User-loaded SlipChip for equipment-free multiplexed nanoliter-scale experiments.
User-loaded SlipChip for equipment-free multiplexed nanoliter-scale experiments.
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DOI：
10.1021/ja908555n
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发表时间：
2010-01-13
影响因子：
15
通讯作者：
Ismagilov, Rustem F.
中科院分区：
文献类型：
--
作者：
Li, Liang;Du, Wenbin;Ismagilov, Rustem F.
本文描述了一种微流体方法，通过将样品与多种不同的试剂（每种试剂以多种混合比例）相结合来执行多重纳升规模的实验。该方法采用用户加载、无需设备的滑动芯片。以稀释荧光染料为特征的混合比可以通过每个组合孔的体积来控制。通过针对 48 种不同试剂筛选来自拟鼻疽伯克霍尔德氏菌的戊二酰辅酶 A 脱氢酶的结晶条件，对滑动芯片设计进行了约 12 nL 规模的验证；每种试剂均以 11 种不同的混合比例进行测试，总共进行了 528 次结晶试验。蛋白质样品的总消耗量约为 10 μL。成功确定了结晶条件。使用滑动芯片中确定的条件，在孔板中成功地扩大了结晶实验的规模。通过 X 射线衍射对晶体进行了表征，并以比传统方法获得的结构更高的分辨率提供了不同空间群的蛋白质结构。在这项工作中，这种用户加载的滑动芯片已被证明可以可靠地处理具有不同物理化学特性（例如粘度和表面张力）的流体。在这些玻璃滑动芯片中可以直接进行荧光强度的定量测量和高分辨率成像。使用氟化润滑液控制表面化学，类似于基于塞的结晶中使用的氟化载液。因此，我们预计这种方法在蛋白质结晶以外的许多领域都很有价值，特别是那些基于液滴的微流体系统已取得成功的领域，包括酶动力学和血液凝固的测量、基于细胞的测定和化学反应。
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 ~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 ~ 10 μL. 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 handle reliably 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.
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