Droplet based screening of molecular libraries in a microfluidic device

描述（由申请人提供）：在第二阶段，我们将继续开发基于RainDance Technologies液滴的微流体仪器平台，基于第一阶段原理验证实验的结果，并开发一个商业系统，包括筛选液滴中化合物库的能力。我们的目标是在第二阶段结束时提供一个完整的仪器平台，能够使用重组酶和已建立的细胞系的测定来筛选由多达100，000个成员组成的化合物文库。最初的努力将集中在化合物管理和条形码液滴的处理上，额外的努力将充分实现微流体分析和一次性芯片设计，我们将在两年资助期结束前完成商业化的集成和工程开发。我们相信，基于液滴的开放平台的完整开发将对研究和制药界产生重大价值，特别是用于使用具有荧光体外蛋白质和细胞测定的高通量筛选（HTS）格式的液滴库。公共卫生相关性：生物学和化学实验室操作的小型化使反应体积从几毫升（试管中）减少到几微升（微量滴定板中）-减少了1000倍。通过测定小型化来增加通量的驱动对于药物筛选特别重要。今天，许多工业筛选程序每天处理100，000种化合物（每秒约1种），是1990年一周处理量的1000倍。尽管基于机器人微量滴定板的基础设施已经能够筛选超过一百万个小分子的集合，但主要筛选方法在很大程度上限于测定每种化合物的单一高浓度。进一步将测试体积减少到低于1536孔板的1-2 μ l容量将产生显著的成本节约并实现更高的通量。此外，例如，与单点筛选相比，包括每种化合物的剂量-反应曲线的初步筛选将显著减少假阳性和假阴性的数量，并且能够识别微妙的药理学，例如部分激动或拮抗作用。此外，还应该可以针对多个靶标同时筛选每种化合物以确定特异性。然而，使用微量滴定板技术用于进一步小型化是有问题的：例如，蒸发在微升体积中变得显著，毛细作用导致液体在威尔斯之间的“芯吸”和桥接，并且这不会减轻与机器人微量滴定板处理相关的时间和消耗品成本。该提案的关键技术是RainDance Technologies基于液滴的微流体平台。使用这个平台，我们相信我们可以通过将每个检测限制在单独的显微液滴中，进一步将每个检测的体积减少103至106倍，并将通量增加> 1，000倍。我们将利用该技术实现基于分子和细胞的分析的高通量筛选，显著降低用于筛选的成本、时间和试剂，并实现高通量原代细胞筛选的前景。

DESCRIPTION (provided by applicant): In Phase II we will continue the development of RainDance Technologies droplet-based microfluidic instrument platform, based on the results from the Phase I proof-of-principle experiments, and develop a commercial system that includes the ability to screen compound libraries in droplets. Our goal by the end of Phase II will be to provide a complete instrument platform able to screen a compound library composed of up to 100,000 members using assays for both a recombinant enzyme and an established cell line. Initial efforts will focus on compound management and handling of barcoded droplets, additional effort will fully enable microfluidic assays and disposable chip designs, and we will complete the integration and engineering development for commercialization before the end of the two year grant period. We believe that significant value to the research and pharmaceutical communities will result from complete development of a droplet-based open platform, specifically for the use of high throughput screening (HTS) formatted droplet libraries with fluorescent in vitro protein and cellular assays. PUBLIC HEALTH RELEVANCE: Miniaturization of laboratory operations in biology and chemistry has reduced reaction volumes from a few milliliters (in test-tubes) to a few microliters (in microtiter plates) - a 1000-fold reduction. The drive to increase throughput by assay miniaturization has been particularly important for drug screening. Today, many industrial screening programs process 100,000 compounds a day (~1 per second), a thousand times as many as were processed in an entire week in 1990. Although robotic microtiter plate based infrastructure has enabled the screening of collections exceeding one million small molecules, the primary screening methodology has largely been restricted to assaying a single high concentration of each compound. Further reducing test volumes below the 1-2 <l capacity of 1536-well plates would create both significant cost savings and enable higher throughput. Furthermore, a primary screen that included a dose-response curve for every compound, for example, would significantly reduce the number of false positives and false negatives compared to a single-point screen, and are able to identify subtle pharmacologies such as partial agonism or antagonism. In addition, it should also be possible to screen each compound simultaneously against multiple targets to determine specificity. However, using microtiter plate technology for further miniaturization is problematic: for example, evaporation becomes significant in microliter volumes, capillary action causes "wicking" and bridging of liquid between wells, and this would not alleviate the time and consumables cost associated with robotic microtiter plate handling. The key technology in this proposal is RainDance Technologies' droplet-based microfluidic platform. Using this platform, we believe we can further reduce the volume of each assay by 103- to 106-fold and increase the throughput >1,000 times, by confining each assay in a separate microscopic droplet. We will use this technology to enable high throughput screening for molecular and cell-based assays, significantly reducing the cost, time, and reagents for screening, and enabling the prospect of high throughput primary cell screening.