1. The Emulsion Sorting Technology Platform
for High-Throughput Screening Assays in Various Fields of Biology
Abstract
Introduction
Cultivation of microorganism is an effective use of emulsion droplets, as it enables
culturing of microorganisms with their clones in small compartments. Moreover, this
technique allows for culturing of microbes that do not form a colony, reactions within the
droplet, and collection of these target droplets. We anticipate this technology will be used
widely, for example, in screening for new species found in environment that produce beneficial
substances and in analysis for microbial flora.
Gene Screening for High Functional Protein
On-chip
Droplet GeneratorTM On-chip SortTM
Clonal Culture and Screening of Microorganisms
Digital PCR and Positive Droplet Screening
Fig. 2 shows droplets that encapsulated
either 0 or 1 GFP-expressing Escherichia
coli , and cultured for 20 hours. The number
of E. coli increased by about 1000 times. Only
the droplets containing E. coli were collected by
sorting GFP expressing droplets.
Generates 20-100μm W/O emulsion
droplets within a microfluidic chip
A “Droplet Sorter” for analysis and
collection of emulsion droplets within
microfluidic channels
Disposable
chip
flow
Forward Scatter
Fluorescentintensity
50µm
80 μm
Fluorescentintensity
Forward Scatter
Fig. 2 E. coli cultivation within emulsion droplets and sorting
Fig. 3 Encapsulation of Euglena gracilis and sorting
High-throughput screening for high functional protein is extremely important in the various fields like pharmaceutics and industry.
Conventional screening technologies, such as Enzyme-Linked ImmunoSorbent Assay (ELISA) and Phase Display, are based on protein affinity, and
thus they are limited to screening for antibodies. The use of emulsion and cell-free translation system enables isolation of genetic information
carrier (nucleic acids) and trait (protein) in one common space per clone, and so genetic screening based on enzymatic activity becomes possible.
a
Yohsuke Bansho*, Jin Akagi, Takahide Ino, Kosuke Osawa, Tomiko Tanaka, Masayuki Ishige, Yuu Fujimura, Kazuo Takeda
On-chip Biotechnologies Co., Ltd.
oil oil
sample
Fig. 4 shows the experimental model of the
protein genetic screening using emulsion. (A) First,
we prepared an DNA library that consist of GFP-
coding DNA and non-coding DNA, biotinylated PCR
primers, and streptavidin beads. The emulsion was
generated using a sample with adjusted DNA
concentration for one-template-per-droplet
encapsulation. After running PCR, DNA bound
beads were collected and trapped inside droplets
with cell-free translation system for further
reaction. (B) These droplets were sorted into three
groups according to the fluorescence intensity. PCR
on the collected beads as template resulted in
enrichment of GFP coding fragment for the sorted
droplet group with high fluorescence intensity.
80 μm
In recent years, high-throughput analysis and screening technologies for drug discovery, microbial inspection and evolutionary engineering
are becoming increasingly important. The combination of single cell (or single molecule) plating on multititer plate by limiting dilution
and screening using plate reader is commonly used in order to perform screening of tremendous number of cells.
However, this method is very costly, time consuming and labor intensive, and thus there is a limit to screening of cells
and biomolecules that could easily exceed the library size of 105.
A “vessel” for high-throughput analysis and screening that is becoming increasing popular is W/O emulsion
(Fig. 1). W/O emulsion is micron-scale droplet suspended in oil. Since it is very easy to prepare a large
number of droplets, it is considered very useful in academic research and technology development that
require high-throughput analysis and screening. Hence, we have developed On-chip Droplet
Generator, which allows for simple generation of monodisperse droplets, and On-chip Sort, a
disposable microfluidic chip based cell sorter that enables measurement and collection of the
emulsion droplets with fluorescence signals. Herein, we introduce some of the applications
achieved using these two instruments.
Microtiter Plate
105 reactions = 260 of
384well plates
Volume: 100’s µL VS A few pL
Time & Cost: Consuming VS Saving
Sample Recovery: Difficult VS Easily by On-chip Sort
W/O Emulsion
105 reaction = Less than
1mL, ready in a few mins
Furthermore, Fig. 3 shows droplets that contain one Euglena gracilis
every 300 droplets on average. We observed Euglena gracilis survive
and actively move within the droplets. Sorting of these droplets
were based on the autofluorescence of chlorophyll led to a
successful enrichment of droplets containing Euglena gracilis.
Fig. 4 Fluorescent protein screening using emulsion and cell-
free translation system
(B)
Digital PCR is a new nucleic acid quantification technique, and related products
from Bio-Rad Laboratory Inc. and Applied Biosystems are currently on the market. Not
only the products of On-chip Biotechnologies can be used for quantification of nucleic acids, but
they also enable recovery of positively expressing droplets after measurement. If PCR together with
detection of fluorescence could be carried out from cells directly, this will lead to the selective
recovery of cell extract with specific mutation or mRNA expression.
Fig. 5 PCR detection and sorting of emulsions
Fig. 5 shows the results of purified human genome quantification PCR within droplets. On-chip
Sort was able to not only measure the number of target DNA within the medium, but also we were
able to selectively enrich the droplets containing target DNA fragment.
Forward scatter
Fluorescence
After Sorting
Microdroplets of water-in-oil (W/O) emulsions are rapidly gaining interest as a tool for analysis and screening of multiple samples. On-chip
Biotechnologies Co., Ltd. have developed On-chip Droplet Generator and On-chip Sort: an emulsion generation device and an emulsion
analyzer/sorter. Using these technologies, we have performed model experiments for clonal culture of microorganism, phenotypic screening,
and digital PCR. We are eager to find collaborative partners whom we could share the experimental results and together develop of new study
cases.
*Correspondence
y-bansho@on-chip.co.jp
Fig. 1 Comparison of miniature reaction “vessel”
Biotinylated Primer
Streptavidin beads
Encapsulation
& PCR
Beads recovery
GFP
translation
GFP coding DNA
Non-coding DNA
Encapsulation
Sorting
Cell-free translation in emulsionEmulsion PCR on beads(A)
Producinggenetic
informationcarriers
GenotypetophenotypeassociationCollectionof
targetgene
Euglena gracilis used in this experiment was provided by the courtesy of Dr. Yonamine of Kyushu University
The results given here suggests that protein
screening incorporating emulsion is plausible if there
is a technique to correspond the protein activity and
fluorescence intensity. We believe this can be applied
a to screening for beneficial protein and
evolutionary molecular
engineering.
100% sorted
Top 50% sorted
Top 10% sorted
100%sorted
Top50%sorted
Top10%sorted
Notemplate
Forward scatter
Fluorescence
Materials used in this experiment were provided by the courtesy of Chugai Pharmaceutical Co.