by Dr. Laurel Coons, Researcher at the National Institutes of Health and Duke University, and Richard Gray, Commercial Director at Dolomite Microfluidics
Phage display is a widely used research and drug discovery technique for creating and screening highly diverse peptide libraries to identify ligands for any target. Affinity selection involves panning of a phage library to enrich target-binding clones, followed by their amplification. Uniform amplification is of utmost importance for all selection experiments and, if it is not achieved, bias is introduced into the selection that favors faster growing clones regardless of the selection pressure applied. This article describes the development and optimization of a microfluidic flow-focusing technology (MFFT) approach as a high throughput method of generating monodisperse, droplet-based compartments to encapsulate individual phage clones and achieve non-competitive, uniform amplification of millions of clones with differing growth characteristics.
Addressing challenges and eliminating bias
Selection from phage display libraries is driven by two independent processes – the panning and amplification steps. Panning enriches clones that bind to the desired target or any other physical moieties present during this stage. Amplification, which is the infection of bacteria by a single phage particle to create multiple copies of genetically identical phage, enriches clones that have a growth advantage or amplify faster during the process – an exponential effect. These selection pressures are independent; increasing one does not eliminate the effect of the other. To eliminate this bias, individual phages must be separated into different growth chambers so they cannot compete for bacterial hosts.
Researchers have accomplished this by using MFFT. This generates monodisperse droplet-based compartments, separated from each other by an immiscible carrier phase, to encapsulate individual phage clones and achieve non-competitive amplification of millions of clones having different growth characteristics. Each droplet is therefore analogous to the traditional chemist’s flask, with the added physical advantages of reduced reagent consumption, rapid mixing and automated handling. The elimination of growth-based competition ensures that the selection of binding clones is driven only by the binding strength of each clone for the target.
A deep dive into water-in-oil droplet formation
Flow focusing is the production of droplets by straightforward hydrodynamic means. The output is a dispersed liquid in the form of a droplet emulsion. The basic principle consists of a continuous phase fluid surrounding the dispersed focused phase to give rise to a droplet in the vicinity of an orifice through which both fluids are extruded.
For droplets to be truly functional microvessels, there must not be any cross-contamination between them. For this, it is attractive to use a fluorocarbon oil as the continuous phase, as these oils are both hydrophobic and lipophobic. These oils are also highly permeable to oxygen, meaning that the growth of bacteria in droplets is not limited by oxygen. The low solubility of biological reagents in the aqueous phase makes these oils well suited for inhibiting molecular diffusion between droplets. However, droplets are still prone to coalesce, so adding surfactant to the continuous phase is critical for ensuring that droplets remain stable. These surfactant molecules have a hydrophilic head and hydrophobic tail, allowing them to align at the water-oil interface.
Researchers used a custom fluorosurfactant PFPE-PEG-PFPE – a triblock copolymer consisting of a polyethylene glycol (PEG) center block covalently bound to two perfluorinated polyether (PFPE) blocks by amide linking groups – to stabilize aqueous droplets in fluorocarbon oils. PFPEs are soluble in fluorocarbon oils and provide good steric stabilization of the water-in-fluorocarbon emulsion. PEGs are soluble in water and prevent adsorption of biological compounds at the droplet interface. PFPE-PEG-PFPE was therefore ideal as it provided stability to the droplets, preventing coalescence, and produced a biologically inert interior surface for the water droplets.
Figure 1: Droplet formation at the X-junction.
Development of the MFFT platform
The development of this novel technology began with demonstrating proof-of-concept. The microfluidic chip – a microfluidic flow-focusing device – is the pivotal part for generating droplets. This chip comprised two separate droplet junctions, an X and a T. The T-junction combined the phage and bacteria streams (aqueous), which were then fed into the X-junction using one central stream with the two outside streams (oil). Syringe pumps were used to inject the fluids into the microfluidic chip (Figure 1).
Using this set-up, researchers were able to demonstrate stable droplet production – with volumes of 3.74 nl – for short periods of time, droplet wall impermeability, long-term droplet stability over 24 hours, bacterial growth and phage infection or lysis within droplets (Figure 2). As the process uses viscous media, pressure-driven pumps with flow rate control were subsequently incorporated into the system.
Implementation of a high throughput droplet system
An innovative high throughput droplet system that is both reliable and versatile was established. Pulseless pressure-driven pumps with flow rate control were ideal for droplet formation where a highly stable flow was required. In addition, closed-loop flow control allowed for improved control of flow rates from as low as 70 nl/min up to 5 ml/min, resulting in long-term droplet monodispersity. Together with flow resistors and flow sensors, these pressure-driven pumps provided maximum flexibility, offering the ability to adjust the pressure for controlled flow rates and optimize the system for different fluids with varying viscosity and droplet sizes.
Figure 2: Isolation of phage in separate droplets containing bacteria.
A two-reagent droplet chip was used, enabling the two reagent streams – phage and bacteria – to remain isolated until right near the junction. The throughput was further enhanced by selecting a seven-junction, two-reagent chip. All seven junctions could be fed from a single input stream, maximizing performance for higher throughput applications, as well as enabling generation of droplets with volumes as small as 564 pl. This set-up enabled efficient, uniform amplification of complete phage libraries, and permitted the production of more than 10,000 stable monodisperse microdroplets per second (Figure 3).
Other applications: single cell genomics using microfluidic technology
Many biologically important processes take place at the single-cell level. However, many traditional techniques involve homogenizing tissue samples, and so only deal with population averages (masking the uniqueness of each cell). Droplet microfluidics offers the unique ability to isolate thousands to millions of cells in individual droplets. Genome-wide expression profiling of individual cells is done by separating thousands of individual cells into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. This results in transcripts from thousands of individual cells that are all identified by their cell of origin.
Figure 3: Stable droplet production.
In the Drop-seq method, tens of thousands of cells are individually encapsulated with uniquely barcoded RNA capture beads and lysis buffer. Encapsulation of single cells with single barcoded beads is achieved by limiting dilution. Once encapsulated, the cells lyse, and the mRNA is captured onto the bead in the cell. The barcoded oligo bead library is constructed such that each bead has a unique DNA barcode sequence but, within a bead, the thousands of copies of oligo all contain an identical barcode sequence. The 3’ end of the oligo has a poly(dT) stretch that captures mRNA and primes reverse transcription. Subsequently, beads are then released from their droplets and subjected to reverse transcription. The cDNA product is a full-length amplicon of the mRNA transcript due to the use of template switching oligonucleotides. The cDNA is then amplified and sequenced using conventional library preparation methods and next generation sequencing.
To accomplish this, the beads and cells are loaded into sample loops, allowing them to be flowed without mechanical stirring to avoid fragmenting the beads or premature lysis of the cells. One pump is loaded with the fluorocarbon oil and surfactant, the cells are loaded with the cell suspension buffer driven by another pump, and the beads are loaded with lysis buffer driven by a third pump. The system is first primed by pumping the buffers until drops appear at the connector, before turning the pumps off and switching the injection valves to the sample injection position. This set-up results in 330 pl droplets at 4 kHz (4,000 droplets/second), at flow rates of 40 μl/min each for the bead and cell suspensions, and 200 μl/min for the oil.
Summary
The user-friendly MFFT platform offers many benefits that can be demonstrated in a wide range of applications. As separate experiments take place in each droplet, this can reveal sample heterogeneity that would otherwise be difficult to observe using traditional techniques, making it easier to characterize rare cell types. This system can also be used to generate large numbers of identical sample droplets, which can be exposed to different compounds or conditions for applications such as drug discovery, highlighting the versatility of taking a microfluidic approach.