Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo

Abstract

The potential of Raman-activated cell sorting (RACS) is inherently limited by conflicting demands for signal quality and sorting throughput. Here, we present positive dielectrophoresis–based Raman-activated droplet sorting (pDEP-RADS), where a periodical pDEP force was exerted to trap fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting. Screening of yeasts for triacylglycerol (TAG) content demonstrated near-theoretical-limit accuracy, ~120 cells min−1 throughput and full-vitality preservation, while sorting fatty acid degree of unsaturation (FA-DU) featured ~82% accuracy at ~40 cells min−1. From a yeast library expressing algal diacylglycerol acyltransferases (DGATs), a pDEP-RADS run revealed all reported TAG-synthetic variants and distinguished FA-DUs of enzyme products. Furthermore, two previously unknown DGATs producing low levels of monounsaturated fatty acid–rich TAG were discovered. This first demonstration of RACS for enzyme discovery represents hundred-fold saving in time consumables and labor versus culture-based approaches. The ability to automatically flow-sort resonance Raman–independent phenotypes greatly expands RACS’ application.

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

“…MADS is efficient for low-concentration substrate and could be an option especially for isomers. Raman-activated cell sorting (RACS) would be a good choice if richer information is required apart from the enzymatic reaction itself (Wang et al, 2020 ); meanwhile, the relative low throughput can be compromised. Moreover, another informative sorting method-NMR-based sorting is molecule-informative while of low throughput (Hale et al, 2018 ).…”

“…Recent breakthroughs have just been made in developing non-resonance-based RADS. A significantly improved rate of 120 cells/min and two variants of an unknown enzyme [algal diacylglycerol acyltransferases (DGATs)] were discovered by applying this RADS setup (Wang et al, 2020 ). A quartz-made chip, with low background signals, could be used for non-resonance signal RADS rather than PDMS.…”

Let the Biocatalyst Flow

“…64 Future detection modes will include fluorescence-based approaches (anisotropy, Förster resonance energy transfer, lifetime) and label-free approaches based on light scattering (including Raman scattering) or droplet morphology. 55 Reports on the application of positive dielectrophoresis-based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo, 65 and droplet sorting by inter- Žnidaršič-Plazl: Let the Biocatalyst Flow facial tension 66 confirm these expectations. A sophisticated Raman-activated droplet sorting device uses periodically applied positive dielectrophoresis force to capture fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting.…”

“…The label-free method of sorting droplets by pH requires no active components and provides a robust platform for enzyme sorting in highthroughput applications. 65 Another promising approach in this regard is the coupling of droplet microfluidics with electrospray ionization -mass spectroscopy (ESI-MS), which provides a label-free high-throughput screening platform. The system also enabled effective in vitro transcription-translation within the droplets analyzed directly by MS, demonstrating opportunities to greatly accelerate the screening of enzyme evolution libraries.…”

The NanDeSyn database for Nannochloropsis systems and synthetic biology

“…Notably, the development of approaches based on single‐cell Raman spectra that characterize the profiles of energy storage molecules in N . oceanica , the ‘ramanome’, has accelerated the time‐consuming microalgal phenotyping processes (Ji et al ., 2014; Wang et al ., 2014b; He et al ., 2017; Llansola‐Portoles et al ., 2017) by unveiling and sorting single‐cell‐level phenomes in a label‐free, non‐invasive manner (Wang et al ., 2017, 2020; Jing et al ., 2018; He et al ., 2019).…”