Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science , Masaryk University , Kamenice 5 , Brno 625 00 , Czech Republic.
International Clinical Research Center , St. Anne's University Hospital , Pekarska 53 , Brno 656 91 , Czech Republic.
Anal Chem. 2019 Aug 6;91(15):10008-10015. doi: 10.1021/acs.analchem.9b01839. Epub 2019 Jul 10.
Functional annotation of novel proteins lags behind the number of sequences discovered by the next-generation sequencing. The throughput of conventional testing methods is far too low compared to sequencing; thus, experimental alternatives are needed. Microfluidics offer high throughput and reduced sample consumption as a tool to keep up with a sequence-based exploration of protein diversity. The most promising droplet-based systems have a significant limitation: leakage of hydrophobic compounds from water compartments to the carrier prevents their use with hydrophilic reagents. Here, we present a novel approach of substrate delivery into microfluidic droplets and apply it to high-throughput functional characterization of enzymes that convert hydrophobic substrates. Substrate delivery is based on the partitioning of hydrophobic chemicals between the oil and water phases. We applied a controlled distribution of 27 hydrophobic haloalkanes from oil to reaction water droplets to perform substrate specificity screening of eight model enzymes from the haloalkane dehalogenase family. This droplet-on-demand microfluidic system reduces the reaction volume 65 000-times and increases the analysis speed almost 100-fold compared to the classical test tube assay. Additionally, the microfluidic setup enables a convenient analysis of dependences of activity on the temperature in a range of 5 to 90 °C for a set of mesophilic and hyperstable enzyme variants. A high correlation between the microfluidic and test tube data supports the approach robustness. The precision is coupled to a considerable throughput of >20 000 reactions per day and will be especially useful for extending the scope of microfluidic applications for high-throughput analysis of reactions including compounds with limited water solubility.
与测序相比，新型蛋白质的功能注释滞后于下一代测序所发现的序列数量。与测序相比，传统检测方法的通量太低；因此，需要实验替代方法。微流控技术作为一种紧跟基于序列的蛋白质多样性探索的工具，具有高通量和减少样品消耗的优势。最有前途的基于液滴的系统有一个显著的局限性：疏水性化合物从水相泄漏到载体相，阻止了它们与亲水性试剂一起使用。在这里，我们提出了一种将底物递送到微流控液滴中的新方法，并将其应用于疏水性底物转化酶的高通量功能表征。底物传递基于疏水性化学物质在油相与水相之间的分配。我们应用了 27 种疏水性卤代烷烃从油相到反应水相的受控分布，对来自卤代烷脱卤酶家族的 8 种模型酶进行了底物特异性筛选。与经典试管测定相比，这种按需分配的微流控系统将反应体积减少了 65000 倍，分析速度提高了近 100 倍。此外，微流控装置还可以方便地分析一组嗜温和超稳定酶变体在 5 至 90°C 范围内的活性对温度的依赖性。微流控和试管数据之间的高度相关性支持了该方法的稳健性。该方法的精度与相当高的通量相关，每天可进行 >20000 次反应，对于扩展微流控应用范围，特别是对于高通量分析包括有限水溶性化合物的反应，将非常有用。
