1PRODUCTION OF WATER-IN-OIL
EMULSIONS USING A DROPLET
GENERATOR CHIPINTRODUCTION
Microfluidic generation of droplets has attracted a lot of interest due to the ability ofthis method to produce highly monodispersed droplets with high frequency (up tohundreds of kHz). Interest in droplet-based microfluidic systems has grown substantiallyin the past decade1,2 as they offer the ability to handle very small volumes (μl to fl) offluids conveniently, providing better mixing, encapsulation, sorting, sensing as well assuitability for high throughput experiments. Microfluidic-based droplets have many
diverse and varied applications such as particle synthesis3 and chemical analysis4. Highly
controlled droplet production also makes single cell analysis, or drug testing possible5,6.
Droplet microfluidic concepts, components and processes are now being adopted andleveraged by end-users to engender completely new science and innovation. Realworld success is now evidenced through a range of mainstream commercial productsthat are being aggressively applied to key biological and healthcare related problems(e.g., 10X Genomics, Drop-seq and nucleic acid quantification via Droplet DigitalTMPCR systems)7. It is fair to say that droplets have now become an indispensable tool inchemical and biological research.
Droplet generation in microfluidics is based on the use of two immiscible phases thatare referred to as the continuous phase (medium in which droplets flow) and dispersed
phase (the droplet). For generating droplets, microfluidic systems generally include a
microfluidic chip, a fluid handling system and tubing. This system is usually connected
to a computer and a microscope to visualize droplet formation.
Fluigent develops, manufactures and supports innovative fluid handling solutions for avariety of applications using microfluidic droplet generation.
The objective of this applicaion note is to demonstrate droplet generation using theFluigent microfluidic system including pressure pumps, chemicals, tubing and a Droplet
Generator Chip obtained from our partner microfluidic ChipShop.

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APPLICATION NOTE
Materials and Method
Figure 1: System setup. Two Flow EZ pressure pumps are connected to the droplet
generator from microfluidic ChipShop.
The tubing passes through Flow units tocontrol pressure and flow. Visualization ofthe chip channels is performed using anoptical microscope.

An external pressure source is connected to two Flow EZ pressure pumps that are connected to the microfluidic ChipShop chip via tubing. The tubing passes through Flow units
to measure pressure and flow rate. Visualization of the chip channels is performed using
an optical microscope. A LINK can connect the Flow EZ module to a PC for monitoringpressure and flow rate in real time.

Microfluidic flow controller
The Flow EZ is the most advanced flow controller for pressure-based fluid control. It can be combined with a Flow Unit tocontrol pressure and flow. It can be used without a PC. A flow EZ
1 bar was used hereReagentsContinuous phase: dSurf 2% diluted in 3M™ Novec™ 7500: dSURF
is a biocompatible fluorosurfactant providing highly reliabledroplet production and stability even under PCR amplification
conditions. Fluorinated oil is used instead of mineral oil as it has
overall better properties (highly biocompatible, immiscible, lowviscosity …).
Dispersed phase: distilled waterFlow Unit
The flow unit is a sensor included in the fluidic system and allows
real-time flow rate measurements. By combining a Flow Unit
with Flow EZ, it is possible to switch from pressure control to flowrate control, allowing enhanced droplet monodispersity over along period of time.

3APPLICATION NOTE
Microfluidic chip
The microfluidic chip used here is the Droplet Generator Chip Fluidic 440, obtained
from our partner microfluidic ChipShop. The chip is made of polycarbonate (PC) andfeatures eight individual droplet generator units and Mini Luer interfaces. Fluidic 440
is designed to generate droplets with channel dimensions at the droplet formation
region of 80 μm (Design 1 and 2), 70 μm (Design 3 and 4), 60 μm (Design 5 and 6) and50 μm (Design 7 and 8) channel width and height. Droplets are generated using a flow
focusing method: the dispersed phase is introduced directly into the main channel while
the continuous phase is injected by two perpendicular channels. The dispersed phaseis then pinched on both sides by the continuous phase, a droplet thus is formed due tothe viscous force and chip geometry, as shown in Figure 2. Accessories to connect thechip to tubing, as well as a variety of different droplet generator chips are available frommicrofluidic ChipShop.

Figure 2: Droplet generator Fluidic 440. General layout of the chip (left). Scheme illustrating droplet generation using flow focusing geometry (middle) and a droplet generator setup with appropriate accessories,
such as connectors and tubing (right)

Droplet generation
A scheme of the microfluidic setup is presented in Figure 3. The design 4 of the Droplet
Generator Chip is used. This consists of 70 μm width and height channel dimensions at
the droplet formation region. Two 15 mL reservoirs containing water and 2% dSurf areconnected to the two inlets of the microfluidic chip via 1/32 in. PEEK tubing of 254 µm
inner diameter. The tubing passes through flow units allowing flow rate measurement
and control. The tubing length from the reservoir to the microfluidic chip is 80 cm.
Pressure is applied using the Flow EZs on the reservoirs containing distilled water anddSurf. Water is injected in the inner channel and dSurf is injected in the surrounding
channel of the microfluidic device. The pressure applied ranges from 100 to 300 mbar
depending on the droplet size and frequency required. Visualization of the chip channels
is performed using an optical microscope. A LINK can be connected to the Flow EZ
module and a PC for monitoring pressure and flow-rate in real time.

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APPLICATION NOTE
To calculate the diameter of the generated droplets, we measured the droplet length
within the microfluidic channel using the software ImageJ and calculated the droplet
volume using the following equation proposed by Musterd et al8:With H and W the channel height and width, and L the droplet length. We next equaled
this volume to the volume of a sphere, and the droplet diameter can be derived.

Figure 3: Schematic of the microfluidic system used for droplet generation

Results
Using the microfluidic system presented in the previous part, droplets were generated.
Table 1 shows water-in-oil droplet generation as a function of the flow rate and thepressure. The oil flow rate remains constant at 5 µL/min while the water flow rate ranges
from 2.5 µL/min to 10 µL/min. The droplet diameter, generation rate and its relatedpicture are also presented in the same table.
We observe that by increasing the water flow-rate and keeping the oil flow rate constant,the droplet diameter increases.

5APPLICATION NOTE
Table 1: Droplet diameter and generation rate as a function of water flow-rate
HFE with 2%dSURF

WaterDroplets
PictureQ[µL/min]P[mbar]

Q[µL/min]P[mbar]

Diameter
[µL]

Rate
[Hz]

2,5152

5160

98

84

5170

5152

111

117

5207

120

139

5270

133

135

7,510252

342

Table 2 shows water-in-oil droplet generation as a function of the flow rate and thepressure. The water flow rate remains constant at 5 µL/min while the oil flow rate isranging from 2.5 µL/min to 10 µL/min. The droplet diameter, generation rate and its
related picture are also presented in the same table.
We observe that by increasing the oil flow-rate and keeping the water flow rate constant,the droplet diameter decreases.

6APPLICATION NOTE
Table 2: Droplet diameter and generation rate as a function of oil flow-rate
HFE with 2%dSURF

WaterDroplets
PictureQ[µL/min]P[mbar]Q[µL/min]P[mbar]Diameter[µL]Rate
[Hz]

5168

2,5136

129

74

5170

5152

111

117

7,5167

100

158

10186

94

190

55171

177

The droplets generated and placed on a glass slide are shown in Figure 4. Figure 5 summarizes droplet diameter and generation rate as a function of the oil and water flow
rates, using flow rates ranging from 2,5 to 10 µL for each solution. This graph demonstrates that the droplet diameter increases when the water flow rate is increased, andthe droplet diameter decreases when the oil flow rate increases. We can also note thatby increasing both water and oil flow rates, the droplet generation frequency increases.
Using this range of flow rates controlled by Flow EZs with the droplet generator frommicrofluidic ChipShop, it is possible to obtain droplets with a diameter ranging from 80µm to 160 µm, and with a generation rate ranging from 50 Hz to 250 Hz.

7APPLICATION NOTE

200 µm200 µmFigure 4: Droplets on a glass slides observed with an optical microscope

Figure 5: Summary of droplet size and generation rate as a function of water and oil flow rates

8APPLICATION NOTE
Droplet Generator Chip for cells encapsulation
We have demonstrated in the previous section that it is possible to generate dropletswith a diameter ranging from 80 µm to 160 µm, and with a generation rate ranging from50 Hz to 250 Hz. In these ranges, one can encapsulate particles, solutes or cells withina droplet. Indeed, Langer and al. demonstrated E. Coli encapsulation using the Droplet
Generator Chip from microfluidic ChipShop. We can observe that about ten bacteria
were encapsulated within a droplet.

Figure 6: Example of E. coli encapsulation in dropletsHence, using the microfluidic setup presented in the technical note, it is possible togenerate monodispersed droplets that are suitable for the encapsulation of particles orcells.

CONCLUSION
We have demonstrated the use of Fluigent pressure pumps
combined with microfluidic ChipShop’s chip for the generation ofmonodispersed water-in-oil droplets. The versatility of the systemallows one to easily produce droplets of different diameters andat several generation rates. Droplets fabricated from this system
are suitable for many applications, including those that requirebiocompatibility.

For additional information, contact us by email : contact@fluigent.com or consult our website : www.fluigent.com
Microfluidic ChipShop website: www.microfluidic-chipshop.com

9APPLICATION NOTE
References
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Reports Prog. Phys. 75, (2012).
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Microfluidic High Throughput Screening (µHTS). Muhsincan Sesen,a Tuncay Alan,aand Adrian Neild*a 10715–10722 (2017) doi:10.1039/b000000x.
3. Galas, J. C., Bartolo, D. & Studer, V. Active connectors for microfluidic drops on demand. New J. Phys. 11, (2009).
4. Jullien, M.-C., Tsang Mui Ching, M.-J., Cohen, C., Menetrier, L. & Tabeling, P. Droplet
break in a low capillary T-junction. in 19th Mechanical French Congress (AFM, Maisonde la Mécanique, 39/41 rue Louis Blanc-92400 Courbevoie, 2009).
5. Yu, L., Chen, M. C. W. & Cheung, K. C . 2010 Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing. Lab Chip 10, 2424–2432 (2010).
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proof-of-concept to real-world utility? Chem. Commun. 55, 9895–9903 (2019).
8. Musterd, M., Van Steijn, V., Kleijn, C. R. & Kreutzer, M. T. Calculating the volume ofelongated bubbles and droplets in microchannels from a top view image. RSC Adv.
5, 16042–16049 (2015).