Name: Video: Production of a Combinatorial Plug Library from a Bilayer Microfluidic Device
Uploaded: 2023-12-01T12:30:00
Duration: 5 min 6 s
Description: 252 Views. Production of a Combinatorial Plug Library from a Bilayer Microfluidic Device

production of a combinatorial plug library from a bilayer microfluidic device

Setup of a Microfluidic Device for Combinatorial Plug  Production

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.&#160;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.

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Bilayer Microfluidic Device for Combinatorial Plug Production

Production of a Combinatorial Plug Library from a Bilayer Microfluidic Device

production-combinatorial-plug-library-from-bilayer-microfluidic

Research

JoVE Journal

Bioengineering

252 Views. Production of a Combinatorial Plug Library from a Bilayer Microfluidic Device

Video: Production of a Combinatorial Plug Library from a Bilayer Microfluidic Device

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.

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 ...