗粒。在沉降速度实验中，可以在多分散样品上以高流体动力学分辨率测量与颗粒的大小、形状、密度和溶剂化有关的流体动力学性质。在最近的一项多实验室基准研究中，包括来自67个实验室的商业分析超临界仪器的数据，发现径向尺寸的校准准确度是限制AUC准确度的主要因素之一。在目前的工作中，我们开发了一个工件，包括一个精确校准的反射图案光刻沉积到一个AUC窗口。当在AUC对照实验中扫描时，其用作十字线，用于径向放大率的绝对校准。在使用不同的光学系统扫描的地标之间的间距的分析后，我们估计，在径向放大率与径向尺度伪影的外部校准后的剩余不确定性是10.2%，外部校准后的其他重要的贡献，如温度和时间的不确定性类似的幅度。之前的多实验室研究发现，许多仪器的径向测量误差为1%至2%，少数仪器的误差超过15%，这意味着使用这里开发的人工制品可以将误差减少5至10倍或更多。因此，采用外部径向校准是确保分子流体力学和AUC粒度测量相关研究准确性的重要因素。
Analytical ultracentrifugation (AUC) is a first-principles based method for studying macromolecules and particles in solution by monitoring the evolution of their radial concentration distribution as a function of time in the presence of a high centrifugal field. In sedimentation velocity experiments, hydrodynamic properties relating to size, shape, density, and solvation of particles can be measured, at a high hydrodynamic resolution, on polydisperse samples. In a recent multilaboratory benchmark study including data from commercial analytical ultracentrifuges in 67 laboratories, the calibration accuracy of the radial dimension was found to be one of the dominant factors limiting the accuracy of AUC. In the present work, we develop an artifact consisting of an accurately calibrated reflective pattern lithographically deposited onto an AUC window. It serves as a reticle when scanned in AUC control experiments for absolute calibration of radial magnification. After analysis of the pitch between landmarks in scans using different optical systems, we estimate that the residual uncertainty in radial magnification after external calibration with the radial scale artifact is ≈0.2 %, of similar magnitude to other important contributions after external calibration such as the uncertainty in temperature and time. The previous multilaboratory study had found many instruments with errors in radial measurements of 1 % to 2 %, and a few instruments with errors in excess of 15 %, meaning that the use of the artifact developed here could reduce errors by 5-to 10-fold or more. Adoption of external radial calibration is thus an important factor for assuring accuracy in studies related to molecular hydrodynamics and particle size measurements by AUC.
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