The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.
Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions – from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.
Microfluidics allows for the manipulation of small amounts of fluids in microchannels1. The scale of operation of typical microfluidic devices is tens to hundreds of micrometers which allows the miniaturization of chemical and biological reactions, thereby enabling such reactions to be carried out with relatively small quantities of reagents. Initially, microfluidic devices were fabricated with materials such as silicon2 and glass3. Although they are still in use4, they pose certain issues, such as solvent compatibility, high cost of manufacture, and difficulties in integrating controls for fluid flow5,6. PDMS-based fabrication methodologies, termed soft-lithography, offer an inexpensive alternative for the rapid prototyping of devices7 and an avenue to fabricate complex multilayered devices8. The addition of valves and pumps to PDMS devices allows for the ability to control the routing and speed of fluids in devices9,10. Several methods to design and actuate microvalves in a reversible or irreversible manner have been developed – for example, bimetallic valves made from silicon and aluminum, which are thermally actuated11 or using gas generated from an electrochemical reaction to deflect a silicon nitride membrane12. Gu et al. demonstrate the use of the mechanical pins of a Braille display to apply pressure on microchannels to regulate flow13. One set of microvalves that has gained popularity is the pneumatic PDMS-based valves pioneered by the group of Stephen Quake14. Typically, such valves are composed of two orthogonal microchannels – a flow channel and a control channel. Upon pressurization of the control channel, a thin PDMS membrane deflects upon the flow channel, closing it off and thereby interrupting fluid flow. Once depressurized, the membrane relaxes, thereby opening the flow channel and allowing the resumption of fluid flow. PDMS valves thereby allow flow regulation in a robust and reversible manner since the control channel can be pressurized and depressurized multiple times15. Additionally, since such valves can be actuated by the application of pressure, they open up avenues for digital control and automation16. Furthermore, as they are of the same material, they can be integrated seamlessly into the fabrication of PDMS-based devices using soft-lithography techniques8,17,18. These features make PDMS valves an attractive choice for flow regulation in microfluidic devices. Thorsen et al. used the principle of such valves to design a fluidic multiplexer – a combinatory array of pneumatic valves – to address nearly a thousand input flow channels with twenty control channels19. This principle has been extended to selectively route fluids to in-chip microfluidic chemostats such that unique reactions can be carried out simultaneously in each reactor20,21,22,23. However, such microreactors, while useful in optimizing the usage of limited reagents, cannot parallelize multiple reactions and are not sufficient for high-throughput studies.
Droplet microfluidics is a subcategory of microfluidics that involves the production of droplets through the manipulation of immiscible, multiphasic liquid flow in microfluidic devices24. Droplet formation involves the breakup of a continuous fluid by the introduction of an immiscible fluid, resulting in a pinch-off due to instability in the interfacial energy and in the formation of an emulsion25. Surfactants aid in the formation of rounded droplets when emulsions leave the microchannel by stabilizing the interfacial energies26. Larger droplets, called plugs, are less stable and can be collected in a holding compartment (such as a length of tubing) as an array of aqueous compartments spaced on either side by one or more immiscible liquids27. In addition to miniaturization and compartmentalization, droplet microfluidics also offers increased throughput of biological reactions, as a large number of monodisperse droplets can be produced – each serving as a nanoreactor28. Droplets, once generated, can also be subjected to further manipulations, such as splitting29,30, fusion31,32, sorting33,34, and assemblage into higher order structures35,36. Droplet microfluidics has revolutionized several scientific fields and technologies – from PCR37 to single-cell transcriptomics38, from drug-discovery39,40 to virology41, from next-generation sequencing42 to chemical synthesis43.
Integration of PDMS-based soft-lithography and microvalves with droplet technology is a potent combination that allows for the regulation of fluid flow in microchannels and subsequent control over droplet contents. Depending on the opening and closing of channels, it is possible to produce distinct populations of droplets, each with a specific composition. Such a platform could miniaturize, compartmentalize, and parallelize biochemical reactions and therefore be a useful technique for combinatorial screening44. Combinatorial screening is a high-throughput method to generate tens of thousands of combinations of selected reagents to produce libraries which consist of individual populations of known composition. Combinatorial screening has been used to discover synergistic effects between drugs and antibiotics for bacterial growth inhibition45. In the field of cancer therapy, combinatorial screening has been used to test combinations of anti-cancer drugs for a given patient thereby advancing personalized therapy46,47. Mathur et al. have built on this technology by integrating a combinatorial DNA barcoding approach to assess transcriptome changes in high-throughput drug screening48. Thus, combinatorial screening is a powerful yet nascent technology, and there is a need for developing diverse microfluidic technologies to execute and facilitate such screening procedures.
The goal of this manuscript is to present a complete set of protocols for the fabrication of a bilayer microfluidic device capable of generating a combinatorial library of water-in-oil plugs and describe the hardware and software needed for the operation of such a device. The fluid flow is regulated using pressure-controlled PDMS-based pneumatic valves, which are in turn controlled by a custom LabVIEW program. Flow of reagents in the device is achieved using commercially available pressure pumps. An eight-inlet prototype is presented wherein a plug is formed by the contents of three inlets, each containing an aqueous reagent. The aqueous phase meets a continuous oil phase, and plugs are produced at a T-junction with a frequency of 0.33 Hz. The functioning of the system is demonstrated by producing a quantitative library containing three distinct populations of fluorescent plugs. This technology and set of protocols will help to expedite the production of combinatorial libraries for high-throughput screening purposes.
In this paper, a set of protocols for the fabrication and operation of a PDMS-based microfluidic device for the automated generation of combinatorial libraries in water-in-oil compartments called plugs has been presented. The combination of microfluidics with droplet technology provides a powerful technique to encapsulate small quantity of reagents in a large number of compartments, therefore opening avenues for large-scale combinatorial screening.
Previously, several technologies have been described to generate chemically distinct compartments using microfluidics, each with its advantages and limitations. Kulesa et al.50, described a strategy to encapsulate cells with barcodes in droplets using microtiter plates and merging these droplets using an electric field to create a combinatorial library. While such an approach can generate a lot of combinations of droplets, it is limited by the need for manual handling steps in the workflow. Tomasi et al.51 developed a microfluidic platform to merge a spheroid (free floating cell aggregates)-containing droplet with a stimulus droplet, thereby allowing the manipulation of the spheroid microenvironment. This method allows for the study of important phenomena such as cell-cell interactions and the effect of drugs, but it is relatively low throughput. Eduati et al.46 and Utharala et al.47 developed a microfluidic valve-based platform that can generate high-throughput combinatorial libraries in an automated fashion. However, in these studies, the valves are operated using a Braille device, which necessitates cumbersome alignment steps between the microvalve and the microfluidic chip. A key feature of the system described in this paper is the implementation of pneumatic PDMS valves to regulate the flow of fluid in the input channels. Since these valves are PDMS-based, they can be incorporated rather smoothly into the fabrication steps of the microfluidic chip. Additionally, they are a relatively straightforward option to control the flow of liquids in the inlet channels, as they can be actuated by applying pressure through an external gas source. Finally, the duration and sequence of pressurization and depressurization of these valves can be programmed, thereby automating the production of distinct populations of plugs in a high-throughput manner. Another important feature is the use of constant pressure regimes for the injection of reagents through the inlet, which allows one to opt out of incorporating waste channels to relieve any pressure accumulation that arises in a constant flow rate regime. This simplifies the device design, reduces the need for additional valves and hardware to control the valves of the waste channel, and minimizes reagent wastage.
While the fabrication of devices with PDMS is relatively uncomplicated, the implementation of such devices does require the usage of extensive hardware paraphernalia such as the pneumatic solenoid valves (to control the actuation of the PDMS valves), pressure pumps (to control the flow of inlet and oil reagents) and software programs (to regulate the solenoid valves). While they represent a significant investment, such a setup provides consistency and reliability for the successful operation of the device. Additionally, the hardware components and architecture outlined in this protocol are set up in a modular way. Therefore, alternatives can be used for some modules to reduce costs or to adapt them to a specific need. For example, there exists a variety of pumps that can be used based on utility, budget, availability, and convenience52,53,54. Additional components such as fluid reservoirs and temperature regulators can be incorporated for sensitive inlet reagents23. Furthermore, this design can be scaled up or down to address specific scientific needs. For example, in this paper, an eight-inlet prototype is described which allows eight unique reagents to be combined to produce plugs. This can be upscaled to a 16-inlet device which allows for a higher number of inlets and larger combinations thereof. Consequently, it will need extra control channels and solenoid valves to address the inlets, but such a prototype allows larger and more diverse combinatorial libraries to be generated. Finally, in this paper, each plug population is produced by the opening of three out of the eight aqueous inlets of the microfluidic device. It was observed that for such a configuration, a pressure of approximately 200 mbar for the oil reagents and 400 mbar for the aqueous reagents corresponded to a regime of plug production, which is driven solely by valve actuation. When higher pressures were applied to the oil(s), a breakup of plugs was observed, and the application of lower pressures led to a fusion of plugs. The optimal pressure regime for plug production depends on a wide range of factors, such as the number of inlets contributing to the formation of a plug, the nature and viscosity of the fluids, and the dimensions of the channels, and should be optimized as and when necessary.
One of the drawbacks of operating in a constant-pressure regime is that fluids with different viscosities have different flow rates under constant pressure. Therefore, it needs to be ensured that the aqueous reagents flowing through the inlets are of comparable viscosities. The use of fluids of different viscosities will affect not only fluid flow in the inlet channels but also plug formation at the T-junction, thereby compromising the composition of the plug populations. Another drawback is the contamination of a plug population from residual reagents at the T-junction. When the device switches between the production of different plug populations, the first/last plug in the sequence of each population tends to be contaminated by the previous or the following population. This can be overcome by producing extra replicates of each population and discounting the contaminated plug during analysis. Finally, there is also the potential for variation between individual devices arising from inconsistencies in fabrication and/or external sources (pressure fluctuations). This issue can be mitigated by reusing a single microfluidic chip multiple times and ensuring that a complete run of a combinatorial library is performed on a single chip to minimize the effect of these inconsistencies.
The microfluidic device and the accompanying set of operational protocols presented in this paper have been used to demonstrate the production of a quantitative combinatorial library of plugs. This platform can, therefore, rapidly generate combinatorial libraries of distinct plug populations in a high-throughput manner. As a result, such technologies can be used for a variety of screening purposes including, but not limited to, combinatorial drug screening on patient biopsy samples – whereby a small number of cells retrieved from a biopsy can be distributed in a large number of droplets and treated with a large combination of the anti-cancer drug to optimize individual therapy for a given patient sample – and therefore accelerate personalized cancer therapy46,48,55.
The authors have nothing to disclose.
We would like to thank Stacey Martina of the NanoLab TuE for help with HMDS vapour deposition. This research was funded by the Institute for Complex Molecular Systems (ICMS) at TU/e and by the Netherlands Organization for Scientific Research (NWO) Gravitation programme IMAGINE! (project number 24.005.009).
1,1,3,3 tetramethyldisiloxane | Merck Life Science NV | MFCD00008256 | |
4 channel digital input/output module | WAGO Kontakttechnik GmbH | 750-504 | |
Acetone | Boom Labs | BOOMSKEUZW3 | |
Analysis Software | Eindhoven University of Technology | https://github.com/SysBioOncology/BilayerMicrofluidicsAnalysis_JoVE | |
AZ 40XT 11D | Merck Life Science NV | 212299 | Positive photoresist |
AZ 726 MIF developer | Merck Life Science NV | 10055824960 | Developer for positive photoresist |
Biopsy Punch, Rapid Core | World Precision Instruments Germany, GMBH | 504529 | 0.75 mm ID, W/Plunge |
Blue food dye | PME | FC1036 | |
Controller end module | WAGO Kontakttechnik GmbH | 750-600 | |
Ethernet Controller | WAGO Kontakttechnik GmbH | 750-881 | |
FC-40 | Merck Millipore | F9755-100ML | |
Fluigent flow unit | Fluigent | FLU-S-D | |
Fluigent pressure system | Fluigent | MFCS-EZ | 0 – 2 bar |
Fluorescein | Merck Life Science NV | MFCD00005050 | |
Hot plate | Torrey Pines Scientific | HP61 | |
Inverted microscope | Nikon Instruments | Eclipse Ti-E | |
Isopropanol | Boom Labs | BOOMSKEUZE3 | |
LabVIEW (Software Version 20) | Eindhoven University of Technology | https://github.com/SysBioOncology/BilayerMicrofluidicsAnalysis_JoVE/tree/main/LabVIEW_8_inlet_device_ VERSION_1 |
All files have been saved for LabVIEW version 20. It is advised to use this version or higher to open the files. |
Luer stubs | Instech Laboratories, Inc. | LS23 | 23 ga, 0.5" |
Male Luer to barb connectors | Cole Parmer | 45505-32 | 3/32" ID |
MasterFlex PTFE tubing | Avator/VWR | 48634 | |
Microscope Slides | VWR | 470150-480 | |
Microscope slides, Plain | Corning | 2947-75X50 | |
Mineral Oil | Merck Millipore | 330760-1L | |
mr DEV 600 | Micro resist Technology | R815100 | Developer for negative photoresist |
Oven | Thermo Scientific | Heraeus T6P 50045757 | |
Oxygen plasma asher | Quorum Technologies | K1050X | |
Photomask | CAD/Art Services, Inc. | ||
Photomask Design | Eindhoven University of Technology (Adapted from Merten Lab, EPFL) | https://github.com/SysBioOncology/BilayerMicrofluidicsAnalysis_JoVE/blob/main/8_inlet_JoVE_device_design.dwg | |
Pneumatic valve array | FESTO | 1x 8 valve array, Normally closed valves | |
Silicon Wafers | Silicon Materials | <1-0-0>, 100 mm diameter, 525 μm thickness | |
Single edge blades | GEM Scientific | ||
Soft tubing | Fluigent | 1 mm ID, 3 mm OD | |
Spin coater | Laurell Technologies Corporation | WS-650MZ-23NPPB | |
Stereo microscope | Olympus Corporation | SZ61 | |
SU-8 3050 | Kayakli Advanced Materials | Y311075 1000L1GL | Negative photoresist |
Sylgard 184 Silicone Elastomer Kit (PDMS) | Dow | 1317318 | |
Syringe | B Braun Injekt – F Fine Dosage Syringe | 10303002 | |
UV-LED exposure system | Idonus | UV-EXP150S-SYS | |
Vacuum pump | Vacuumbrand GmbH | MD1C | |
Weighing scales | Sartorius | M-prove |