“Droplets” can be easily generated in large quantities and have the ability to encapsulate microorganisms or cells individually, allowing for the analysis of a wide variety of samples simultaneously and in parallel. Therefore, Droplet has been used in various fields, including high-throughput screening (HTS), making use of various advantages such as accelerated generation and analysis, small-bioreactor culture, and single-cell analysis.
Advantages of droplet technology
- Each droplet (picoliter-scale) functions as each well (microliter-scale) of a well-plate.
- Over one million droplets (reaction compartments) can be stored in a single tube.
- Significant savings can be achieved in analysis time, reagent consumption, and incubation space.
- Target droplet can be screened by On-chip Sort/On-chip Droplet Selector
| Micro-titer plate (Conventional method) | Droplet (Next generation method) | |
|---|---|---|
| One reaction volume | 100μL/1well | 100pL ～ 1 drop |
| For analysis of one million samples | 2,600 plates (384-well) | One tube only |
| Analysis Speed | Long (several days) | Short (several hours) |
| Cost | High | Low |
Applications
Water-in-oil (W/O) droplets
- ・Structure:
- Emulsion in which water droplets (e.g. the medium) are dispersed in oil and stabilized by surfactants.
- ・Features:
- Microorganisms/cells grow and metabolites accumulate in each droplet as they are compartmentalized by oil. The addition of substrates allows for the detection of metabolites along with the cultivation.
- ・Use:
- Microbial culture, screening of environmental microbes/mutant strains, directed evolution of enzymes, etc.
Single-cell cultivation
Single-cell encapsulation and cultivation allow massively parallel screening based on the growth, protein expression, and secretion amount.
Luu, X. C., Shida, Y., Suzuki, Y., Sato, N., Nakamura, A., Ogasawara, W.. New Biotechnology 2022 72, 149-158.
Novel microorganisms screening
As a tool for isolating novel microorganisms from diverse environments, including soil, hydrosphere, and extreme environments.
Nakamura, A., Honma, N., Tanaka, Y., Suzuki, Y., Shida, Y., Tsuda, Y., Hidaka, K., Ogasawara, W., Analytical Chemistry 2022 94 (5), 2416-2424.
Cell-cell interaction
Droplet Library can be easily prepared containing combinations of various types of bacteria by taking advantage of the feature of high-throughput droplet generation.
The results are obtained in conjunction with Dr. Yasunori Ichihashi and Dr. Megumi Narukawa, RIKEN BioResource Research Center
Gel microdrops (GMD)
- ・Structure:
- Droplets with gelled interior.
- ・Features:
- Substances are highly permeable both inside and outside the droplet structure because the droplets can be dispersed in an outer aqueous phase, such as a culture medium, rather than in oil, while relatively large materials such as cells, beads, and genomes are retained by the gel as a scaffold.
- ・Use：
- Cell culture, screening of mutant strains/antibody-producing cells, microbial interaction studies, etc.
Isolation of Protein High-Producing Strains
High-throughput screening of high-producing strains from a large number of candidates such as UV mutant libraries
Fujitani, H., Tsuda, S., Ishii, T., Machida, M., bioRxiv 2019
Screening based on substance production
Staining of microorganisms after GMD cultivation allows screening based on substance production such as lipids.
Tanaka, Y., Nakamura, A., Suzuki, Y., Maruta, K., Shida, Y., Ogasawara, W. , Journal of Biotechnology 2022 358, 46-54.
Elucidation of the gut microbiota function
High-throughput screening to elucidate the function of diverse gut microbiota
The results are obtained in conjunction with Prof. Shinji Fukuda, Institute for Advanced Biosciences, Keio University
Droplet Sorting by Microfluidics Chip
Detection and Screening of Droplets Encapsulating Microorganism
A major challenge in obtaining pure cultures of environmental microorganisms has been detecting and isolating droplets containing microorganisms with various growth rates from millions of droplets. To solve this problem, Dr. Noda and his team at the National Institute of Advanced Industrial Science and Technology (AIST) developed FNAP-sort (fluorescent nucleic acid probe in droplets for bacterial sorting), a method that utilizes fluorescence to detect droplets containing grown microorganisms. In this method, a fluorescent nucleic acid probe, which is quenched by FRET (fluorescence resonance energy transfer), is cleaved by RNase generated during microbial growth, resulting in the elimination of FRET and an increase in fluorescence emission.
FNAP-sort workflow and proof of principle
(A) FNAP-sort workflow, (B) microscopic images of droplets immediately after generation, (C) microscopic images of droplets after one day of incubation, (D) fluorescence histogram of droplets after one day of incubation, and (E) microscopic images of sorted droplets. Upper panels: dark-field observation; lower panels: fluorescence observation for (B), (C), and (E).
Ota, Y., Saito, K., Takagi, T., Matsukura, S., Morita, M., Tsuneda, S., Noda, N., PLOS One 2019 14(4), e0214533.
Saito, K., Ota, Y., Tourlousse, D. M., Matsukura, S., Fujitani, H., Morita, M., Tsuneda, S., Noda, N., Scientific Report 2021 11(1), 9506.
Sample List
Sorting of w/o droplets or GMD encapsulating cells
| Microorganisms | Escherichia coli |
|---|---|
| Bacillus subtilis | |
| Mycorrhizal fung | |
| Actinobacteria | |
| Aspergillus | |
| Filamentous fungi | |
| Yeast | |
| Oil-producing algae | |
| Tetrahymena |
| Environmental samples | Soil |
|---|---|
| Seawater and lake water | |
| Gut | |
| Microplankton | |
| HTS screening | CHO cells |
| Hybridoma | |
| Cell-free protein synthesis | |
| GPCR reaction system |
| Gel Microdrop | Beads |
|---|---|
| PEG | |
| Gelatin | |
| Low melting point agarose | |
| Alginic acid | |
| Collagen |
