Fu Xiaozhi, Zhang Yueying, Xu Qiang, Sun Xiaomeng, Meng Fanda
Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
Department of Clinical Laboratory Medicine, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, Shandong Medicine and Health Key Laboratory of Laboratory Medicine, Jinan, China.
Front Chem. 2021 Apr 23;9:666867. doi: 10.3389/fchem.2021.666867. eCollection 2021.
Droplet-based microfluidics has been widely applied in enzyme directed evolution (DE), in either cell or cell-free system, due to its low cost and high throughput. As the isolation principles are based on the labeled or label-free characteristics in the droplets, sorting method contributes mostly to the efficiency of the whole system. Fluorescence-activated droplet sorting (FADS) is the mostly applied labeled method but faces challenges of target enzyme scope. Label-free sorting methods show potential to greatly broaden the microfluidic application range. Here, we review the developments of droplet sorting methods through a comprehensive literature survey, including labeled detections [FADS and absorbance-activated droplet sorting (AADS)] and label-free detections [electrochemical-based droplet sorting (ECDS), mass-activated droplet sorting (MADS), Raman-activated droplet sorting (RADS), and nuclear magnetic resonance-based droplet sorting (NMR-DS)]. We highlight recent cases in the last 5 years in which novel enzymes or highly efficient variants are generated by microfluidic DE. In addition, the advantages and challenges of different sorting methods are briefly discussed to provide an outlook for future applications in enzyme DE.
基于液滴的微流控技术因其低成本和高通量，已在细胞或无细胞系统的酶定向进化（DE）中得到广泛应用。由于分离原理基于液滴中的标记或无标记特性，分选方法对整个系统的效率起着主要作用。荧光激活液滴分选（FADS）是应用最广泛的标记方法，但面临目标酶范围的挑战。无标记分选方法显示出极大拓宽微流控应用范围的潜力。在此，我们通过全面的文献调研综述了液滴分选方法的发展，包括标记检测[FADS和吸光度激活液滴分选（AADS）]和无标记检测[基于电化学的液滴分选（ECDS）、质量激活液滴分选（MADS）、拉曼激活液滴分选（RADS）和基于核磁共振的液滴分选（NMR-DS）]。我们重点介绍了过去5年中通过微流控DE产生新型酶或高效变体的最新案例。此外，还简要讨论了不同分选方法的优缺点，以为酶DE的未来应用提供展望。
