Monodisperse droplet generator: On-chip Droplet Generator

On-chip Droplet Generator allows mass production of micrometer-sized water-in-oil (W/O) emulsion droplets and gel microdrops (GMDs) with a unique microfluidic chip. The individual droplets function as miniaturized vessels for reactions such as microbial/cell cultivation and genetic analysis. Droplet technology is now recognized as a prominent high-throughput screening method allowing single cell analysis or simultaneous assay of more than 1 million samples.

Applications

Exploration of novel microorganisms

Encapsulation, cultivation and isolation of a variety of microorganisms including soil microorganisms, intestinal microbiota and marine microorganisms.

 

Development of highly productive strains

Detection and separation of enzymes and metabolites secreted in droplets.

 

Screening of antibody-producing cells

Development of stable and productive antibody forming cell lines.

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Features

Generation of stable droplets

On-chip Droplet Generator allows production of stable and monodisperse droplets.

 

Easy adjustment of droplet size

Droplet size can be easily adjusted between 60 µm and 120 µm in diameter by regulating the pressure applied to the sample and oil reservoirs.

 

Real-time monitoring of droplet generation

Droplet size can be viewed and monitored real-time with the supplied monitoring device.

On-chip Droplet Generator can produce two types of droplets: water-in-oil (W/O) emulsion droplets and gel microdrops (GMDs).

Utilization of On-chip Droplet Generator and On-chip® Sort

Workflow of On-chip Droplet Generator and On-chip® Sort

Droplets produced by On-chip Droplet Generator can be screened at a high throughput manner by our microfluidic chip-based cell sorter, On-chip® Sort. On-chip® Sort is capable of using oil as the sheath fluid for analyzing and sorting of W/O emulsion droplets.

 

Sorting of droplets containing proliferated cells

W/O and GMD droplets containing proliferated cells encapsulated by On-chip Droplet Generator can be sorted on On-chip® Sort. Examples are:

(1) Sorting of E. coli encapsulated and incubated in W/O droplets based on the fluorescence of GFP (expression within E. coli as an indicator).

(2) Sorting of Tetrahymena encapsulated and incubated in W/O droplets based on the autofluorescence as an indicator.

(3) Sorting of A549 cells encapsulated and incubated in GMDs based on the fluorescence of GFP as an indicator.

 

Detection of microorganisms proliferated within W/O droplets

A simple and reliable method that allows bacteria containing droplets to be isolated on On-chip® Sort has been developed. W/O droplets containing bacteria were sorted based on the fluorescence produced as a result of the interaction between the bacteria and fluorescence resonance energy transfer (FRET)-based RNA probe encapsulated inside the droplets. This method is named Fluorescent Nucleic Acid Probe in droplets for Sorting bacteria (FNAP-sort). An increase in the fluorescence intensity of E. coli, Bacillus subtilis, Streptomyces aureofaciens or Bradyrhizobium japonicum cultivated as model microorganisms were identified in the droplets in which these microorganisms proliferated. The use of this system allows screening based on the proliferative activity in the environment and other cellular events.

 

Screening for high enzyme producing bacteria from soil

Microorganisms from the soil were encapsulated and incubated with the substrate which responded to enzyme (peptidase) in W/O droplets. One million droplets were analyzed and droplets with higher fluorescence intensity (i.e., high enzymatic activity) were sorted on On-chip® Sort. Collected droplets were transferred to agar plate. For those that formed colonies, supernatant assay was performed after re-incubation. Successful isolation of highly peptidase-producing microorganism was achieved using On-chip Droplet Generator, followed by On-chip® Sort.

 

Cultivation within W/O droplets maintains microbial diversity

Bacteria from soil were cultivated in liquid media and in W/O droplets over 7 days. DNA was extracted and analyzed by NGS to study the bacterial flora. The bacterial flora in the droplets showed to be much higher in individual Operational Taxonomic Units (OUTs) compared to those cultivated conventionally in liquid media.

 

Screening of high protein producer yeast with GMD

Yeast transformant that produces secreted luciferase were UV irradiated for random mutagenesis. They were then encapsulated and cultured within GMDs. GMDs with high fluorescence intensity derived from luciferase were sorted using On-chip® Sort and transferred to agar medium. For colonies formed, the supernatant was collected, and an assay was performed on the supernatant after re-incubation. As a result of screening, strains with higher producing ability than that of the parent strain were successfully collected.

 

Specifications of On-chip Droplet Generator

The On-chip Droplet Generator set up contains of the main unit for supplying pressure, monitoring unit for real-time monitoring of droplets, and temperature controller for precise temperature adjustment.

 

 

Specifications		On-chip Droplet Generator Monitoring unit with temperature control
Main unit	Size	270 x 270 x 280 mm (W x D x H)
	Liquid delivery pressure	7 – 80 kPa
	Maximum liquid delivery line	3 lines (sample, oil, other reagents)
	Droplet size generated	Diameter 60 – 120 μm (can be adjusted discretionally)
	Generation rate	Approx. 100,000 droplets/3 min (for generation of 100 μm droplets)
	Regulation	Laptop PC (Windows 10)
	Power input	AC 100-240V, 50/60Hz
	Power consumption	1.2A typ (ACIN 100V)
Monitoring unit	Size of monitoring unit	215 x 310 x 430 mm (W x D x H)
	Accessories	Monitor camera, Peltier unit, Controller
	Temperature range	-5℃ – 70℃
	Ambient environment	Temperature 5 – 40˚C, humidity ≤ 85% RH
	Power input	DC24V
	Power consumption	6.5A max

 

Product No.	Product name	Product description
60003	On-chip Droplet Generator (Monitoring unit with temperature control)	Main unit, chip holder, monitoring unit with temperature control

Consumables

Microfluidic chip

Specifications	2D chip-1060DG
Samples applied	Aqueous solution, culture medium, gel solution etc.
Oils applied	Fluorinated oil
Chip material	COP
Fluid channel size	60 μm x 60 μm
Reservoir volume	Sample- 640 μL max. Oil- 2,300 μL max.
Output	Approx. 100,000 droplets (diameter 100 μm in approx. 3 min)

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Reagents

Fluorinated oil is used for producing stable droplets.