Droplet-based microfluidic analysis and screening of single plant cells

Abstract

Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.

Chemical Analysis of Single Cells and Organelles

“…Yu et al also employed a T-junction in a PDMS-glass device to generate water-in-oil droplets. 185 Here, yellow fluorescent protein (YFP)-modified M. polymorpha protoplasts (plant cells without a cell wall) were encapsulated. The autofluorescence of chlorophyll or the YFP reporter protein was quantified using a confocal microscope with a throughput of 115 200 individual cells per hour.…”

Recent Advances in Droplet-based Microfluidic Technologies for Biochemistry and Molecular Biology

“…This application of DMF technology on plant cells analysis can open the opportunity for on-chip fluorescence-based selection of individual plant cells that can be used in targeted regeneration. Combining DMF technology with RNA libraries and gene editing tools like CRISPR-cas9 endonuclease can also improve the academic and industrial research in plant biotechnology [116].…”

The Usual Suspects 2019: of Chips, Droplets, Synthesis, and Artificial Cells

“…Microfabricated fluorescence-activated cell sorter (µFACS) systems with inline droplet generators offer similar advantages as FACS; however, they eliminate the need for expensive equipment and minimize the probability of channel clogging. One recent example of employing droplet microfluidics for cell sorting has been shown by Yu et al for plant protoplasts, as shown in Figure 1B [21]. Protoplast fluorescence was detected on-chip by coupling a laser-based optical detection setup with electrodes generating a dielectric force dependent on fluorescent readout.…”

“…Overall, microfluidics proves to be an important tool for synthetic biologists in manipulating cellular systems. The devices offer great advantages such as automated feedback control [18,19], sorting of engineered cells based on protein yield [21,22,25], and on-line detection of cell growth [25,26]. Digital microfluidic devices additionally allow for novel fluid handling operations and process automation.…”

“…( B ) Droplet-based microfluidic analysis system that can screen single plant cells in a controllable soluble microenvironment. (Reprinted with permission from [21]; Copyright 2019 American Chemical Society).…”