Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Lab Chip. 2019 Apr 23;19(9):1589-1598. doi: 10.1039/c9lc00125e.
To address current limitations in adapting solid phase sample capture and washing techniques to continuously flowing droplet microfluidics, we have developed the "Coalesce-Attract-Resegment Wash" (CAR-Wash) approach. This module provides efficient, high-throughput magnetic washing by electrocoalescing magnetic bead-laden input droplets with a washing buffer flow and magnetophoretically transporting beads through the buffer into a secondary droplet formation streamline. In this work, we first characterized the technology in terms of throughput, sample retention, and flow-based exclusion of waste volume, demonstrating >500 Hz droplet processing with >98% bead retention and >100-fold dilution in final droplets. Next, we showed that the technique can be adapted to alternative commercially available magnetic beads with lower magnetite content per particle. Then, we demonstrated the CAR-Wash module's effectiveness in washing away a small molecule competitive inhibitor to restore the activity of magnetic bead-immobilized β-galactosidase. Finally, we applied the system to immunomagnetically enrich a green fluorescent protein-histone H2B fusion protein from cell lysate while washing away mCherry and other lysate components. We believe this approach will bridge the gap between powerful biochemical and bioanalytical techniques and current droplet microfluidic capabilities, and we envision future application in droplet-based immunoassays, solid phase extraction, and other complex, multi-step operations.
为了解决当前将固相样品捕获和洗涤技术应用于连续流动液滴微流控技术的局限性，我们开发了“聚并-吸引-再分割洗涤”（CAR-Wash）方法。该模块通过电聚合并将载有磁珠的输入液滴与洗涤缓冲液流合并，同时通过磁泳将珠体输送到缓冲液中的二次液滴形成流线，从而实现高效、高通量的磁洗涤。在这项工作中，我们首先从处理速度、样品保留和基于流动的废物体积排除等方面对该技术进行了特征描述，结果表明，该技术可实现>500 Hz 的液滴处理，磁珠保留率>98%，最终液滴的稀释倍数>100 倍。接下来，我们证明了该技术可以适应具有较低每颗粒磁铁矿含量的替代市售磁珠。然后，我们展示了 CAR-Wash 模块在洗涤掉小分子竞争抑制剂以恢复固定在磁珠上的β-半乳糖苷酶的活性方面的有效性。最后，我们将该系统应用于从细胞裂解物中免疫磁分离绿色荧光蛋白-组蛋白 H2B 融合蛋白，同时洗涤掉 mCherry 和其他裂解物成分。我们相信这种方法将缩小强大的生化和生物分析技术与当前液滴微流控技术之间的差距，我们设想未来在基于液滴的免疫分析、固相萃取和其他复杂的多步骤操作中应用该系统。
