Published: March 9th, 2021
Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.
Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.
Droplet microfluidics leverages compartmentalization in picoliter droplets to increase the sensitivity and accuracy of assays compared to bulk reactions, and have numerous applications in chemical screening, protein engineering, and next generation sequencing1,2,3. For example, digital droplet polymerase chain reaction (ddPCR) affords increased accuracy compared to bulk quantitative polymerase chain reaction (qPCR), with applications for genetic variation in cancers, detection of disease causing mutations, and prenatal diagnostics4,5,6. A challenge of droplet microfluidics, however, is the requirement of microfluidic devices to partition samples; while microfluidics afford excellent control over droplet properties, they require specialized expertise to build and operate7,8. Consequently, droplet-based methods are largely limited to expert labs or, in rare instances, applications in which a commercial instrument is available9,10. To broaden the use of droplet assays, the requirement for specialized microfluidic instrumentation is a hurdle that must be overcome.
In this article, we describe Particle Templated Emulsification (PTE), a microfluidic-free method for performing reactions in monodispersed droplets. In PTE, templating particles engulf the sample into droplets in carrier oil by simple vortexing (Figure 1). As the system mixes, the aqueous portion fragments into droplets of reducing size until the droplets contain single particles, at which point further fragmentation is not possible because it requires breaking the particles. The engulfed sample surrounds the particles as a shell in the droplets, thereby encapsulating any dispersed cells, reagents, or functional moieties (Figure 1D). Thus, PTE requires no equipment or expertise to perform droplet reactions beyond a common vortexer. Additionally, droplet generation takes seconds compared to minutes or hours with microfluidics, and the amount produced is proportional to the container volume, not device operation time, making it supremely scalable. These benefits make PTE ideal for conducting droplet assays in a variety of circumstances in which microfluidics are impractical. Here, we demonstrate PTE and use it to conduct ddPCR.
Figure 1. Overview of particle templated emulsification process. (A) Templating particles are mixed with reagents. (B) Excess reagents are removed following centrifugation. (C) The addition of template molecules occurs before the addition of oil. (D) Vortexing produces droplets containing a single template molecule. (E) Subsequent thermocycling and imaging allows for digital droplet analysis of target template. Please click here to view a larger version of this figure.
1. Preparation of hydrogel particles for particle templated emulsification.
Hydrogel particles used for particle templated emulsification can be prepared using two different methods.
2. Particle templated emulsification.
Following the preparation of templating particles, PTE is used to encapsulate the sample and reagents in droplets.
|Particles (450 particles / μL)
|2x PCR master mix
|10 μM forward primer
|10 μM reverse primer
|10 μM probe
|Nuclease free water
Table 1. Preparation of the PCR master mix used with PTE for digital droplet PCR.
3. Digital droplet PCR and analysis.
|Repeat x34 steps 2 to 4
Table 2. Thermocycling conditions for digital droplet PCR using PTE emulsions.
Figure 2. Encapsulation of sample into droplets using particle templated emulsification. (A) templating particles used for particle templating emulsification. (B) Separation of templating particle pellet from supernatant following centrifugation. (C) Droplets resulting from particle templated emulsification with (D) identifiable aqueous shell. Please click here to view a larger version of this figure.
In PTE, the monodispersity of the emulsions is dictated by that of the templating particles, because the droplets have a diameter slightly larger than the particles. Thus, uniform particles are central to controlled PTE encapsulation11. A variety of methods exist for generating uniform templating particles, including chemical (sol-gel, emulsion polymerization), hydrodynamic (membrane emulsification, homogenization), and filtration methods. Microfluidic approaches in particular, afford superb monodispersity (Figure 2A) and allow additional particle engineering to enhance their functionality in PTE12. Alternatively, templating particles can be purchased, although their uniformity, while adequate, is typically less than with microfluidic generation11.
To perform PTE, the particles are mixed with the sample to be encapsulated (Figure 1A), and the excess supernatant is removed by centrifugation and pipetting (Figure 1B), as illustrated by a photograph of a particle pellet at the bottom of a PCR tube (Figure 2B). The encapsulating oil containing a stabilizing surfactant is then added (Figure 1C), and the sample gently pipetted before vortexing for 30 seconds (Figure 1D), to generate the emulsion (Figure 2C). The resultant droplets contain a particle core and aqueous shell comprising the initial sample, within which reside the reagents, target molecules, and cells necessary for the reaction (Figure 2D). Just as in droplet microfluidic encapsulation, discrete entities like small beads or cells are encapsulated randomly and in accordance with a Poisson distribution, although nearly all droplets contain a templating particle due to the nature of PTE physics.
Figure 3. Identification and cleanup of particle templated emulsification droplets. (A) Example of non-uniform droplet generation with multiple particles per droplet from insufficient vortexing. (B) Expected presence of satellites and droplets following particle templated emulsification and (C) the water-in-oil fractionation. (D) Resulting emulsion following oil washing. (E) Excessive satellite generation resulting from residual supernatant during particle templated emulsification. Please click here to view a larger version of this figure.
Even in successful PTE, double or triple core droplets exist, though they generally contribute negligibly to the reaction, provided they are rare. Achieving a low frequency of multicore droplets while retaining adequate shells requires optimization of process parameters, including surface tension, inter-particle adhesion forces, sample viscosity, container size, and vortexing power and time. For example, a poorly optimized emulsification may contain polydispersed droplets with many templating particles (Figure 3A), indicating that the vortexing was insufficient to fully emulsify the sample. In such instances, detergents can be added to reduce inter-particle adhesion and lower surface tension, or vortexing power or time can be increased. Another common issue is generation of excessive satellites, which are small empty droplets (Figure 3B). Satellites can be unavoidable in PTE emulsions depending on the interfacial tension and rheological properties of the sample and carrier oil. However, they often result from not adequately removing excess sample prior to emulsification (Figure 2B), or vortexing with too much power, stripping the shells from the droplets. In a successful PTE emulsification, satellites should comprise no more than ~10% of the total encapsulated sample volume (Figure 3C)11. At this level, they usually contribute negligibly to the reaction and can be ignored. For aesthetic purposes, they can be cleared from the emulsion by washing with fresh oil (Figure 3D).
Figure 4. Evaluation of particle templated emulsification digital droplet PCR. (A) Fluorescent imaging of the droplets identifies positive fluorescent droplets and negative non-fluorescent droplets. (B) Identification of rare template or low concentrations of template with digital droplet PCR. (C) Over abundant template encapsulation resulting in a variable number of template molecules per droplet. Please click here to view a larger version of this figure.
To demonstrate the utility of PTE, we used it to perform microfluidic-free digital PCR11. Using the process, we encapsulated a sample comprising S. cerevisiae genomic DNA, and thermocycled it. In digital PCR, droplets containing amplified targets become fluorescent, while those without remain dim. Thus, a fluorescent droplet indicates a target, allowing direct quantitation of targets by counting positive droplets (Figure 4A). The number of fluorescent droplets thus scales with the target molecules, yielding few positives when the target is rare (Figure 4B) and many when it is abundant (Figure 4C). As with encapsulation of other discrete components, target encapsulation follows a Poisson distribution, allowing the positive droplet fraction to be transformed into the target concentration (Figure 4D), thereby demonstrating the ability to perform digital PCR with PTE11.
Figure 5. Demonstration of digital droplet PCR using commercially available PAA. (A) Fluorescent imaging of the droplets identifies negative non-fluorescent droplets. (B) Identification of low concentrations of template with digital droplet PCR. (C) Identification of high concentrations of template with digital droplet PCR. Please click here to view a larger version of this figure.
These results are repeatable using commercially available polyacrylamide particles (Figure 5) and demonstrate the ability of PTE to perform standard digital PCR with commercially available polyacrylamide particles, achieving accurate measurements over the same range.
Supplemental File. Please click here to download this file.
PTE uses particles to encapsulate samples in monodispersed droplets by vortexing. In addition to its simplicity and accessibility, PTE provides several additional benefits, including allowing large volumes of droplets to be generated instantaneously. Moreover, the process can be conducted in an isolated tube, obviating the need to transfer samples to microfluidic devices, streamlining the overall workflow and limiting opportunities for sample contamination or loss. The templating particles also provide a means by which to engineer the contents of the resultant droplet reactions. For example, particle size, chemistry, and wettability can be engineered for targeted biomolecule or cell capture, while functional moieties such as enzymes, actives, or nucleic acids, can be displayed on particle to facilitate reactions, such as for single cell sequencing or functional characterization. While the approach is flexible, there are nevertheless important constraints to its use. For example, it is not currently possible to perform droplet additions as are often conducted with microfluidics, requiring that all reaction components be introduced before encapsulation; this requires that reagents be compatible and stable until the droplets can be generated and, in the case of troublesome combinations, can often be addressed by quickly mixing and emulsifying the sample on ice. Alternatively, reactive components that can be triggered externally with light or heat can be used13. PTE thus provides a flexible and scalable method for conducting droplet assays accessible to non-experts. This, coupled with its innate simplicity and flexibility, makes PTE ideal for the execution and development of numerous droplet applications.
Authors have nothing to disclose.
This work developing this protocol was supported by the National Institutes of Health (R01-EB019453-02), the Office of the Director of National Intelligence, Intelligence Advanced Research Projects Activity through Raytheon BBN Technologies Corp (N66001-18-C-4507), the Chan-Zuckerberg Biohub Investigator Program, Defense Advanced Research Projects Agency through Texas A&M University (W911NF1920013), and Centers for Disease Control and Prevention through Johns Hopkins University Applied Physics Laboratory (75D30-11-9C-06818 (CDC3)). The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the above organizations or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation therein.
|0.22 um syringe filter
|0.5M EDTA, pH 8.0
|0.75 mm biopsy punch
|World Precision Instruments
|1 mL syringes
|1M Tris-HCI, pH 8.0
|27 gauge needles
|3" silicon wafers, P type, virgin test grade
|3D-printed centrifuge syringe holder
|Acrylamide solution,40%, for electrophoresis, sterile-filtered
|Aquapel (fluorinated surface treatment)
|Pittsburgh Glass Works
|FC-40 fluorinated oil
|Novec-7500 Engineering Fluid (HFE oil)
|Platinum Multiplex PCR Master Mix (Taq Master Mix)
|Specialty Coating Systems
|Span 80 (sorbitane monooleate)
|SU-8 3025 photoresist
|Triton X-100 (octylphenol ethoxylate)
|Tween 20 (polysorbate 20)
|Platinum Multiplex PCR Master Mix (Taq Master Mix)
|EVOS FL AUTO
|EVOS LED Cube, GFP
|SYLGARD 184 KIT 1.1 LB (PDMS base and curing reagents)
|Saccharomyces cerevisiae genomic DNA
|Expanded plasma cleaner (plasma bonder)
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