Sequential Production and Trapping of Droplets

The microfluidic chip Fluidic 719 consists of a flow focusing droplet generator connected to a channel with 2261 microfluidic droplet traps of 173 μm diameter. The microfluidic traps work by floatation, profiting from the difference in density of the two phases of an emulsion. If droplets of the same size as the traps are produced, they will remain individually trapped over time and can be identified according to their position. Combined with the multi-injection valve MUX distributor, the dispersed phase can be seamlessly changed to produce batches of different droplets in the same chip to be observed and compared under the same conditions. This Application Note describes the procedure and equipment involved to achieve sequential production of different droplets and their storage in microfluidic traps for in situ optical analysis.

Sequential Production and Droplet Trapping Applications

• Comparative chemical reactions
• Single cell encapsulation
• Antimicrobial screening
• Chemical-cell interaction testing
• Diffusion studies
• High-throughput experiments
• Concentration experiments
• Study of emulsion stability

Experiment setup

Flow controller OB1

Flow sensor

MUX Distribution Valve

Fluidic 719 – ChipShop

Figure 1: Setup schematic

Materials

Hardware

• OB1 flow controller with at least 2 channels (0-2000 mbar)
• Flow sensors (MFS3, 2.4-80 μL/min & MFS2, 0.4-7 μL/min)
• Tubings (1/32” OD PTFE tubing with sleeves; 20 cm of 100 µm ID resistance tubing), fittings and reservoirs
• 9-port manifold
• Fluidic 719 droplet generation and storage chip from Microfluidic ChipShop 
• 1 pack of mini Luer connectors and male Luer plugs from Microfluidic ChipShop
• 1 pack of Fluidic 438 male low volume displacement mini Luer plugs from Microfluidic ChipShop
• MUX distributor

Chemicals

• HFE-7500 + 1 % FluoSurf surfactant (Emulseo, France)
• Filtered water
• Color dyes

Software

• ESI software

Design of the Chip

There is one flow focusing device per chip to produce the droplets to be trapped. There is one inlet for the continuous phase (1) and up to three inlets (2-4) for the dispersed phase (Detail A). Two of these can be used to mix different substances right before droplet formation, however, in this application we will only use one of them to generate a variety of different droplets. There are two other inlets in the opposite side that can be used to produce double emulsions that will not be used in this experiment (6&7). Once produced, the droplets are transported though a long serpentine channel with seventeen straight sections. Each one of these sections has 133 droplet traps of 173 μm diameter. The traps are positioned alternatively in a zigzag pattern along the channel, allowing flow around the trap if occupied by a droplet. Therefore, the droplet size is critical in this experiment, as bigger droplets will not be trapped and smaller droplets will be pushed away. By making droplets in the 150-180 μm diameter range, we ensure there will be only one droplet per trap and it will remain stable in that position. There are two different outlets, one right before the serpentine channel that can be used to extract droplets after production (8) and another one at the end of the serpentine channel (5). Connections to the chip is done using Microfluidic ChipShop Mini Luer connectors, while the inlets and outlets that are not in use are sealed using low volume displacement male Luer plugs.

Quick start guide

Instrument Connection

1. Connect your OB1 pressure controller to an external pressure supply using pneumatic tubing and to a computer using a USB cable. For detailed instructions on OB1 pressure controller setup, please read the “OB1 User Guide”.
2. Connect the flow sensor to the OB1. For details, refer to “MFS user guide”.
3. Turn on the OB1 by pressing the power switch.
4. Launch the Elveflow software. The Elveflow Smart Interface’s main features and options are covered in the “ESI User Guide”. Please refer to the guide for a detailed description.
5. Press Add instrument choose OB1 set as MK4, set pressure channels if needed, give a name to the instrument and press OK to save changes. Your OB1 should now be on the list of recognized devices.
6. OB1 calibration is required for the first use. Please refer to the “OB1 User Guide”. 
7. Add the flow sensor: press Add sensor select flow sensor analog or digital (choose the working range of flow rate for the sensor if you have an analog one), give a name to the sensor, select to which device and channel the sensor is connected and press OK to save the changes. Your flow sensor should be on the list of recognized devices. For details refer to “MFS user guide”.
8. Add the MUX distributor: press Add instrument select MUX distributor and give a name to the instrument. Your MUX distributor should be in the list of recognized devices.
9. Open the configuration of the flow sensor measuring oil (MFS3) and in the calibration tab change the settings from H2O to Isopropyl.
10. Open the OB1 and Mux Distributor Windows.
11. In the OB1 window, in each of the operating channels, open the sensor settings, and in the PID values, change both responsiveness and smoothness from 0.001 to 0.008 for the water (dispersed phase) and to 0.012 for the oil (continuous phase).

Tips from the expert. These parameters are adapted to the oil used and dispersed phase with the properties of water using 20 cm of 100 µm ID resistance tubing. If phases of different properties, viscosities or different resistance tubing are used, these values should be adjusted for reliable flow control.

Set-Up Preparation

1. Fill two reservoirs with perfluorinated oil (reservoirs A), and up to 7 with the desired aqueous solutions (reservoirs B).
2. Connect the cap of one A reservoir directly to the OB1 with pneumatic tubing; this is the continuous phase. Branch all reservoir B caps along with the other reservoir A to a 9-port manifold as shown in the schematic and connect to the OB1. Any unused channels in the manifold must be sealed with the included plugs.
3. Connect reservoirs B and A from the manifold to consecutive MUX Distributor inlets using 1/32” OD tubing. Connect another piece of tubing to the central position, serving as the outlet.
4. Connect reservoir A to the flow sensor (MFS3) inlet with 1/32” tubing. Add a piece of tubing to the outlet of the flow sensor and add a mini Luer male fitting to the free end of tubing (destined for the chip inlet).
5. Connect the tubing from the MUX distributor outlet to the MFS2 flow sensor inlet. Add 20 cm of 100 µm ID resistance tubing to the outlet of the MFS2, then another piece of 1/32” tubing using using a union connector. Add a mini Luer male fitting to the free end (destined for the chip inlet, step 16 below).

Tips from the expert. For more details on the use of resistance to obtain the best performances in terms of flow rate control please refer to the “Flow control tuning” document.

6. Secure another piece of 1/32” tubing to a waste reservoir (using a cap, a piece of tape…) and connect the opposite end to a mini Luer male fitting.

Tips from the expert. The chip is connected in later steps to make sure it is completely filled with oil before the dispersed phase sequence reaches the flow focusing nozzle. This will aid generation of stable droplets of the right size and sequence, as well as limiting air bubbles.

Experiments: Dispersed Phase Sequence Production

The following steps detail the formation of a sequence of aqueous solutions in the tubing, separated by small plugs of oil to prevent them from mixing, before the droplet generation chip. Preparation of the dispersed phase sequence before connecting the tubing to the droplet generation and storage chip aids in the downstream sequential production of droplets with different contents because of the very small volumes involved. 

1. Open the MUX distributor control window.
2. The channels inside the MUX distributor must be purged of air. To do so, separate the tubing connected to the MUX distributor outlet from the MFS2. Set pressure (200 mbar for example) of the dispersed phase and consecutively flow each one of the solutions until they can be seen in the outlet tubing. This way you make sure all the working volume is filled.
3. Substitute the piece of tubing filled with reagent and air by a new empty one, and connect it to the MFS2.
4. In the MUX control window, choose the valve corresponding to the simplest aqueous solution in the experiment (for example water or buffer), and specifically, do not choose the oil phase connected to the MUX.

Tips from the expert. The channels and resistance should be filled with liquid to obtain an accurate reading of the flow rate and use flow control as opposed to pressure control. Therefore, the liquid needs to have the properties of the dispersed phase corresponding to water, as specified in the sensor parameters.

5. Set pressure (200 mbar for example) of the dispersed phase (MFS2) until the solution starts dripping out of the end of the tube connected to the mini Luer male fitting (i.e. that will be connected to the chip inlet later).

6. Switch the control settings from pressure control to flow control (“sensor” toggle) and set the flow rate to 0.5 μl/min. Then, prepare the sequence in the MUX distributor interface.

7. Select the reservoir containing the oil from the MUX distributor window, and let it flow at 0.5 µl/min for 1 minute to generate a 0.5 μl oil plug. The oil will act as a spacer to prevent the subsequent solutions from mixing inside the tubing.

Tips from the expert. The volume of the oil plug is not crucial and does not need to be exhaustive. However, bigger plugs will considerably affect the flow control and smaller plugs can fail to reliably separate consecutive plugs of reagents inside the 1/32” OD tubing.

8. Sequentially change the MUX distributor valve position according to the sequence of droplets to be produced later. Each of these aqueous solutions must be separated by 0.5 μl of oil to avoid cross-contamination of the dispersed phases.

Tips from the expert. The ideal droplet size is equivalent to 1.5-2 nl of volume. This means that the total volume of dispersed suspension for 2261 droplets, not counting oil spacer volume, is approximately 4 μl. Thus, to prepare a dispersed phase sequence of 4 different aqueous reagents, ~1 μl of each reagent is required. Using a constant dispersed phase flow rate of 0.5 μl/min, prepare an injection sequence where each reagent flows for 2 min. See the Sequential Injection User Guide for tips on creating injection sequences.

9. The whole sequence should be finished by another plug of oil. Many sequences can be prepared to fill multiple chips consecutively. Depending on the stability of reagents, even pieces of tubing containing the desired sequence can be produced, sealed and stored to later produce the droplets as long as the air is expelled before connecting the chip

Tips from the expert. In case a previously prepared sequence needs to be injected, just fill the whole circuit after the MUX distributor with neutral solution and then substitute the tubing piece at the end, connected after the microfluidic resistance with the prepared piece.

Experiments: Droplet Generation and Storage

10. Select an aqueous solution with the MUX distributor and use it to push the sequence through the tubing connected to the MUX outlet until the first plug of oil reaches the end of the tube connected to the mini Luer male fitting.

Tips from the expert.At this stage, the flow rate can be changed to up to 4 μl/min to accelerate the waiting time. However, higher flow rates might affect the prepared sequence and the flow should be progressively slowed down as it nears the end of the tubing.

11. Stop the flow, and make sure the sequence stays still in the tube by placing it gently on a flat surface.

12. Set pressure (200 mbar for example) of the continuous phase (MFS3) until the solution starts dripping out of the tubing and then connect the mini Luer male fitting to inlet 1 of the Fluidic 719 chip.

13. Connect the mini Luer male fitting from the waste to outlet 5.

14. Seal all remaining inlets and outlets with low volume displacement plugs.

Tips from the expert.Other microfluidic plugs can also be used. However, once all traps are filled, big volume fluctuations may push or displace the trapped droplets.

15. Fill the microfluidic chip with oil and switch the control settings from pressure control to flow control to a set flow rate of 25 µl/min.

16. Remove the plug from inlet 3 and connect the mini Luer male fitting from the MFS2.

17. Restart the flow of the dispersed phase, now connected to the chip, at 0.7 µl/min.

18. The droplets will then be produced sequentially. It is convenient to supervise the droplet production process and adapt the dispersed phase flow rate in case of fluctuations in droplet size.

Tips from the expert. The oil flow rate is carefully chosen in tune with the dispersed phase for droplet production and to optimize droplet trapping. A faster flow rate might push droplets out of the traps, while a slower flow rate might cause droplets to get stuck outside the traps, where they are squeezed and not stable over time. If any of these happen, the flow rate can be adjusted, but it is recommended to stay within the 20-30 µl /min range. If the droplets move too slowly even at such flow rates check for leaks in the circuit.

19. Once the sequence is finished, stop the channel corresponding to the dispersed phase (MFS2), and after a minute, substitute the corresponding mini Luer male fitting for a low displacement mini Luer plug.

20. Keep the flow rate of the continuous phase stable and running until all droplets are stabilized in a trapped and none of them are circulating along the serpentine channel.

21. Remove the mini Luer male fitting from the outlet and leave the outlet unstoppered (open).

22. Decrease the flow of the continuous phase progressively until it reaches zero.

23. Carefully remove the corresponding last mini Luer male fitting (from port 1) and quickly seal both the inlet (port 1) and the previously unplugged outlet (port 5) with low displacement mini Luer plugs.

Tips from the expert. The oil used in this application note evaporates fast, hence the need for sealing the inlets and outlets of the chip as soon as possible when they are not in use. If air or a bubble reaches the serpentine channel, it will likely displace the droplets due to its lower density and ruin the sequence.

Tips from the expert.In the case of cell culture, given the affinity of oil for oxygen, the droplet environment will remain aerobic for a few hours. Dynamic culture can be done by continuously circulating oil, but this will progressively reduce the droplet size over time.

24. The chip is now ready for observation and will be stable for days.

Results of Sequential Production and Droplet TRapping

Droplets were generated using the droplet generation and storage chip (Figure 1). A quick indicator of the right size of the droplets (150-180 µm diameter) can be visualized right after production. Immediately after the dispersed phase is pinched off, the droplet should be circular and almost tangential to the channel walls.

Figure 1. Sequential droplet generation viewed by bight field microscopy. Droplets are generated at the flow focusing junction of the chip. The dispersed (aqueous) phase is entering from the left. The continuous phase (oil) is entering perpendicular to the dispersed phase from both sides (visible left hand side of image) to steadily pinch the dispersed phase into droplets

Droplet contents were changed by preparing sequentially alternating aqueous solutions using a rotary valve positioned before the chip. Following this protocol, the liquids do not mix inside the tube or the in the nozzle during droplet production. Oil plugs between the aqueous solutions become part of the continuous phase when they meet at the nozzle of the droplet generator. Thus, there is no transition gradient between aqueous reagents and droplet boundaries are sharp (Figure 2). 

In the case that reagents cannot be differentiated under the microscope (e.g. if they are the same color), a reservoir of colored water can be connected to the MUX distributor and used to indicate the end of one reagent and start of the next. In order to create a spacer of colored droplets, instead of using only a plug of 0.5 μl of oil for separation, a sequence of 0.5 μl oil, 0.5 μl colored water, 0.5 μl oil should be used.

Figure 2. Stabilized droplets trapped inside the microfluidic chip. The limit between four different solutions (here shown as droplets made from green, red, blue and yellow dye) was sharp and easily discerned.

Droplets are stable in the chip for days for in situ analysis.

Application note written by Jesús Manuel Antúnez Domínguez 

Acknowledgements

This application note is part of a project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 812780.