Li Liang, Mustafi Debarshi, Fu Qiang, Tereshko Valentina, Chen Delai L, Tice Joshua D, Ismagilov Rustem F
Department of Chemistry and Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2006 Dec 19;103(51):19243-8. doi: 10.1073/pnas.0607502103. Epub 2006 Dec 11.
High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a "hybrid" droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as approximately 140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in approximately 10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 microl of protein solution, approximately 1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.
高通量筛选和优化实验对于包括化学、结构生物学和分子生物学在内的许多领域至关重要。这两个步骤的分离可能会引入假阴性结果，并在初始筛选和后续优化之间造成时间延迟。尽管将这两个步骤结合起来的混合方法可能解决这些问题，但需要小型化以尽量减少样品消耗。本文报道了一种基于液滴的“混合”微流控方法，该方法将筛选和优化步骤整合到一个简单的实验中，并使用纳升大小的塞子来尽量减少样品消耗。许多不同的试剂以大约140纳升的塞子形式依次引入微流控装置中，并与一种底物和一种稀释缓冲液混合。在含有不同浓度试剂的大约10纳升塞子中进行测试。开发了形成可控浓度、索引浓度塞子的方法，并能廉价且无蒸发地孵育数千个塞子。为了验证这种混合方法并证明其对具有挑战性问题的适用性，展示了模型膜蛋白的结晶以及去污剂和粘性沉淀剂溶液的处理。通过使用10微升蛋白质溶液，一名研究人员在20分钟内设置了大约1300次结晶试验。通过获得高分辨率衍射图像并解析晶体结构证明，该方法与膜蛋白高质量晶体的生长、操作和提取兼容。这种稳健的方法需要廉价设备和耗材，应该特别适合在单个实验室中使用，并且可以在许多需要化学、生物化学和生物学筛选及优化的领域找到应用。
