Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

Abstract

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.

A Lab-in-a-Fiber optofluidic device using droplet microfluidics and laser-induced fluorescence for virus detection

“…So called optofluidic devices offer a way to synergistically combine key microfluidics sample processing capabilities with optical functionality, opening up promising avenues for bio- and chemical-sensing 14 . Established optical techniques such as fluorescence-activated droplet sorting 15 , 16 , optical microscopy 17 , 18 , or laser-induced fluorescence (LIF) 19 – 21 , can be incorporated into microfluidic chips.…”

Deformability-Based Microfluidic Microdroplet Screening to Obtain Agarolytic Bacterial Cells

“…Droplet microfluidics has been widely applied in the discovery and directed evolution of enzymes because of its low cost and high throughput. 44 Microbial cells and enzymes with the desired activities have been isolated based on the characteristics of the microdroplets, such as fluorescence, 9,11 absorbance, 14,15 Raman scattering, 16 electrochemical detection, 17,18 and mass spectrometry. 19,20 In this study, we demonstrated that enzymatic hydrolysis of agarose can be assayed in microdroplets based on their deformability (Figures 3 and 4).…”

High-throughput bacterial co-encapsulation in microfluidic gel beads for discovery of antibiotic-producing strains

“…Fluorescence Activated Droplet Sorting (FADS) is a technique that enables the selection of W/O droplets based on the fluorescent signals emitted by the droplet contents as they travel through microchannels. 20 It is a high-throughput methodology that has been used for selecting enzyme mutants with higher activity in directed evolution projects [21][22][23] The technique has also been modified for label-free droplet detection. 24 In microbial interaction studies, FADS has been used to analyze the diversity of antibiotic-producing bacteria from different natural habitats.…”