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6 Top Tips Resolving Challenges of Droplet Generation

Updated: Mar 3, 2022

Double-Encapsulation:  Single-cell droplet in oil shell
Double-Encapsulation: Single-cell droplet in oil shell

Droplet generation studies have been growing considerably in the past few years mainly due to the significant advantages they bring, particularly for high-throughput single cell analysis: millions of single-cell reaction droplets in one eppendorf tube is the equivalent of >10,000 96-well plates with a single cell per well.  

Applications for droplet generation are far-reaching and go beyond drug discovery and diagnostics into such areas as food and cosmetics production; and industrial applications such as paints.  

Microfluidic droplet generation can offer significant cost savings compared to more traditional production techniques and it's an exciting field which is under continuous development.   But as with the evolution of any new field, there are also challenges so we've put this blog together to highlight some of the most common questions we get asked by our customers when setting up their own droplet generation experiments.

Cellix's Top Tips:

1. Protocol:  The order in which you fill the microfluidic channel with different solutions is critical! Flow sensors (which are connected to the pumps) are calibrated for different liquid types; e.g. for oil or aqueous-based solutions. It is a common mistake to connect both pumps (oil and aqueous phase) to the chip at the start of the experiment! If you then try to prime the chip by pre-filling with the oil phase while the chip is connected to the pump containing the aqueous phase; oil is likely to back-flow into the sensor connected to the pump for the aqueous phase.  The flow sensor connected to the aqueous phase is only calibrated for aqueous-based solutions.  If a non-aqueous solution, such as oil, flows into this flow sensor, it will result in incorrect flow rates being delivered for the aqueous phase once you start your experiment. 

Cellix's top tips:

  • Disconnect the chip from the pump which contains the water phase

  • Pre-coat the microfluidic channels ​of your chip with solution to ensure it is hydrophobic.  Cellix recommends DropGen PreCoat for use with our DropChips.  

  • Pre-fill the microfluidic channels of your chip with the oil phase - remember to keep the pump for the aqueous phase disconnected!

  • Connect your microfluidic pump for the water phase to the flow sensor and pre-fill with the aqueous phase until you see a droplet coming out at the end of the tubing.  You may now connect this tubing to your microfluidic chip.  


2. Achieving uniform, stable droplets:  Two of the top factors affecting droplet stability are channel surface chemistry (hydrophobic for water-in-oil droplets and hydrophilic for oil-in-water droplets) and addition of surfactant in the oil phase which stabilises the interface between the oil and water phases.  After this, it comes down to tweaking the ratio of the flow rates of the oil and water phases.  

Cellix's top tips:

  • Water-in-oil droplets need a hydrophobic channel: Ensure the surface of the microfluidic channel is hydrophobic. A lot of our customers make their own chips in PDMS which is naturally hydrophobic but is often sealed with a glass coverslip which is hydrophilic.  To ensure you have a completely hydrophobic surface, the first thing you should do is fill the microfluidic channel with a solution such as Cellix's DropGen PreCoat.  Leave this in the channel for at least 10 minutes, then flush it out with air or immediately prime with the oil phase before starting your experiment. 

  • Prime the channels of your microfluidic chip: Also make sure that you fill your channel with your continous phase liquid first; i.e. oil for water-in-oil droplets or water phase for oil-in-water droplets.  

  • You can also try increasing the concentration of your surfactant in oil.  


3. Droplet generation stops mid-way through experiment and both oil and water-based phases become laminar flow:  The flow is unstable.  There can be a number of reasons for this.  

Cellix's top tips:

  • Water-in-oil droplets need a hydrophobic channel: Did you coat the microfluidic channels first with DropGen PreCoat or other similar reagent to ensure hydrophobicity?  If not, start again...

  • Check for leaks or blockages: If you did coat the microfluidic channels with a reagent to ensure hydrophobicity (for water-in-oil droplets only); then you should check to ensure that there are no leaks or blockages (e.g. air bubbles) in your system - this can often result in a change in flow rate​ which disrupts the formation of droplets.  

  • Optimise the flow rates: If there are no leaks or blockages, try keeping the flow rate of the oil phase constant while slowly ramping up the flow rate of the aqueous phase but make sure you do not allow the oil phase to back-flow into the sensor controlling the water phase.  See our tip #1 above about protocol.


4. Controlling Droplet Size:  The most important factor affecting droplet size is the geometry of the microfluidic chip, in particular, the junction where the oil and water phase meet.  

Cellix's top tips:

  • Geometry:  Select the channel geometry (particularly the junction where the oil and water phases meet) of the microfluidic chip such that it can easily create the droplet sizes that you want.  Check out our DropChip page for a table with details of the droplet diameter and volumes that can be achieved with Cellix's DropChip. 

  • Surfactant:  To a lesser extent than geometry, the surfactant concentration (normally added to the oil phase) will affect the size of the droplets.  By altering the surfactant concentration, you will slightly alter the ratio of flow of the different phases enabling you to achieve very slightly different sizes; i.e. you can achieve more water in your droplet, thereby making it a slightly bigger size.  Cellix's recommends DropSurf surfactant for droplet generation studies.

  • Ratio of flow rates of oil and water phases:  If the geometry of the microfulidic chip is fixed, altering the ratio of flow rates of the 2 phases has a much lesser affect on droplet sizes.  However, it can still produce a minor change but there is typically only a very small range of flow rates for a specific fixed geometry that will work so this will only give you limited flexibility.


5. Biocompatible oil and surfactants for droplet generation:  This is a common question for those interested in applications related to cell-based work.  

Cellix's top tip:  Mineral oil works very well for the continuous phase and is widely available but biocompatible surfactants are not as widely available and tend to be quite expensive.  Cellix recommends DropOil for droplet generation studies.


6. Measuring droplet stability (size, distribution) and monodispersity:  There are lots of software packages available which work well, including Image-Pro Premier offered by Cellix.  However, it should be noted that all of these software programs work in 2D and so precision is often compromised.  Even a very small change in diameter of the droplet can have a significant effect on the volume of the droplet. 

Cellix's top tip:  When developing our DropChip and characterising the droplets which were generated, we used scattering (3D) - this enables us to accurately measure the size, volume and frequency of droplet generation.  There is a significant amount of work associated with characterisation of droplets but if you want to be absolutely confident, we recommend using a 3D method such as scattering.


If you're struggling with your droplet generation set-up and would like some advice,

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