Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
J Am Chem Soc. 2010 Jan 13;132(1):106-11. doi: 10.1021/ja908555n.
This paper describes a microfluidic approach to perform multiplexed nanoliter-scale experiments by combining a sample with multiple different reagents, each at multiple mixing ratios. This approach employs a user-loaded, equipment-free SlipChip. The mixing ratios, characterized by diluting a fluorescent dye, could be controlled by the volume of each of the combined wells. The SlipChip design was validated on an approximately 12 nL scale by screening the conditions for crystallization of glutaryl-CoA dehydrogenase from Burkholderia pseudomallei against 48 different reagents; each reagent was tested at 11 different mixing ratios, for a total of 528 crystallization trials. The total consumption of the protein sample was approximately 10 microL. Conditions for crystallization were successfully identified. The crystallization experiments were successfully scaled up in well plates using the conditions identified in the SlipChip. Crystals were characterized by X-ray diffraction and provided a protein structure in a different space group and at a higher resolution than the structure obtained by conventional methods. In this work, this user-loaded SlipChip has been shown to reliably handle fluids of diverse physicochemical properties, such as viscosities and surface tensions. Quantitative measurements of fluorescent intensities and high-resolution imaging were straighforward to perform in these glass SlipChips. Surface chemistry was controlled using fluorinated lubricating fluid, analogous to the fluorinated carrier fluid used in plug-based crystallization. Thus, we expect this approach to be valuable in a number of areas beyond protein crystallization, especially those areas where droplet-based microfluidic systems have demonstrated successes, including measurements of enzyme kinetics and blood coagulation, cell-based assays, and chemical reactions.
本文描述了一种通过将样品与多种不同的试剂组合并以多种混合比例进行多重纳升级实验的微流控方法。该方法采用用户加载、无需设备的 SlipChip。通过稀释荧光染料来控制混合比例，可以通过组合井的每个井的体积来控制。通过在大约 12 nL 的规模上对 SlipChip 设计进行验证，筛选出 Burkholderia pseudomallei 中的戊二酰辅酶 A 脱氢酶结晶的条件，针对 48 种不同的试剂进行测试；每种试剂在 11 种不同的混合比例下进行测试，总共进行了 528 次结晶试验。蛋白质样品的总消耗量约为 10 微升。成功确定了结晶条件。使用在 SlipChip 中确定的条件在微孔板中成功放大了结晶实验。通过 X 射线衍射对晶体进行了表征，得到了一个与传统方法获得的结构不同的空间群和更高分辨率的蛋白质结构。在这项工作中，已经证明这种用户加载的 SlipChip 能够可靠地处理具有不同物理化学性质的流体，例如粘度和表面张力。在这些玻璃 SlipChips 中，可以轻松进行荧光强度的定量测量和高分辨率成像。表面化学通过使用类似于基于插件的结晶中使用的氟化载流液的氟化润滑剂来控制。因此，我们预计这种方法将在除蛋白质结晶以外的许多领域具有价值，特别是在那些已经证明基于液滴的微流控系统取得成功的领域，包括酶动力学和血液凝固、基于细胞的测定和化学反应的测量。
