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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins
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Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

机译：纳升微流体混合方法用于同时筛选和优化，并通过膜蛋白结晶验证

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摘要

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 approximate to 140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in approximate to 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 mu l of protein solution, approximate to 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.
机译：高通量筛选和优化实验对于许多领域至关重要，包括化学，结构和分子生物学。这两个步骤的分离可能会引入假阴性，并在初始筛选和后续优化之间产生时间延迟。尽管结合了两个步骤的混合方法可以解决这些问题，但仍需要进行小型化以最大程度地减少样品消耗。本文报道了一种基于“混合”液滴的微流控方法，该方法将筛选和优化步骤结合到一个简单的实验中，并使用纳升大小的塞子来最大程度地减少样品消耗。依次将许多不同的试剂（约140 nl塞子）引入微流控设备中，并与底物和稀释缓冲液合并。在含有不同浓度试剂的大约10-nl塞子中进行测试。开发了形成浓度可控，指数浓度的塞子的方法，并廉价且无蒸发地孵育了数千个塞子。为了验证该混合方法并证明其对挑战性问题的适用性，证明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，一位研究人员在20分钟内进行了大约1300次结晶试验。通过获得高分辨率的衍射图并解决晶体结构，该方法与膜蛋白高质量晶体的生长，处理和提取兼容。这种稳健的方法需要廉价的设备和耗材，应该特别适合在单个实验室中使用，并且可以在需要化学，生化和生物筛选和优化的许多领域中找到应用。

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