Harvard School of Engineering and Applied Sciences, Cambridge, MA 02138, USA.
Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4004-9. doi: 10.1073/pnas.0910781107. Epub 2010 Feb 8.
The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify approximately 100 variants comparable in activity to the parent from an initial population of approximately 10(7). After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen approximately 10(8) individual enzyme reactions in only 10 h, using < 150 microL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.
我们对基因组、蛋白质组和代谢组的认识呈爆炸式增长，这推动了对生命生物化学的理解不断深入，使人们能够对复杂的生物系统及其进化进行全新的研究。这一知识还推动了现代生物技术的发展，如分子工程和合成生物学，这些技术具有巨大的潜力，可以解决包括开发强效新药和提供环保能源在内的紧迫问题。然而，许多此类研究最终受到需要对生化反应进行更高通量测量的限制。我们提出了一种基于液滴的微流控高通量筛选平台，该平台克服了这些限制，彻底改变了筛选的规模和速度。我们使用分散在油中的水滴作为皮升级别的反应容器，并以每秒数千个的速度对其进行筛选。为了展示其威力，我们将该系统应用于定向进化，鉴定出了新的辣根过氧化物酶突变体，其催化速率比其母体快 10 多倍，而母体已经是一种非常高效的酶。我们利用超高通量进行初始纯化筛选，去除无活性的突变体；从初始群体中筛选出约 100 个与母体活性相当的变体，该群体约为 10^7。经过第二代诱变和严格筛选，我们鉴定出了几个显著改进的突变体，有些接近扩散限制效率。总共，我们在仅 10 小时内筛选了约 10^8 个单个酶反应，使用的总试剂体积<150 μL；与最先进的机器人筛选系统相比，我们的整个测定速度提高了 1000 倍，成本降低了 100 万倍。
