Droplet Microfluidics: How Droplets Are Made On Chip

Key takeaways

Droplet microfluidics generates and manipulates micron-sized, highly monodisperse droplets by bringing two immiscible liquids, usually an aqueous solution and an oil, together in microchannels.

Three chip geometries are widely used: co-flow with concentric capillaries, the T-junction with perpendicular channels, and flow-focusing, where two side streams of the carrier phase pinch the dispersed phase.

Three regimes govern break-up: squeezing gives plugs that fill the channel, dripping gives spherical droplets close to the channel width, jetting gives much smaller droplets at very high frequency.

Droplet size is set mainly by the flow rates of the two liquids, the chip geometry and materials, and the balance between viscous forces and interfacial tension.

Each droplet can act as an isolated reactor, which is the basis of single-cell sequencing methods such as Drop-Seq and of droplet-based digital PCR.

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What is droplet microfluidics?

Droplet microfluidics is the generation and manipulation of micron-sized, monodisperse droplets of one liquid inside a second, immiscible liquid as both flow through microchannels. Because each droplet stays separated from the next by the continuous phase, it can be handled as an individual biochemical reactor, from particle synthesis to single-cell analysis.

Microfluidic-based droplet control and generation allows for:

Highly monodisperse droplet production through “in-line”, continuous generation, unlike batch emulsion methods

Single droplet manipulation as an individual pL scale biochemical reactor

Miniaturization of production and bioanalytical devices via droplet generation

Microfluidics-based droplets have many diverse and varied applications, including particle synthesis [1] and physicochemical analysis [2].

Good control of droplet production can also make single-cell analysis [3] or drug testing possible [4,5].

This page explains the methods and technology used to make droplets on chip. For a survey of the wider field, see our droplet-based microfluidics guide.

alginate bead generation monodispercity — *Figure 1 Water in oil droplets produced on chip monodisperse droplets packed in a regular monolayer*

How are droplets generated in microfluidics?

The droplet generation method in microfluidics is based on the use of two immiscible fluids, usually oil and an aqueous solution. The droplets are made in devices called microfluidic chips. Different physical aspects are involved in the droplet production method, depending on the design and materials of the microfluidic chip. Among all chip designs to generate droplets, three are widely used in the field of microfluidics.

A co-flow, a T-junction and Flow Focusing design [6,7,8,9].

Droplet production in a co-flow design

The co-flow design for droplet production consists of two concentric capillaries with the inner capillary carrying the dispersed phase and the outer capillary carrying the continuous phase. As the dispersed phase enters the main channel, the viscous stresses created by the continuous phase stretch the interface until it breaks forming a droplet [7][10] [11][12] [13]

One of the main advantages is the simple design of the co-flow.

Nevertheless, the droplet size and frequency remain limited in this kind of design.

Figure 2 Co flow design the dispersed phase flows from an inner channel into the co flowing continuous phase which stretches the interface until droplets detach

Droplet production in a T-junction design

First highlighted by Thorsen et al in 2001 [14], this technique is the simplest and most commonly used to generate droplets in a controlled way. In a T-junction, the dispersed/internal phase is injected perpendicularly to the flow of the continuous/external phase to generate microfluidic droplets.

When the immiscible fluids arrive at the T-junction of the two channels, the dispersed phase progressively enters the main channel. The continuous phase shears at this point. The dispersed phase then forms a bend at the interface of the two fluids. The more the dispersed phase advances in the main channel, the more the elbow narrows to a break in the continuity of the interface. The break generates the detachment of a drop that continues to move in the direction of the flow of the channel. [15,16,17]

The advantages of this method of droplet production are the simplicity of the design and the great knowledge of the physical phenomena involved in droplet “break-up”, which allows for better understanding and control of the droplet generation process. In the T-junction design, it is very easy to control the droplet frequency and size. However, the frequency and size ranges remain limited by the chip design and materials.

Figure 3 T junction design the dispersed phase enters perpendicularly to the continuous phase flow and the interface narrows until a droplet detaches

Droplet production in a flow-focusing design

As demonstrated by Anna et. al. in 2003 [14]

In a flow-focusing design, the dispersed phase is introduced directly into the main channel, while the continuous phase is injected by two branches placed perpendicularly. The dispersed phase is then pinched on both sides by the continuous phase, and a droplet is formed due to the competition between the viscous force and the surface tension at the interface between the two phases [19]. One of the big differences with the T-junction design is the symmetry of the flow-focusing design.

The advantage is that the symmetric design and physics effect allows one to have more flexibility in terms of droplet size and droplet frequency. The symmetric design allows for droplet generation which is more sensitive to the flow of the two phases. The knowledge and control of droplet “break up” phenomena remain limited.

Figure 4 Flow focusing design the continuous phase injected from two perpendicular branches pinches the dispersed phase on both sides to form droplets

The three geometries compared

The three geometries differ in how the dispersed phase meets the carrier phase, and that sets the trade-off between simplicity, control and operating range. Co-flow and the T-junction both rest on a simple design; the T-junction adds the best-understood and most easily controlled break-up, while flow-focusing gives the most flexibility in droplet size and frequency.

Table 1. Co-flow, T-junction and flow-focusing geometries compared

Geometry	How the droplet forms	Main strength	Size and frequency range	Choose it when
Co-flow	Dispersed phase in an inner capillary, carrier phase in a concentric outer capillary; viscous stresses stretch the interface until it breaks [7,10]	Simple design	Limited	Design simplicity matters more than a wide operating range
T-junction	Dispersed phase injected from a side channel at right angles to the main channel; shear narrows the interface until a drop detaches [14,15]	Simple design; break-up physics well known, so generation is easy to control	Easy to adjust, within limits set by chip design and materials	Predictable, controlled generation is the priority
Flow-focusing	Dispersed phase in the main channel, pinched from both sides by two perpendicular side channels; viscous force competes with surface tension [18,19]	Symmetric design	Most flexible; more sensitive to the flow of both phases; break-up less well understood	A wide range of sizes or frequencies is needed and the feed of both liquids can be held steady

Whichever geometry is chosen, the shape of the droplets depends on the production regime it runs in, covered in the next section. Hybrid geometries also exist: the RayDrop co-flow-focusing device, developed by Secoya Technologies, combines co-flow and flow-focusing in a non-embedded design.

Which production regime fits my needs?

In a microfluidic droplet production method, several regimes of droplet formation are observable, depending on the experimental conditions:

The “squeezing” regime leads to a plug-shaped drop flow that occupies the entire width of the channel with a length-to-width ratio greater than 1. The size of plugs obtained depends mainly on the flow rates of the dispersed and continuous phases. The rupture of these plugs is due to the pressure drop in the main channel resulting from the presence of the dispersed phase.

The “dripping” regime leads to a flow of drops of spherical shape and dimension close to the width of the channel. In this case, the viscous shear forces cause the detachment of the drops.

The jetting regime is characterized by the generation of drops far from the intersection of the two phases. The drops generated are much smaller than the width of the channel and are generated at a very high frequency.

Figure 5 Droplet production regimes dripping jetting and stable co flow shown for co flow top flow focusing middle and T junction bottom geometries The squeezing regime described in the text is not represented

In short: choose squeezing for plugs whose length you set through the two flow rates, dripping for spherical droplets close to the channel width, and jetting when small droplets at high frequency matter most.

Table 2. Squeezing, dripping and jetting regimes: how to choose

Regime	What drives break-up	Droplet character	When to choose it
Squeezing	Pressure drop in the main channel caused by the dispersed phase; plug size depends mainly on the flow rates of both phases	Plug-shaped, filling the channel width, length-to-width ratio greater than 1	You need plugs, sized through the flow rates
Dripping	Viscous shear forces	Spherical, with a dimension close to the channel width	You need spherical droplets sized close to the channel width
Jetting	Break-up far from the intersection of the two phases	Much smaller than the channel width, generated at very high frequency	Small droplets at high frequency are the priority; check the size distribution, since flow-focusing experiments have produced both monodisperse and polydisperse emulsions across the range of flow rates [18]

The Droplet Starter Pack is the packaged route into these regimes: it supplies the pressure controllers, flow sensors, droplet generation chip and continuous phase with surfactant needed to generate droplets, as detailed further down this page.

What determines droplet size in droplet microfluidics

Droplet size in a microfluidic chip is set by the flow rates of the two liquids and the ratio between them, by the geometry and materials of the chip, and by the balance between viscous forces in the carrier fluid and the interfacial tension holding the dispersed fluid together.

Each lever shows up in the methods above. In the squeezing regime, plug size depends mainly on the two flow rates. In a T-junction, size is easy to control, but its range stays limited by chip design and materials. In flow-focusing, the competition between viscous force and surface tension sets break-up [19], and drop size has been mapped against the flow rates and their ratio, down to drops much smaller than the orifice [18].

To estimate the size a given geometry and set of flow rates should give, use the droplet size calculator. Because the flow-rate ratio sets the size, holding that ratio steady helps keep the size steady over a run: a Flow EZ pressure controller paired with a flow sensor regulates each fluid, the combination used in the Droplet Starter Pack. When generation drifts, our 10 tips for reliable generation cover the practical fixes.

set up Pressure based droplet generation

Figure 6. Pressure-based droplet generation: each phase is driven by a Flow EZ pressure controller with a Flow Unit flow sensor, holding the flow-rate ratio that sets droplet size.

Overview of Drop-Seq application in microfluidics

DNA and its expression are at the heart of cellular mechanisms, but our knowledge of cells and their diversity remains limited and incomplete. Study is essential to understand the development, functioning and reproduction of living beings, especially since mutations are at the origin of numerous pathologies (cancer, autoimmune disease, diabetes …). The currently available methods to determine and sequence the genome do not allow us to fully understand the functioning of DNA.

Drop-Seq and inDrop are methods and technologies that use microfluidics to tackle this issue by providing the ability to target thousands of individual cells simultaneously by encapsulating them in tiny droplets for parallel analysis [3].

Figure 7 Droplet generation with the Fluigent Droplet Starter Pack monodisperse droplets forming at the junction of a Fluigent chip

Recent advances in dPCR for microfluidic application

PCR (Polymerase chain reaction) is a well-known and widespread technique in molecular biology. It allows for amplification (with a multiplication factor of the order of one billion) of a known DNA or RNA sequence.

Currently, microfluidics has enabled a new technique which consists of single cell and PCR mixture encapsulation into microdroplets. The high droplet generation frequency and the low volume of the microfluidic system allow one to increase the number of amplifications possible while reducing the cost of reagent consumption.

single cell analysis droplets microfluidics — *Figure 8 Single cell encapsulation in droplets a droplet forming at a microfluidic junction the encapsulation step that isolates single cells and PCR mixtures in individual droplets*

Analysis of a commercial surfactant for digital PCR assay

The Fluigent Droplet Production Starter Pack

Figure 9 The Fluigent Droplet Starter Pack system two Flow EZ pressure controllers connected to the software through the LINK pressurize the water and dOIL + dSURF reservoirs each flow is measured by a Flow Unit before reaching the Fluigent EZ Drop chip

The Fluigent Droplet generation pack provides an easy droplet production method with total control of droplet size and frequency. The droplet generation pack provides all items needed to generate droplets, including pressure controllers, the chip and the surfactant.

In this pack, Fluigent gives you:

Pressure controllers: Flow EZ
Flow sensors: Flow Unit
Droplet generation chip: EZ-Drop
Continuous phase with surfactant: dSURF
Tubing pack with reservoir

Complex emulsion production platform

The Complex emulsion production platform is a platform dedicated for droplet production and complex emulsion such as double emulsion, microcapsules and more.

Figure 10. The Fluigent Complex emulsion production platform.

Figure 11. RayDrop single emulsion and double emulsion devices compared. Device: (a) single emulsion device, (c) double emulsion device. Droplet generation: (b) single emulsion droplets forming at the outlet of the injection nozzle, (d) a double emulsion droplet forming at the nozzle outlet.

For the generation step itself, Fluigent and Secoya Technlogies offer a single emulsion device and a double emulsion device.

This easy-to-use platform allows users to control droplet production to target multiple applications:

Double emulsion

Figure 12. Double emulsion droplets (scale bar: 100 µm).

Chitosan microcapsule

Figure 13. Chitosan microcapsules.

PLGA microcapsule

Figure 14. PLGA microcapsules.

UV-polymerised microcapsule

Figure 15. UV-polymerised resin microcapsules.

Frequently asked questions about droplet microfluidics

What is droplet microfluidics used for?

Droplet microfluidics is used wherever many small, isolated volumes of identical size are useful. Research applications include single-cell sequencing, digital PCR, cell encapsulation and drug testing, and the synthesis of particles, capsules and vesicles. Each droplet acts as its own reactor, so the analysis of thousands of cells or reactions can run in parallel.

Single-cell sequencing: Drop-Seq profiles gene expression in thousands of individual cells by encapsulating them in nanolitre droplets [3].

Digital PCR: encapsulating the PCR mixture in microdroplets increases the number of amplifications while reducing reagent consumption; see digital PCR.

Cell encapsulation and drug testing: droplet systems have been used for multicellular tumour spheroid formation and anticancer drug testing [5] and in drug discovery [4].

Particles, capsules and vesicles: double emulsions and chitosan, PLGA or UV-crosslinked microcapsules, GUV production, and oil-in-water emulsions.

How monodisperse are microfluidic droplets?

Microfluidic droplets are highly monodisperse when generation is stable, because microfluidic systems produce them in-line, one at a time at a single junction, rather than by batch emulsification. Monodispersity is not automatic, though: flow-focusing experiments have produced both monodisperse and polydisperse emulsions depending on the flow rates [18]. Holding the flow rates of both phases steady, in a deliberately chosen regime, helps keep droplet size uniform over a run.

Related Resources

References

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Droplet Microfluidics: How Droplets Are Produced On Chip