Particle-containing droplet systems with monodisperse fluid volumes

Abstract

To solve the problem in which, although particles contained in droplets can act as templates to define a minimum droplet size, fluid surrounds each particle only in a thin layer and thus this approach has significant drawbacks.SOLUTION: Systems and methods are described herein that create discrete volumes associated with solid-phase particles (e.g., drop-carrier particles) suspended in an immiscible phase (e.g., dropicles). One embodiment of the system includes a plurality of hydrogel-based drop-carrier particles containing a microscale voids or cavities that hold an aqueous phase droplet of fluid within each drop-carrier particle. The hydrogel drop-carrier particles associated with the aqueous phase are suspended as individual elements in an immiscible oil phase. The microscale hydrogel drop-carrier particles containing the voids or cavities may be manufactured using microfluidic droplet generators. The dropicles may be used to analyze single entities (e.g., single molecules and single cells) and analytes.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 719,476, filed Aug. 17, 2018, which is incorporated herein by reference in its entirety. Priority is claimed pursuant to 35 U.S.C. § 119 and other applicable law.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under Grant No. GM126414 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003]

[0003] The technical field generally relates to small sub-millimeter particles having defined voids or cavities formed therein that hold a fluid and are suspended in a separate immiscible fluid. More specifically, the technical field relates to dropicle structures formed from droplet carrier particles that hold a fluid within the voids or cavities formed therein. In a preferred embodiment, the droplet carrier particles are formed from a hydrophilic hydrogel material and hold an aqueous solution within the voids or cavities. [Background technology]

[0004]

[0004] Microfluidics is the gold standard approach for forming monodisperse emulsions (suspensions of dispersed droplets of immiscible fluids in another continuous phase of fluid). Although microfluidic techniques are becoming more available, the devices and equipment are expensive and require expertise. In some applications, it is also advantageous to generate droplets that contain particles (e.g., to perform solid-phase reactions or to grow cells that adhere to a surface). Generating droplets that contain a single particle per droplet is currently difficult because particle encapsulation in the droplet is a stochastic process. An alternative approach for generating aqueous droplets with increased monodispersity compared to randomly mixed emulsions was described by Novak et al. in "Single-Cell Multiplex Gene Detection and Sequencing with Microfluidically Generated Agarose Emulsions," Angewandte Chemie International Edition, 50(2), 390-395 (2011).

[0005] In Novak et al., droplets were formed using simple agitation (vortex / pipette) of the aqueous phase containing dispersed particles in an oil-water suspension. Novak et al. utilized re-emulsification of cell-encapsulated agarose beads to form isolated compartments for PCR amplification. This work relied on a microfluidic device for encapsulation of cells and primer-functionalized beads in agarose gel droplets. Downstream analysis after PCR was performed by cytometry on the released primer beads but not on the larger agarose beads.

[0006]

[0006] The particles contained in the droplets can act as a template to define the minimum droplet size. However, since the fluid only surrounds the particles in a thin layer, this approach has significant drawbacks. The droplets formed by this approach have a large variation in the "thin" volume formed around the hydrogel particles, do not provide space for encapsulation of microscale objects (e.g., cells, beads), and are not ideal for reactions with large molecules that cannot diffuse freely within the particle matrix. Summary of the Invention

[0007]

[0007] In one embodiment, the use of microparticles containing voids or cavity regions connected to or communicating with the particle surface can act as significantly improved particle templates to generate uniform distribution of droplets while also containing open spaces for carrying out reactions or encapsulating cells, beads and other small microscopic objects. These cavity-containing particles also improve the ability to encapsulate larger volumes of liquid samples (per droplet and total sample volume for multiple particles) compared to non-hollow containing particles, which is important for cell culture, cell secretion analysis and diagnostic analysis of large volumes of samples.

[0008]

[0008] The shape of the microparticles and the design of the voids are important parameters to control in achieving uniform volumetric emulsions templated by these particles. The shape of the particles can aggregate and nest / connect with each other, leading to aggregated particles that reduce the uniformity of the droplets formed upon mixing with a two-fluid phase system. In a preferred embodiment, the shape of the microparticles is defined by a spherical envelope with a void volume inscribed within the spherical envelope. The voids or cavities taken up can take the shape of a sphere that connects, communicates with or opens onto the outer surface of the particle, generating a final particle with a crescent-shaped cross section. The inscribed voids intersect the spherical envelope at its surface to generate a passage for fluid filling. In a preferred aspect, the voids intersect the spherical envelope with a narrow opening (i.e., a small fraction of the surface area of ​​the spherical envelope). In some embodiments, the area of ​​this fraction defined by the openings is less than 33%, in other embodiments less than 10%, and in further embodiments the area of ​​the fraction is less than 5% of the total spherical envelope of the particle. Alternatively, in some embodiments, the removed voids do not intersect the surface of the spherical envelope. In such embodiments, cells or large molecules cannot be isolated by particle-templated droplets, but diffusion of water and small molecules is possible for microparticles formed from hydrogels or other porous materials, allowing for filling of the voids and molecular analysis or other downstream assays. In related embodiments, the void volume comprises a polymeric material with high porosity / molecular diffusivity compared to the microparticle material. Note that in other embodiments, the particle envelope shape may be elliptical or other shapes without a larger contact surface area. The void volume may comprise one or more void regions removed from the particle volume.

[0009]

[0009] The surface properties and materials of the microparticles should be controlled to support the formation of droplets. For example, microparticles with hydrophilic surfaces (e.g., low interfacial tension with the water phase compared to the oil phase) can be used to template aqueous-based droplets. Alternatively, hydrophobic / fluorophilic particles (e.g., low interfacial tension with the oil / fluoro-oil phase compared to the water phase) can be used to template oil-based droplets of a separate immiscible continuous phase. The oil phase in the oil-based droplets may include fluoro-oil, mineral oil, silicone oil, vegetable-derived oil, animal-derived oil, crude oil, hydrocarbons or fuels, organic solvents, etc. In a preferred embodiment, the microparticles are formed from hydrophilic hydrogel materials that template the formation of aqueous droplets of uniform volume based on the inscribed void volume contained in the fluoro-oil continuous phase.

[0010] In an exemplary embodiment, hollow or void-containing hydrogel particles are fabricated using an aqueous two-phase system combined with droplet microfluidics to generate uniform cavities. In an exemplary embodiment, a PEG / dextran aqueous two-phase system is disclosed, but other aqueous two-phase systems such as PEG / polyvinyl alcohol or PEG / high ionic strength salt systems (or three-immiscible phase systems) can also be used. In a particular embodiment, PEG and dextran are co-flowed in a flow-focusing droplet generator to generate an emulsion from the mixed material that phase separates to generate two distinct regions in each droplet. The outer PEG regions are crosslinked (e.g., via ultraviolet light excitation and the presence of photoinitiators and crosslinkers) and the inner dextran layer is washed away, leaving large void spaces within the microparticle. By adjusting the relative concentrations of both PEG and dextran, the morphology and / or volume of the void spaces or cavities within the microgel particles can be adjusted. Initial tests of emulsification of cavity-containing particles into oil show a uniform range of droplet sizes. Furthermore, it has been shown that the void spaces or cavities within the particles are freely accessible to large molecules, such as high molecular weight FITC-dextran solutions (500 kDa), which enter the voids or cavities.

[0011] In one embodiment, a droplet-based system using a volume bound to solid phase particles suspended in an immiscible fluid includes a plurality of three-dimensional hydrophilic droplet carrier particles formed from a crosslinked hydrogel, each hydrophilic droplet carrier particle having a void or cavity formed therein. An aqueous fluid is bound to the three-dimensional hydrophilic droplet carrier particles and disposed within the voids or cavities of the plurality of three-dimensional hydrophilic droplet carrier particles. The plurality of three-dimensional hydrophilic droplet carrier particles bound to the aqueous fluid are further disposed with an oil phase. In an embodiment, the aqueous fluid disposed within the voids or cavities of the three-dimensional hydrophilic droplet carrier particles have substantially the same volume (e.g., substantially monodisperse volume). Thus, the system includes a plurality of solid particles with defined voids or cavities formed therein that retain a first fluid therein and are suspended in a second distinct immiscible fluid. In an embodiment, the first fluid may be an aqueous fluid, while the second distinct immiscible fluid is an oil-based fluid. In other embodiments, the first fluid is an oil-based fluid while the second, separate, immiscible fluid is an aqueous fluid.

[0012]

[0012] In another embodiment, a droplet-based system using a volume bound solid phase particle suspended in an immiscible fluid includes a plurality of three-dimensional hydrophilic droplet carrier particles formed from a crosslinked hydrogel, each hydrophilic droplet carrier particle having a void or cavity formed therein. An aqueous fluid is bound to the three-dimensional hydrophilic droplet carrier particles and disposed within the voids or cavities of the plurality of three-dimensional hydrophilic droplet carrier particles. The plurality of three-dimensional hydrophilic droplet carrier particles bound to the aqueous fluid are further disposed or suspended in an oil phase to form a droplet emulsion. Preferably, substantially all of the droplet emulsions formed include a single droplet carrier particle therein.

[0013]

[0013] In another embodiment, a method for producing hydrophilic droplet carrier particles includes providing a microfluidic droplet generator device having multiple inlets. A fluid solution including a crosslinkable first component of an aqueous two-phase system including a photoinitiator is flowed into one of the multiple inlets, the crosslinkable first component being poly(ethylene glycol) PEG or a PEG derivative. A solution of a second component of the aqueous two-phase system including a crosslinker is flowed into another of the multiple inlets. An oil phase is flowed into the device at another of the multiple inlets, thereby forming droplets in the microfluidic device, each droplet separating into separate regions within each droplet, the separate regions including a phase rich in the crosslinkable first component (e.g., PEG or a PEG derivative) and a phase rich in the second component of the aqueous two-phase system (e.g., a polymer such as dextran). The phase rich in the crosslinkable first component is then crosslinked by exposure to light to form hydrophilic droplet carrier particles.

[0014] In another embodiment, a method for producing hydrophilic droplet carrier particles includes providing a microfluidic droplet generator device having multiple inlets. A solution of a crosslinkable first component of an aqueous two-phase system including a crosslinking agent is flowed into the droplet generator device at one inlet of the multiple inlets, the crosslinkable first component being poly(ethylene glycol) PEG or a PEG derivative. A solution of a second component of the aqueous two-phase system is flowed into the droplet generator device at another inlet of the multiple inlets. An oil phase is flowed into the droplet generator device at another inlet of the multiple inlets, whereby droplets are formed in the microfluidic device, each droplet splitting into distinct regions within the droplet that include a rich phase of the crosslinkable first component (e.g., PEG or a PEG derivative) and a rich phase of the second component of the aqueous two-phase system. The rich phase of the crosslinkable first component is then crosslinked by increasing the pH to form hydrophilic droplet carrier particles.

[0015]

[0015] In another embodiment, a method of performing a cellular secretion assay using droplet carrier particles includes the steps of providing a plurality of three-dimensional hydrophilic droplet carrier particles, each particle having a void or cavity formed therein, loading cells into the voids or cavities of the plurality of three-dimensional hydrophilic droplet carrier particles, adding an affinity agent specific to the cellular secretions of a subject, emulsifying the plurality of three-dimensional hydrophilic droplet carrier particles containing the cells and the affinity agent to form a plurality of droplets, incubating the plurality of droplets, breaking the emulsion of the droplets to regenerate three-dimensional hydrophilic droplet carrier particles containing the cells in an aqueous solution, adding a stain, dye or other secondary affinity agent specific to the secretions of a subject to one or more of the plurality of three-dimensional hydrophilic droplet carrier particles, and analyzing the plurality of three-dimensional hydrophilic droplet carrier particles of the previous step for a signal formed or generated by the stain, dye or other secondary affinity agent specific to the cellular secretions of a subject to one or more of the plurality of three-dimensional hydrophilic droplet carrier particles. The plurality of three-dimensional hydrophilic droplet carrier particles may optionally be washed prior to adding the affinity agent. [Brief description of the drawings]

[0016] [Figure 1A] FIG. 1A shows one embodiment of a drop. [Figure 1B]

[0017] FIG. 1B shows another embodiment of the drops. [Diagram 2]

[0018] Figure 2 shows a general schematic of droplet formation. Dry or suspended droplet carrier particles are taken and the dispersed phase is added or exchanged along with an optional surfactant. The continuous phase is added along with an optional surfactant and the suspension is agitated (e.g., via vortex, pipette, etc.) to produce a size-reduced emulsion. After sufficient agitation, uniformly sized droplets are formed along with satellite droplets. [Diagram 3]

[0019] Figure 3 shows the formation of "droplets" with 5 μM FITC dextran (500 kDa) solution as the dispersed phase and Novec™ 7500 fluorinated oil with 0.5% Pico-Surf™ as the continuous phase. The fluorescent image shows a distinct signal in the cavities of the droplets. [Figure 4A]

[0020] FIG. 4A shows an image of an emulsion formed by a microgel particle suspension emulsified in Novec™ 7500 oil + 0.5% Pico-Surf™ with spherical droplet carrier particles shown in the TRITC channel (right). [Figure 4B]

[0021] FIG. 4B shows a graph of droplet number as a function of particles / droplet showing that nearly all droplets contain either 0 or 1 particle. [Figure 4C]

[0022] FIG. 4C shows a graph depicting droplet size distribution showing a range of non-uniform satellite droplets along with uniform regions of droplets formed by encapsulated spherical particles. [Figure 4D]

[0023] FIG. 4D shows a graph depicting droplet size distribution showing uniform regions of droplets formed by encapsulated crescent-shaped droplet carrier particles, as well as a range of non-uniform satellite droplets. [Figure 4E]

[0024] 4E shows a graph of droplet fraction as a function of the number of droplet carrier particles (crescents) per droplet (n=1207). Nearly all droplets contain a single droplet carrier particle. [Diagram 5]

[0025] 5 shows a schematic of the separation of a droplet from a satellite droplet. In one embodiment, an external force or combination of external forces (e.g., magnetic, gravitational, buoyant, drag, centripetal, etc.) is applied such that the droplet and the satellite droplet experience different forces (magnitude and / or direction). [Figure 6]

[0026] Figures 6A and 6B show images of PEG-vinylsulfone microgel particles gelled in the presence of thiolated magnetic particles (1 μm) and then emulsified in fluorinated oil. Figure 6A shows that the initial emulsification resulted in aqueous droplets encapsulating the magnetic microgel particles in a large background of empty satellite droplets. Figure 6B was taken after the application of a magnetic field that allowed the emulsified magnetic microgel particles to be separated from the background of empty droplets. [Figure 7A]

[0027] Figure 7A shows one embodiment of the process used to fabricate cavity-containing microparticles via an aqueous two-phase system. In this embodiment, PEG (e.g., 4-arm 10 kDa PEG-norbornene) and dextran (e.g., 40 kDa) phases are co-flowed in a microfluidic droplet generator device (Figure 7B) to generate a monodisperse emulsion in oil (e.g., Novec™ 7500 + 0.25% Pico-Surf™). The PEG and dextran phases separate once droplets are formed, after which the PEG phase is crosslinked. In one embodiment, ultraviolet light excitation is used to crosslink the gel. More specifically, a photoinitiator (2% w / v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)) is predispersed in the PEG phase and a crosslinker (DTT) is predispersed in the dextran phase. After phase separation, UV light is used to generate radicals that induce a thiol-ene reaction between the PEG-norbornene and the DTT crosslinker to generate a gel matrix. After crosslinking, the particles are washed to remove the oil and dextran phases. Microscopic images of droplet generation, phase separation, and washing are shown below each region of the microfluidic droplet generation device. [Figure 7B]

[0028] FIG. 7B illustrates a microfluidic droplet generation device that may be used to form droplet carrier particles using the method of FIG. 7A. [Figure 8A]

[0029] FIG. 8A shows a phase diagram of an example of a PEG-dextran aqueous two-phase system. In this embodiment, 20 kDa 8-arm PEG vinylsulfone and 40 kDa dextran solutions were used. The emulsion was formed using the flow focusing / droplet generation microfluidic device shown in FIG. 7B. The morphology of the PEG and dextran domains can be adjusted by varying their relative concentrations. At dilute concentrations, the dextran and PEG phases remain mixed. At less dilute concentrations, the PEG and dextran undergo phase separation. As the dextran to PEG concentration ratio increases in the final droplet, the volume fraction of the internal dextran domains increases. As the total concentration of PEG and dextran increases, the interfacial tension between the two phases causes the dextran domains to protrude (top right image). The diameter of the droplets shown is about 100 micrometers. [Figure 8B]

[0030] Figure 8B shows the phase diagram of an exemplary PEG-dextran aqueous two-phase system. In this embodiment, a 10 kDa 4-arm PEG-norbornene and 40 kDa dextran solution was used. By adjusting the concentrations of PEG and dextran, both the droplet morphology and the resulting UV-crosslinked particles can be tuned. [Figure 9]

[0031] FIG. 9 shows the range of droplet carrier particles produced using the aqueous two-phase approach. After cross-linking and washing steps, the droplet carrier particles swell to approximately 130% of their original diameter. For the conditions shown here, 20% w / v of 40 kDa dextran and 15% w / v of 4-arm PEG-norbornene (10 kDa) were used. Droplet carrier particles 12 with diameters of 100 micrometers and 80 micrometers were produced using a microfluidic droplet generating device with a channel height of 70 micrometers, a PEG flow rate of 4 microliters / min, a dextran flow rate of 1 microliters / min, and oil flow rates of 10 and 20 microliters / min, respectively. Droplet carrier particles 12 with diameters of 55, 45, and 10 micrometers were produced using a microfluidic droplet generating device with a channel height of 18 micrometers, a PEG flow rate of 2 microliters / min, a dextran flow rate of 0.5 microliters / min, and oil flow rates of 5, 10, and 20 microliters / min, respectively. [Figure 10A]

[0032] Figure 10A shows a PEG-dextran emulsion formed in fluorinated oil. Image analysis (right) shows high homogeneity of both the PEG (CV=0.75%) and dextran phases (CV=1.45%). CV is the coefficient of variation. [Figure 10B]

[0033] Figure 10B shows crosslinked droplet carrier particles dispersed in water and imaged using a fluorescent microscope (left two images). The particles were conjugated with a TRITC-maleimide dye to view them in the fluorescent channel - a similar fluorophore conjugation process can be used for fluorescent barcoding. The particle size distribution is uniform (CV<5%) as seen in the graph of droplet carrier particle number versus diameter located on the right side of Figure 10B. [Figure 10C]

[0034] FIG. 10C shows droplet carrier particles dispersed in water and imaged using a fluorescent microscope. [Figure 10D]

[0034] Figure 10D shows a droplet carrier particle dispersed in water and imaged using a fluorescent microscope. Figure 10D shows a magnified area of ​​Figure 10C. [Figure 10E]

[0035] Figure 10E shows histograms of aperture size and particle size as a function of particle number. High uniformity was achieved by UV crosslinking of phase-separated droplets while they remained within the microfluidic chip (external diameter CV = 1.5%, aperture size CV = 2.1%). [Figure 11A]

[0036] FIG. 11A shows theoretical calculations of droplet volume variation. Here, droplets templated by spherical particles and crescent-shaped or hollow droplet carrier particles (i.e., void or cavity-containing particles) are considered, and the droplets are compared to droplets (without particles) formed by a microfluidic device. The droplet carrier particles are restricted to a certain size, and the outer diameter of the droplet is varied relative to the particle diameter. The graph shows that by using particles with cavities, the variation of the dispersed phase volume relative to the total volume decreases with increasing diameter. More specifically, the variation of the dispersed phase volume decreases as the ratio of the cavity inner diameter to the particle outer diameter increases, which is advantageous for carrying out a uniform reaction in the droplets. [Figure 11B]

[0037] Figure 11B shows theoretical calculations of reagent encapsulation efficiency in droplets, where droplets templated with spherical particles and crescent-shaped or hollow droplet carrier particles (i.e., void or cavity-containing particles) are considered. Figure 11B demonstrates that encapsulation efficiency increases for particles with relatively large cavities and for droplets with a thicker outer aqueous layer. [Figure 11C]

[0037] Figure 11C shows a theoretical calculation of the reagent encapsulation efficiency in droplets. Here, droplets templated with spherical particles and crescent-shaped or hollow droplet carrier particles (i.e., void or cavity-containing particles) are considered. Figure 11C shows that increasing the concentration of droplet carrier particles (reducing the porosity) prior to droplet formation increases the encapsulation efficiency. [Figure 12]

[0038] Figure 12 shows a schematic of how droplet carrier particles can be modified with many different reactive moieties to allow for particle functionalization and droplet compatibility with many standard assays. Three methods of particle conjugation include orthogonal reactive chemistry, biotin-streptavidin binding, or cell adhesive peptides. [Figure 13]

[0039] 13A and 13B show how digital nucleic acid amplification assays such as PCR (FIG. 13A) and digital ELISA (FIG. 13B) can be performed in droplets. Droplet carrier particles functionalized with nucleic acids or antibodies are mixed with appropriate assay reagents in aqueous solution. Analytes of interest preferentially self-bind to the surface of the droplet carrier particles, allowing subsequent washing to remove background signal. The particles are then emulsified through mechanical agitation to form droplets isolating reactions and forming accumulated signals that are attached within the droplet volume or to the droplet carrier particles themselves. The droplets can then be analyzed through standard microscopy, flow cytometry or plate readers. [Figure 14]

[0040] Figures 14A-C show that droplet emulsification does not affect cell viability. Jurkat cells stained with Hoescht and calcein were suspended in aqueous solution with droplet carrier particles. The droplet carrier particles were emulsified in fluorinated oil through mechanical agitation to form droplets containing the cells. The droplets were imaged via fluorescence microscopy in bright field (cell morphology) (Figure 14A), DAPI (Hoescht - nuclear stain) (Figure 14B) and FITC (calcein - cell viability) channels (Figure 14C). [Figure 15A]

[0041] Figure 15A shows an overview of an exemplary embodiment of barcoding. It shows how the size of the internal cavity is maintained while changing the outer diameter of the droplet carrier particle. The droplets are identified by a size range. [Figure 15B]

[0041] Figure 15B shows an overview of an exemplary embodiment of barcoding. In Figure 15B, droplet carrier particles are decorated with one or more dyes of various intensities. Unique particle types are defined by the intensity or ratio of multiple dye intensities. [Figure 15C]

[0041] Figure 15C shows an overview of an exemplary embodiment of barcoding, in which magnetic particles of various numbers or magnetic loadings are embedded / bridged on / within the particles, allowing separation by relative magnetic forces. [Figure 15D]

[0041] Figure 15D shows an overview of an exemplary embodiment of barcoding, in which light scattering particles of various numbers and sizes are embedded / bridged onto or within droplet carrier particles, allowing separation based on the relative amount / intensity of light scattering or scattering angle. [Figure 16]

[0042] FIG. 16 shows an exemplary schematic workflow for cell secretion analysis and optional sorting. Droplet carrier particles are seeded in a well plate, flask, etc., and then cells are seeded in the gaps or cavities of the droplet carrier particles. After the cells attach, the droplet carrier particles and bound cells are formed into droplets for compartmentalization by pipetting or other mixing action with an oil-based continuous phase. The cells are incubated and the secreted molecules are captured on the bound droplet carrier particles. The droplet carrier particles with bound cells are returned to the aqueous phase and the captured secretions are sorted, for example, by fluorescent molecules through a second affinity interaction. The droplet carrier particles and bound cells can then be analyzed and sorted using a flow cytometer. [Figure 17A]

[0043] 17A shows a schematic of one embodiment in which antibodies secreted from cells are captured on droplet carrier particles bound to the cells. In this example, anti-IL-8 secreting CHO cells are incubated in droplets and secreted antibodies are captured by Protein A coupled to the droplet carrier particles. After returning to the aqueous phase, the captured antibodies are labeled with fluorescent anti-IgG for visualization. [Figure 17B]

[0044] 17B shows exemplary microscopy images showing a high fluorescent signal above a threshold on droplet carrier particles bound to cells, and no or low fluorescent signal for droplet carrier particles without cells attached. The bright field image (left) shows a droplet carrier particle and a cell within the cavity of one droplet carrier particle. The center image shows a fluorescent microscope image of cells using a fluorescent stain indicative of live cells. The right image shows a fluorescent microscope image of anti-IgG staining of secreted antibodies coating the droplet carrier particle. [Figure 18]

[0045] FIG. 18 shows high throughput sorting of droplet carrier particles with bound secretory cells using a fluorescence activated cell sorter. The top row of images shows the droplet carrier particles and bound cells before sorting. Bright field imaging is shown on the left, fluorescent imaging showing live cells stained with Calcein AM is shown in the middle, and fluorescent imaging in a separate channel showing captured antibody secretions sorted with a Cy5-conjugated secondary antibody specific for IgG is shown on the right. All three (top panel) images are of the same field of view. The droplet carrier particles and bound cells are passed through a flow sorter. Forward scatter (FSC) and side scatter (SSC) for droplet carrier particle events in the cytometer are shown in a 2D plot. The top right quadrant of the events was gated, and the fluorescence intensity in the far red channel for those events is shown in a histogram (right graph showing fluorescence intensity). Microscopic imaging shows the accumulation of droplet carrier particles with cells and high concentrations of captured proteins by sorting off high fluorescent signal events above a certain threshold, shown as the "sorting gate" in the images. All scale bars are 200 microns. [Figure 19]

[0046] FIG. 19 shows an exemplary workflow for performing sorting / analysis of single cells or single cell colonies based on total secretion. According to the approach described above, single cells can be isolated in droplet carrier particles and emulsified into droplets where secretions accumulate without crosstalk and are captured in the droplet carrier particles. The droplet carrier particles can then be placed back in water, stained to indicate the amount of secretions, and analyzed / sorted together with the attached cells. The sorted subpopulation of cells can then be expanded and subjected to iterative selection steps. Screening can be performed over multiple cycles to improve the selection of the desired subpopulation. In a related embodiment, single cells seeded in droplet carrier particles can be grown to form clonal colonies attached to the droplet carrier particles before emulsification. This allows for combined analysis and sorting based on clonal growth and secretion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017]

[0047] FIG. 1A shows one embodiment of droplets 10. As used herein, the term "droplets" refers to solid phase particles or droplet carrier particles 12 contained within, containing or bound to a separate volume of a dispersed (e.g., water) phase solution or fluid 14 suspended in an immiscible phase 16 (e.g., oil). The droplet carrier particles 12 (or sometimes referred to as particles 12) are small, sub-millimeter scale (in their longest dimension), spherical or ellipsoidal particles formed from a cross-linked hydrogel material, which in a preferred embodiment is hydrophilic. Typical size ranges (diameter or longest dimension) of the droplet carrier particles 12 are from about 10 μm to about 500 μm, more preferably 20 μm to 200 μm, and even more preferably 40 to 120 μm. Each droplet carrier particle 12 has a void volume or cavity 18 formed therein. The voids or cavities 18 define three-dimensional volumes that hold at least a portion of the dispersed phase solution or fluid 14 (e.g., aqueous phase). Typical fluid volumes held within the voids or cavities 18 include volumes in the range of about 100 fL and about 10 nL. The linear dimensions of the voids or cavities 18 within the droplet carrier particle 12 (e.g., diameter of a spherical void) are a few microns, typically greater than about 5 μm and less than about 250 μm. In some embodiments, the droplet carrier particle 12 is contained within a complete droplet of the aqueous phase solution or fluid 14, and a portion of the solution or fluid 14 is also disposed within the voids or cavities 18. This is shown in FIG. 1A.

[0018]

[0048] In one embodiment, a droplet-based emulsion system is provided that employs separate volumes associated with solid-phase droplet carrier particles 12 suspended in an immiscible fluid. In this system, according to one embodiment, a plurality of aqueous droplets 10 form an emulsion that is enclosed in oil 16. The oil 16 acts as the continuous phase (i.e., the outer phase of the emulsion) while the aqueous droplets 10 act as the dispersed phase (i.e., the inner or encapsulated phase of the emulsion). The oil 16 surrounds the droplets 10 to produce a monodisperse droplet 10 emulsion. Monodisperse refers to the ability of the droplets 10 to retain substantially the same volume of fluid within each of the droplets 10.

[0019]

[0049] In another embodiment, a plurality of oil droplets 10 form an emulsion contained within an aqueous fluid 16. The aqueous fluid 16 acts as the continuous phase (i.e., the outer phase of the emulsion) while the oil droplets 10 act as the dispersed phase (i.e., the inner or encapsulated phase of the emulsion). The aqueous fluid 16 surrounds the droplets 10 to produce a monodisperse droplet 10 emulsion. This embodiment is the opposite of the previous embodiment in which the droplet carrier particles 12 are hydrophobic and the surrounding continuous phase is aqueous.

[0020]

[0050] 1A shows one embodiment of a droplet carrier particle 12 that includes a void or cavity 18 that interacts with, communicates with, or opens onto the outer surface of the droplet carrier particle 12. As described herein, the void or cavity 18 may be formed as a removed void or cavity that takes the shape of a sphere, producing a final droplet carrier particle 12 with a crescent-shaped cross-section such as that illustrated in FIG. 1A. The inscribed void or cavity 18 intersects with the spherical or elliptical envelope at its surface to form a pathway for fluid filling (as well as access to cells, beads, and other micro objects). In a preferred aspect, the void or cavity 18 intersects with the spherical or elliptical envelope at a narrow opening (i.e., a small fraction of the actual surface area of ​​the spherical or elliptical envelope of the droplet carrier particle 12). In some embodiments, the area of ​​this fraction defined by the openings is less than 33% of the total spherical / elliptical envelope or surface area of ​​the droplet carrier particle 12, in other embodiments less than 10%, and in further embodiments less than 5%.

[0021]

[0051] Alternatively, in some embodiments, the removed voids or cavities 18 do not intersect with the surface of the spherical envelope. For example, FIG. 1B shows one such embodiment of a droplet carrier particle 12 including voids or cavities 18 disposed entirely within the three-dimensional hydrophilic droplet carrier particle 12 and that do not intersect with the surface of the three-dimensional hydrophilic droplet carrier particle 12. In this embodiment, the material forming the droplet carrier particle 12 may be made permeable or semi-permeable to allow fluids, reagents and / or samples to diffuse through the droplet carrier particle 12 and into the voids or cavities 18. Similarly, reaction products in some embodiments may be able to diffuse outward from the droplet carrier particle 12 depending on their size. The particular size cutoff for diffusion may be adjusted by adjusting the porosity of the underlying material forming the droplet carrier particle 12. Typically, fluids and small molecules and other species can diffuse through the droplet carrier particle 12, while larger species such as cells, beads or other micro objects cannot diffuse through the droplet carrier particle 12. In a related embodiment, the voids or cavities 18 hold or carry a porous polymeric material that allows for molecular diffusion of sample molecules and reagents from the surrounding fluid (e.g., non-crosslinked dextran). Preferably, in this embodiment, the porous polymeric material disposed within the voids or cavities 18 is more porous than the underlying material forming the droplet carrier particles 12.

[0022]

[0052] In certain embodiments, the surface of the droplet carrier particle 12 may be modified with one or more reactive or binding moieties 20 to allow for functionalization of the droplet 10 and compatibility of the droplet 10 with many standard assays, as seen in Figures 1A, 12, 13A, 13B, 16, 17A. For example, reactive or binding moieties 20 may be formed on the surface of the droplet carrier particle 12 within the void or cavity 18. The binding or reactive moieties 20 may include, by way of example, nucleic acids, peptides, cell adhesion peptides, catalysts, enzymes, antibodies, primers, antigens, aptamers, biotin, or biotin / streptavidin complexes. Orthogonal reactive chemistries known to those skilled in the art may be used to conjugate the reactive or binding moieties 20 to the droplet carrier particle 12.

[0023]

[0053] As described herein, in embodiments using hydrophilic droplet carrier particles 12, the formation of droplets 10 is achieved by combining a suspension of droplet carrier particles 12 in an aqueous phase with oil (and optional surfactant) and mixing (e.g., vortexing, pipetting, etc.) as shown in FIG. 2. The agitation and hydrodynamic shear of the mixing produces an emulsion with a small size. After continued agitation, the droplet carrier particles 12 contained within the droplets act as a size constraint that prevents further shrinkage of the droplets. Solid template droplets in which the droplet carrier particles 12 contain voids or cavities 18 can also be thermodynamically stabilized. With increasing temperature or time, the droplets 10 do not coalesce in the same way that unsupported droplets of the dispersed phase in the aqueous phase coalesce due to the reduction in the interfacial energy of the system upon coalescence. Both pipetting and vortexing may be used to form droplets 10. By using pipette and / or vortex mixing, a uniform emulsion of droplets 10 can be achieved with smaller satellite droplets that do not include droplet carrier particles 12. In one embodiment of a uniform particle-templated emulsion, substantially all particle-containing droplets (e.g., >95%, >99%, or >99.9%) are each associated with a single droplet carrier particle 12. Due to their inherent size range, the droplets 10 can be easily identified using image analysis or easily filtered from the surrounding smaller satellite droplets (i.e., background droplets generated during the formation of the droplets 10) using standard filtration techniques. The satellite droplets are also attached to the droplet carrier particles 12 and therefore do not include reactive moieties 20 that do not proceed with reactions as they occur within the droplets 10. If the total water volume of the sample is less than or equal to the sum of the volumes that can be supported by each of the droplet carrier particles 12 mixed with the sample, the satellite droplets can be significantly reduced or eliminated.

[0024]

[0054] In general, it has been found that pipetting performs better than vortexing for forming droplets 10, to form monodisperse droplets 10 with a large fraction of aqueous emulsion containing only single droplet carrier particles 12 (see, e.g., Figures 4B and 4E). Droplet formation can be improved by first breaking up the initial suspension of droplet carrier particles 12 in the oil, either by mechanically agitating / tapping the reagent tube containing the sample, or by using a pipette with a larger diameter opening (e.g., a 1000 μL pipette), and then breaking up into a finer emulsion by vortexing or by pipetting with a pipette with a smaller diameter (e.g., a 100, 200 μL pipette).

[0025]

[0055] The concentration of surfactant present in the organic phase also affects the monodispersity of the droplets 10 formed and the fraction of the volume that contains a single droplet carrier particle 12. As a general trend, increasing the concentration of surfactant in the oil phase resulted in better dispersion of the droplet carrier particles 12 into single particle-containing droplets 10. A Pico-Surf concentration of greater than 0.5% v / v in Novec 7500 resulted in high monodispersity. In a preferred embodiment, 2% v / v of Pico-Surf in Novec 7500 was used as the continuous phase, resulting in near complete monodispersity of the droplets 10 formed. Further improvement in the monodispersity of the droplets can be achieved through the addition of aqueous surfactants (e.g., Pluronic F-127, Triton X-100) in the aqueous phase. In one embodiment, 0.1% Pluronic F-127 is included in the aqueous phase to reduce particle aggregation, reduce interfacial tension, and result in more uniform droplets 10. In another embodiment, the addition of 1% w / v PEG-5000 to the aqueous dispersed phase reduced aggregation of particles 12 and resulted in more uniform droplets 10.

[0026]

[0056] Additionally, it should be noted that factors such as the polymer composition of the droplet carrier particles 12 or the salt concentration of the dispersed phase affect the affinity of the droplet carrier particles 12 for each other in solution. For example, spherical droplet carrier particles 12 formed from a higher weight percent PEG were preferred. 6 weight percent PEG was preferred over 3 weight percent. 12 weight percent PEG droplet carrier particles 12 were even more preferred. Thus, the polymer backbone formulation can affect the surfactant concentration required to adequately disperse the droplet carrier particles 12 in solution to form monodisperse droplets 10. Additionally, the monodispersity of the droplets was increased by using a low salt solution such as deionized (DI) water.

[0027]

[0057] The choice of oil phase may also affect the functionality of the resulting droplets 10. For example, Novec-7500 infused with Pico-Surf surfactant was preferred in forming uniform droplets 10 over alternative oil phases such as Fluorinert™ FC-40 oil with RAN surfactant, although this condition was also able to form droplets 10. However, Fluorinert™ FC-40 with RAN surfactant had improved thermal stability when compared to Novec-7500 with Pico-Surf. Thus, certain applications may benefit from an exchange of oil phases. For example, droplets 10 may be formed in Novec-7500 to produce a monodisperse emulsion, and then the Novec oil may be removed and replaced with Fluorinert™ FC-40 for high temperature applications such as thermocycling for DNA amplification.

[0028]

[0058] FIG. 2 shows one method of forming droplets 10. First, droplet carrier particles 12 are provided, either dried or suspended in a dispersed phase. These may be contained in a holder or container 22 (e.g., an Eppendorf tube is shown, but other holders or containers may be used). If no dispersed phase is present, a dispersed phase (e.g., an aqueous phase) is added to the holder or container 22. In another embodiment, the initial dispersed phase can be added or replaced with another dispersed phase, as seen in step 100 of FIG. 2. FIG. 2 shows droplet carrier particles 12 contained in dispersed phase 14. Then, as seen in step 110, a continuous phase 26 is added (e.g., an oil phase) along with an optional surfactant. The holder or container 22 then undergoes an agitation step 120, which may include a vortex, a pipette, or the like. The agitation step 120 produces droplets 10 and possibly satellite droplets 28. The satellite droplets 28 may be removed, for example, using filtration of the mixture. The formation of satellite droplets 28 may be minimized by controlling the volume of dispersed phase added during the formation of the droplets 10 .

[0029]

[0059] Figure 3 shows the formation of droplets 10 with 5 μM FITC dextran (500 kDa) solution as the dispersed phase 14 and Novec™ 7500 fluoro oil with 0.5% Pico-Surf™ surfactant added as the continuous phase 16. Figure 3 shows droplet carrier particles 12 contained within the dispersed phase 14 before emulsification (left) and after emulsification to form droplets 10. Corresponding images before (left image) and after (right image) emulsification are provided below. Fluorescence images show clear signals within the voids or cavities 18 of the droplets 10 (right image).

[0030]

[0060] FIG. 4A shows an image of the resulting emulsion formed (using spherical particles 12 to template continuous phase aqueous droplets composed of Novec™ 7500 fluorinated oil with 0.5% Pico-Surf™ surfactant). Droplets 10 are shown in a bright field image (left image) and spherical droplet carrier particles 12 are shown in a fluorescent channel (right image). Both highly variable sized satellite droplets 28 are shown, as well as uniform droplets 10 overlapping with fluorescently stained droplet carrier particles 12. FIG. 4B shows a graph showing the number of droplets as a function of droplet carrier particles 12 per droplet. Of the droplets that contain droplet carrier particles 12, nearly all contain only a single droplet carrier particle 12. FIG. 4C shows a graph showing droplet size distribution showing the range of non-uniform satellite droplets 28 along with uniform regions of droplets formed by encapsulated droplet carrier particles 12 (i.e., droplets 10).

[0031]

[0061] FIG. 4D shows a graph depicting droplet size distribution showing a range of non-uniform satellite droplets 28 along with uniform regions of droplets formed by encapsulated crescent-shaped droplet carrier particles 12 (i.e., droplets 10). Crescent-shaped droplet carrier particles 12 were used to template continuous phase water droplets composed of Novec™ 7500 fluorinated oil with 0.5% Pico-Surf™ surfactant. FIG. 4E shows a graph depicting the number of droplets as a function of droplet carrier particles 12 (crescent shape) per droplet. Of the droplets that contain droplet carrier particles 12, nearly all contain only a single droplet carrier particle 12.

[0032]

[0062] 5 illustrates separation of droplets 10 from satellite droplets 28 according to alternative embodiments. In these embodiments, an external force or combination of external forces (e.g., magnetic, gravitational, buoyant, drag, centripetal, etc.) are applied such that droplets 10 and satellite droplets 28 experience different forces (magnitude and / or direction). For example, droplet carrier particles 12 may include a magnetic material with an externally applied magnetic field used to separate droplets 10 from satellite droplets 28 that are not responsive to the applied magnetic field.

[0033]

[0063] For example, Figures 6A and 6B show images of PEG-vinylsulfone microgel particles that were gelled in the presence of thiolated magnetic particles (1 μm) and subsequently emulsified in fluorinated oil and then separated. Figure 6A illustrates that the initial emulsification resulted in aqueous droplets (AD) that encapsulate the magnetic microgel particles in a large background of empty satellite droplets 28. Figure 6B shows that after application of a magnetic field, the emulsified magnetic microgel particles can be separated from the background of empty satellite droplets 28.

[0034]

[0064] To produce droplet carrier particles 12 having voids or cavities 18, a microfluidic droplet generator device 30 is provided that is used to form a monodisperse emulsion of an oil phase, whereby the internal dispersed phase comprises an aqueous two-phase system. One part of the aqueous two-phase system is a crosslinkable hydrogel precursor, such as poly(ethylene glycol) (PEG) or its derivatives. Another part of the aqueous two-phase system is a polymer such as dextran. The two phases of the aqueous two-phase system are then dispersed into distinct regions within the droplets formed. One component of the two-phase system (i.e., the crosslinkable component) is then crosslinked to form the droplet carrier particles 12. FIG. 7A shows a schematic of a process for producing droplet carrier particles 12 having voids or cavities 18 formed therein. As seen in FIG. 7A, an aqueous two-phase system is used to form the droplet carrier particles 12. In this particular embodiment, the aqueous two-phase system includes the crosslinkable components PEG or PEG derivative (e.g., 10 kDa 4-arm PEG-norbornene) and uncrosslinked dextran (e.g., 40 kDa) (using a crosslinker). A microfluidic droplet generator device 30 is used to generate an emulsion of the aqueous two-phase system within the oil phase. Droplets containing the two aqueous phase components (e.g., PEG and dextran) separate after droplet formation in a phase separation step. After phase separation, the PEG or PEG derivative components are crosslinked into a gel. For example, a crosslinker such as dithiothreitol (DTT) in the presence of a photoinitiator (e.g., Irgacure® 2959, LAP, etc.) within the PEG or PEG derivative components is then exposed to light (e.g., UV excitation) to initiate crosslinking. Of course, other crosslinkers such as cysteine-containing peptides or other dithiols or multi-arm crosslinkers may be used. In another embodiment, the thiol-ene reaction between the multi-arm PEG-norbornene+dithiol crosslinker is initiated via UV light and a photoinitiator. In a related embodiment, either the PEG and / or polymer phase can include a combination of one or both of a photoinitiator and a crosslinker. After crosslinking, the droplet carrier particles 12 can be washed to remove the oil and dextran phases.

[0035]

[0065] 7B shows the layout of a microfluidic droplet generator device 30 used to generate droplet carrier particles 12 having voids or cavities 18 using flow focusing. The microfluidic droplet generator device 30 includes multiple microfluidic channels 32, 34, 36 that converge / intersect into a droplet generation zone or region 38 where the emulsion (droplets) are generated. The generated droplets travel down another microfluidic channel 40 that can be directed to a chamber or region 42 where the droplets can accumulate and be temporarily stored. An outlet 44 of the device 30 may be used to remove the droplets / droplet carrier particles 12. Each microfluidic channel 32, 34, 36 includes a respective inlet 46, 48, 50 for inputting the various components. In this example, a PEG (e.g., 4-arm 10 kDa PEG-norbornene) phase and a dextran (e.g., 40 kDa) phase are run together in the microfluidic device 30 to generate a monodisperse emulsion in oil (e.g., Novec™ 7500 + 0.25% Pico-Surf™). In one embodiment, a first inlet 46 leading to the microfluidic channel 32 is used to deliver the oil phase with a surfactant. A second inlet 48 leading to the microfluidic channel 34 is used to deliver the dextran. A third inlet 50 leading to the microfluidic channel 36 is used to deliver the PEG as well as a crosslinker and a photoinitiator. In another embodiment, the first inlet 46 leading to the microfluidic channel 32 is used to deliver the oil phase with a surfactant. A second inlet 48 leading to the microfluidic channel 34 is used to deliver the dextran with a crosslinker. A third inlet 50 leading to the microfluidic channel 36 is used to deliver the PEG and a photoinitiator. Separation of the polymer precursor and crosslinker prior to droplet generation is particularly useful when using highly reactive chemicals that may begin to crosslink within the sample syringe or channel inlet, stopping the flow. In another embodiment, the crosslinker is not included in the PEG or dextran phase, but instead is injected into its own additional inlet and connecting channel. This is useful when the crosslinker does not readily dissolve in the dextran phase, for example the larger thiolated PEG crosslinkers.

[0036]

[0066] The PEG and dextran phases separate once the droplets are formed and the PEG phase is subsequently crosslinked. As mentioned above, in one embodiment, UV excitation is used to crosslink the gel. More specifically, a photoinitiator (2% w / v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)) is predispersed in the PEG phase and a crosslinker (DTT) is predispersed in the dextran phase. After phase separation, UV light is used to generate radicals that induce a thiol-ene reaction between the PEG-norbornene and the DTT crosslinker to generate a gel matrix that forms the droplet carrier particles 12. Crosslinking can occur in the microfluidic droplet generator device 30 or "off-chip" after the droplets exit through the outlet 44 of the microfluidic droplet generator device 30. After crosslinking, the droplet carrier particles 12 are washed to remove the oil and dextran phases.

[0037]

[0067] In another embodiment, a change in pH level is used to crosslink the droplet carrier particles 12. For example, PEG-vinylsulfone / maleimide + dithiol crosslinker is used at low pH to prevent gelation prior to droplet formation in the microfluidic droplet generator device 30. The pH is then increased in the dispersed phase by adding an organic base (e.g., triethylamine) to the downstream oil phase to initiate the crosslinking reaction. In a related embodiment, a parallel step emulsion droplet generator microfluidic device 30 operates on a prepolymer phase mixed with a dextran phase in a single solution to generate monodisperse droplets (e.g., via UV light or pH change) prior to phase separation and polymerization, as described in de Rutte, JM, Koh, L, Di Carlo, D., "Scalable High-Throughput Production of Modular Microgels for In Situ Assembly of Microporous Tissue Scafols," Adv. Funct. Mater. 2019, 29, 1900071, incorporated herein by reference. In all of the above described embodiments, after cross-linking of the PEG, the dextran phase may be removed and the desired aqueous phase added or replaced to form droplets 10. In some embodiments, as illustrated in Figure 1B, for example, when forming hollow droplet carrier particles 12, the internal dextran phase may be maintained within voids or cavities 18.

[0038]

[0068] FIG. 8A shows a phase diagram of an example of a PEG-dextran aqueous two-phase system. By adjusting the relative concentrations of both PEG and dextran, the morphology and / or volume of the void space or cavity 18 within the droplet carrier particle 12 can be adjusted. Here, a 20 kDa 8-arm PEG vinylsulfone and 40 kDa dextran solution was used. The emulsion was formed using the microfluidic droplet generator device 30 of FIG. 7B. The morphology of the PEG and dextran regions can be adjusted by varying their relative concentrations. At dilute concentrations, the dextran and PEG phases remain mixed. At less dilute concentrations, the PEG and dextran undergo phase separation. As the dextran to PEG concentration ratio increases in the final droplet, the volume fraction of the internal dextran regions increases. As the total concentration of PEG and dextran increases, the interfacial tension between the two phases causes the dextran regions to protrude (top right image). The diameter of the droplet shown is about 100 micrometers. At high PEG and dextran concentrations, phase separation was found to be rapid (<1 s), whereas near-binodal conditions showed longer phase separation (>1 s).

[0039]

[0069] FIG. 8B shows a phase diagram of an example of a PEG-dextran aqueous two-phase system. In this embodiment, a 10 kDa 4-arm PEG-norbornene and 40 kDa dextran solution was used. By adjusting the concentration of PEG and dextran, both the droplet morphology and the resulting UV-crosslinked droplet carrier particles 12 can be tuned. At dilute concentrations, the PEG and dextran phases remain mixed as a single phase (below the binodal line). At higher concentrations (above the binodal), the PEG and dextran phases separate into separate PEG-rich and dextran-rich phases within the droplet. As the total concentration of PEG and dextran increases, the interfacial tension between the two phases increases, forcing the dextran phase into contact with a larger portion of the oil interface (top right image). After UV-crosslinking, the resulting droplet carrier particles 12 (except for one example that did not undergo phase separation) maintain a similar cavity volume (relative to the overall size of the droplet carrier particles 12) but increase the cavity opening size. As the relative concentration ratio of dextran to PEG increases (bottom right), the volume of the dextran-rich phase also increases. After UV cross-linking, droplet carrier particles 12 with a larger volume of the dextran-rich phase had proportionally larger cavities or voids 18. Furthermore, it was found that for conditions near the binodal curve, especially at high amounts of dextran, the cavities remained completely enclosed (the voids did not contact the envelope of the outer particle 12) after cross-linking.

[0040]

[0070] FIG. 9 shows a range of droplet carrier particles 12 produced using an aqueous two-phase approach using a microfluidic droplet generator device 30. After cross-linking and washing steps, the droplet carrier particles 12 swell to about 130% of their original diameter. For the conditions shown here, 20% w / v of 40 kDa dextran and 15% w / v of 4-arm PEG-norbornene (10 kDa) were used. Using a microfluidic droplet generator device 30 with a microfluidic channel height of 70 micrometers and width of 60 micrometers at the junction, a PEG flow rate of 4 microliters / min, a dextran flow rate of 1 microliters / min, and oil flow rates of 10 and 20 microliters / min, droplet carrier particles 12 with diameters of 100 micrometers and 80 micrometers, respectively, were produced. Droplet carrier particles 12 with diameters of 55, 45 and 40 micrometers were produced using a microfluidic droplet generator device 30 having a microfluidic channel height of 18 micrometers and a width of 35 micrometers at the junction, a PEG flow rate of 2 microliters / min, a dextran flow rate of 0.5 microliters / min, and oil flow rates of 5, 10 and 20 microliters / min, respectively.

[0041]

[0071] FIG. 10A shows an image of a non-crosslinked emulsion and a graph of droplet count as a function of the diameter of the outer PEG envelope and the dextran inner component of a PEG-dextran emulsion formed in fluorinated oil. Image analysis showed high uniformity for the diameter of both the PEG (CV=0.75%) and dextran phases (CV=1.45%). CV is the coefficient of variation. FIG. 10B shows an image of crosslinked droplet carrier particles 12 dispersed in water and imaged using a fluorescent microscope. The droplet carrier particles 12 were conjugated with a TRITC-maleimide dye to view them in the fluorescent channel - a similar fluorophore conjugation step can be used for fluorescent barcoding of the droplet carrier particles 12. FIG. 10B also shows a graph of particle count as a function of the diameter of the crosslinked droplet carrier particles 12. The particle size distribution is uniform (CV<5%).

[0042]

[0072] 10C and 10D show droplet carrier particles 12 dispersed in water and imaged using a fluorescent microscope. The droplet carrier particles 12 were cross-linked with UV light while passing through the exit area of ​​the droplet generator device 30. The droplet carrier particles 12 were modified to have biotin groups through the addition of biotin-PEG-thiol during the manufacturing process. The droplet carrier particles 12 were then labeled with Alexa Flour™ 568 streptavidin bound to the biotin on the surface of the droplet carrier particles 12 to fluorescently visualize them. Analysis of the fluorescent images showed high uniformity (outer diameter CV=1.5%, orifice diameter CV=2.1%), as seen in FIG. 10E.

[0043]

[0073] FIG. 11A shows a theoretical calculation of the volume variation of the droplet 10. Here, a droplet 10 templated by a spherical droplet carrier particle 12 is considered with a crescent-shaped droplet carrier particle 12 (i.e., a void or cavity-containing particle) and compared to a droplet formed by a microfluidic device 30 without a particle 12 contained therein. This analysis is based on the result that the volume within the cavity 18 can be well controlled while the outer diameter of the fluid surrounding the droplet carrier particle 12 is hardly controlled during the emulsification process. The droplet carrier particle 12 is limited to a certain size and the outer diameter of the droplet varies with the particle diameter. By using a droplet carrier particle 12 with a cavity or void 18, the deviation of the dispersed phase volume compared to the total volume decreases as the diameter increases. More specifically, the variation of the dispersed phase volume decreases as the ratio of the diameter of the internal cavity 18 to the outer diameter of the particle increases, which is useful for carrying out a uniform reaction in the droplet 10. For example, D in / D out For ρ = 0.50 and 0.75, as the porosity of the crescent-shaped particle 12 increases, the behavior approaches the ideal situation of a spherical droplet without inner particles.

[0044]

[0074] 11B and 11C show theoretical calculations of the reagent encapsulation efficiency in droplets 10. Here, droplets 10 templated by spherical droplet carrier particles 12 and crescent-shaped droplet carrier particles 12 (i.e., void or cavity-containing particles) are considered. It is assumed that the encapsulated reagent or sample does not freely diffuse within the particle matrix. The encapsulation efficiency is defined as the fraction of the sample volume in the initial concentrated suspension of droplet carrier particles 12 that is bound to the droplet 10 after droplet formation. Reagents that are not encapsulated are expected to form satellite droplets 28. The porosity of the initial particle suspension is defined as the fraction of the total volume (sample + particles) that occupies the area outside the envelope of the droplet carrier particles 12. To normalize the calculation between crescent-shaped and spherical particles 12, the volume of the internal cavities 18 is not considered as part of the initial porosity. FIG. 11B demonstrates increased encapsulation efficiency for particles 12 with larger cavities and droplets 10 with thicker outer aqueous layers. In either case, the particles 12 take up a smaller fraction of the volume of the final droplet 10, allowing for encapsulation of a larger sample volume. FIG. 11C demonstrates that increasing the concentration of droplet carrier particles 12 (reducing porosity) prior to droplet 10 formation increases encapsulation efficiency. When porosity is increased, there is more reagent or sample in the system and the droplets 10 quickly become saturated, resulting in the formation of satellite droplets 28 with larger volumes and therefore reducing encapsulation efficiency.

[0045]

[0075] 12 illustrates how the droplet carrier particles 12 can be modified with many different reactive moieties to enable particle functionalization and compatibility of the droplets 10 with many standard assays. For example, reactive or binding moieties 20 can be formed on the surface of the droplet carrier particles 12 within the voids or cavities 18. The binding or reactive moieties 20 can include, by way of example, nucleic acids, peptides, cell adhesion peptides, catalysts, enzymes, antibodies, primers, antigens, aptamers, biotin or biotin / streptavidin complexes. Orthogonal reactive chemistries known to those skilled in the art can be used for conjugation of the reactive or binding moieties 20 to the droplet carrier particles 12.

[0046]

[0076] In one embodiment, peptides or larger proteins containing free cysteine ​​groups can be added with a thiol-based crosslinker in the dextran phase. These peptides can be covalently attached to thiol reactive sites on the norbornene-containing polymer backbone as they fuse with the hydrogel phase. To promote CHO cell adhesion to the droplet carrier particles 12, this approach has been used to include cell adhesion peptides during the manufacture of the droplet carrier particles 12. The integrin-binding peptide RGD at a concentration of at least 4 mg / mL was used in the dextran phase and showed sufficient adhesive strength to maintain cell attachment to the droplet carrier particles 12 even in the presence of strong mechanical agitation, centrifugation, and droplet carrier particle sorting. In another embodiment, peptides or larger proteins containing free cysteine ​​groups can be added to the PEG phase and crosslinked before or after mixing with the dextran phase.

[0047]

[0077] In a second exemplary embodiment, the droplet carrier particles 12 were directly modified with biotin by incorporating anywhere from 0.5 mg / mL to 5 mg / mL of 5 kDa biotin-PEG-thiol within the PEG phase during the manufacture of the droplet carrier particles 12. In the presence of a photoinitiator upon exposure to UV light, as described in various embodiments disclosed herein, the extra thiol groups on these molecules bind to thiol reactive moieties on the hydrogel backbone, resulting in droplet carrier particles 12 uniformly modified with biotin groups. Biotinylation sites are typically modified with streptavidin groups to allow conjugation to a secondary biotinylated molecule. The amount of streptavidin used can be adjusted based on the desired number of binding sites per particle. In many embodiments centered on the capture of secreted proteins from individual cells, it has been experimentally determined that 8-10 μg / mL of streptavidin is sufficient to coat the exterior of the droplet carrier particles 12 and produce a secretion signal over a wide dynamic range. However, in other embodiments in which cells are attached to the particles 12 through a biotin-streptavidin reaction and secretions are simultaneously captured on the surface of the droplet carrier particles 12 using a biotinylated capture antibody, higher concentrations of streptavidin were used, ranging from 10 μg / mL to 250 μg / mL. Streptavidin is typically added to the droplet carrier particles 12 in PBS, notably because exogenous biotin is a common additive in cell culture media and can presaturate many of the available binding sites.

[0048]

[0078] 13A and 13B show how digital assays such as PCR (FIG. 13A) and ELISA (FIG. 13B) can be performed in droplets 10. Droplet carrier particles 12 functionalized with nucleic acids or antibodies are mixed with the appropriate assay reagents (e.g., target DNA, analyte, reporter dye, secondary antibody, primers, DNTPs, polymerase) in aqueous solution. The analyte of interest preferentially self-binds with the surface of the droplet carrier particles 12, allowing subsequent washing to remove background signal. The droplet carrier particles 12 are then emulsified through mechanical agitation to form droplets 10 that isolate the analyte and the signal generated (e.g., through enzymatic amplification) within the droplet volume or on the droplet carrier particles 12 themselves. In embodiments where the signal is captured directly on the droplet carrier particles 12, the emulsion can be broken and readout can be performed directly by observing the fluorescent or colorimetric signal on the droplet carrier particles 12 through fluorescence microscopy or flow cytometry (e.g., using flow cytometer 150). In embodiments where the signal accumulates within the droplets 10 but is not directly associated with the droplet carrier particles 12, the emulsion of the droplets 10 can be directly analyzed by observing changes in fluorescent or colorimetric signal through a fluorescent microscope or other fluorescent imaging method, or through several commercially available flow cytometry systems compatible with the oil continuous phase 16.

[0049]

[0079] 14A-C show that emulsification of droplets does not affect cell viability. Jurkat cells stained with Hoechst and calcein were suspended in aqueous solution along with droplet carrier particles 12. The droplet carrier particles 12 were emulsified through mechanical agitation in fluorinated oil to form droplets 10 containing the cells. The droplets 10 were imaged in multiple channels via fluorescence microscopy including bright field to visualize cell morphology (FIG. 14A), DAPI to visualize Hoechst stained nuclei (FIG. 14B), and FITC to visualize calcein labeled live cells (FIG. 14C).

[0050]

[0080] 15A-15D show schematics of representative embodiments of barcoding incorporated within droplet carrier particles 12. FIG. 15A shows how the size of the internal voids or cavities 18 is maintained while varying the external dimensions of the droplet carrier particles 12. Droplets 10 with unique chemistry or properties are identified by size ranges. In FIG. 15B, the droplet carrier particles 12 are modified with one or more dyes of various intensities (two dyes are shown in this embodiment at various intensities). Unique droplet carrier particle types are defined by the intensity or ratio of multiple dye intensities (e.g., Dye1:Dye2 or Dye2:Dye1). In FIG. 15C, magnetic particles 52 of various numbers or magnetic loadings are embedded / bridged on / within the droplet carrier particles 12 to allow separation by relative magnetic forces. In FIG. 15D, light scattering particles 54 of various numbers and sizes are embedded / bridged on or within the droplet carrier particles 12 to allow separation or separate analysis of unique droplet carrier particle types 12 based on the relative amount / intensity of light scattering or scattering angle.

[0051]

[0081] Advantages of the Droplet System

[0082] A distinct advantage of a system using droplets 10 is the ability to generate large numbers of uniform droplets in a short time (~1 min). For example, consider the case where a sample of 10 million cells is to be encapsulated for single cell analysis. A single Poisson distribution of inputs into the microfluidically generated droplets is 10 8 A typical microfluidic droplet generator device 30 generates droplets at a rate of 1000 Hz and requires 28 hours to encapsulate the entire cell population. In some cases, secondary bead / particle encapsulation is required (e.g., droplet sequencing). In this situation, a double Poisson injection requires 10 droplets to ensure single bead and single cell encapsulation (1% bead+cell encapsulation efficiency). 910 droplets. In this case, the time for encapsulation is about 11.7 days. However, the droplet carrier particles 12 can be produced in advance on a short scale in large quantities, stored, and shipped to the user. The user can then perform an emulsification process to form droplets 10 in parallel on a large scale using a simple mixing process in the bulk solution allowing for the encapsulation of huge numbers of cells.

[0052]

[0083] Use of surfactants, surfactant mixtures and oils

[0084] The amount of continuous phase surfactant can be adjusted to adjust the interfacial tension between the immiscible phases to optimize the breakup of droplets and the formation of drops 10. Dispersed phase surfactants (non-ionic, e.g., Pluronic or ionic) may be added to further adjust the interfacial tension and reduce the aggregation of the dispersed phase droplet carrier particles 12. The oil used to form the drops 10 can be adjusted depending on the intended application. For example, fluorinated oils (Novec™ 7500 3M, FC-40 3M, etc.) can be used with equivalent surfactants (Pico-Surf™, RAN surfactants, etc.) for the formation of drops. These oils are particularly suitable for the analysis of biological samples due to their high biocompatibility. In other embodiments, different oils (e.g., silicone oils, mineral oils, vegetable-derived oils, animal-derived oils, long-chain hydrocarbons, organic solvents) may be desired to allow transfer of reagents between drops 10, change of pH within the drops 10, etc. In other embodiments, the droplet carrier particles 12 are made from a hydrophobic material (e.g., poly(propylene glycol) diacrylate, trimethylolpropane ethoxylate triacrylate, hexanediol diacrylate, hexanediol diacrylate plus lauryl acrylate, 1H,1H,6H,6H-perfluoro-1,6-hexanediol diacrylate, or methacryloxypropyl terminated polydimethylsiloxane) and dispersed in an oil phase, followed by formation of an oil-in-water emulsion of droplets 10.

[0053]

[0085] Droplet Carrier Particle to Dispersion Volume Ratio

[0086] The relative number of droplet carrier particles 12 in the dispersed phase 14 can be optimized to achieve efficient and consistent droplet 10 formation as well as prevent excessive formation of satellite droplets 28. At higher dispersed volume ratios, there is a lower fraction of dispersed volume associated with the droplets, leading to the formation of more satellite droplets. At lower volume ratios, the efficiency of capture of reagents and particles (fraction of microscale objects from the sample associated with the droplet 10) into the droplet 10 is improved. Note that unlike microfluidic droplet encapsulation, where the fraction of analyte analyzed from a volume depends on the fraction of the volume that is encapsulated, the presence of a solid phase within the droplet 10 allows for a concentration of analyte on the surface (or within the matrix) of the solid phase that separates the volume being analyzed from the fraction of analyte that can be analyzed.

[0054]

[0087] Transfer of droplets to the dispersed phase

[0088] In some cases, it may be desirable to return the droplet carrier particles 12 to the bulk dispersed phase 14 (e.g., aqueous phase) in order to perform additional washing steps, secondary conjugation, flow the droplet carrier particles 12 through a flow cytometer 150 (FIGS. 13A, 13B, 16, and 18) for analysis / sorting, etc. In systems using hydrogel-based droplet carrier particles 12, water, and fluoro-oil, a second surfactant (perfluoro-octanol) may be added to the oil phase 16 to destabilize the emulsion and recover the droplet carrier particles 12 in the aqueous phase 14. More specifically, excess oil and surfactant are removed from the emulsion suspension via a pipette without removing the droplets 10. A 20% solution of perfluoro-octanol in Novec™ 7500 fluoro-oil (3M Corporation) is then added to the suspension in a volume approximately equal to the remaining suspension volume. Additional aqueous phase 14 is added to dilute the sample, making the suspension easier to handle and / or to prevent crosstalk between droplet carrier particles 12. The suspension is gently mixed and after a few minutes the droplets coalesce. The particle suspension can be removed directly from the top of the emerging immiscible phase. If desired, an additional washing step can be performed with a low density organic phase that is miscible with the fluoro oil. The addition of hexane allows the fluoro oil to separate above the aqueous phase and can be removed via pipette. Other methods such as centrifugation and destabilization via electric fields have been utilized to coalesce emulsions and could be potential alternatives.

[0055]

[0089] Washing of droplet carrier particles for biological assays.

[0090] Efficient labeling or detection of biological samples requires incubation in the presence of reagents such as antibodies or fluorescent small molecules. Furthermore, assays that require sequential addition of reagents, such as sandwich ELISA, rely on intermediate washing steps to remove residual reagents from previous steps. Here, we describe different methods for washing / isolation of droplets 10 as well as sorting in both oil and aqueous phases.

[0056]

[0091] After returning the droplet carrier particles 12 to the aqueous phase, a washing step can be performed to remove free analytes in the solution and prevent crosstalk. In one embodiment, the particle suspension is diluted with the aqueous phase and centrifuged to concentrate the droplet carrier particles 12 at the bottom of the container 22 (for particles with a higher density than water). In one embodiment, the droplet carrier particles 12 are centrifuged at 2000 g for 1 minute. In another embodiment, the droplet carrier particles 12 are centrifuged at 300 g for 3-5 minutes. A lower centrifugation speed is advantageous when sensitive cells are used. The supernatant is removed and replaced with a new solution, and the droplet carrier particles 12 are remixed in this new solution. This process of centrifugation and removal can be repeated multiple times to increase the level of washing. The new solution may include a washing solution or a new reagent solution. In another embodiment, the droplet carrier particles 12 are modified with magnetic particles (e.g., embedded within a gel matrix of the droplet carrier particles 12) to impart magnetic properties and allow for magnetic manipulation / accumulation of the droplet carrier particles 12 at a location within the container 22 to enable washing or solution replacement procedures. In another embodiment, the droplet carrier particles 12 are modified with microbubbles or hollow glass particles to reduce their density and can be washed / separated using, for example, controlled buoyancy using conjugated microbubbles (BACS).

[0057]

[0092] Isolation of droplets from satellite droplets

[0093] To reduce background signal from the satellite droplets 28, the droplets 10 can be separated from the satellite droplets 28 using various methods as described herein. In one embodiment, a magnetic field is applied to separate the droplets 10 having magnetically modified droplet carrier particles 12 from the satellite droplets 28, which are non-magnetic (see, e.g., FIG. 5). The magnetic particles can be embedded within the matrix of the droplet carrier particles 12 by placing them in solution during microfluidic fabrication. An external magnetic field can be used to attract the magnetically labeled droplet carrier particles 12 to a location in the container 22 or slide them away from the satellite droplets 28, where they can be collected with high purity, imaged, or otherwise analyzed. In another embodiment, the larger droplets 10 can be separated from the smaller background satellite droplets 28 using well-known size / deformability-based filtration schemes known in the art. This can include the use of well-known microfluidic particle separation techniques that allow for size-based separation of particles and droplets. Exemplary approaches include inertial microfluidic separation devices, deterministic lateral displacement devices, tangential flow filtration devices, and acoustic or sedimentation-based separation devices that exploit size and / or density differences of particles. Differences in buoyancy can also be exploited for separation. For example, droplets 10 tend to accumulate at the top of an Eppendorf tube due to the increased ratio of buoyancy to drag forces, while satellite droplets 28 accumulate at the bottom and can be partially removed by a pipette.

[0058]

[0094] An aqueous two-phase approach to producing droplet carrier particles

[0095] Microgel droplet carrier particles 12 may be fabricated using an aqueous two-phase system (ATPS) in combination with droplet microfluidics (FIGS. 7A and 7B). These microgel droplet carrier particles 12 have internal voids or cavities 18. In general, one phase of the ATPS should contain a crosslinkable component while the other phase should not include a crosslinkable component. In one embodiment, a crosslinkable PEG phase and a dextran phase are co-flowed in a microfluidic droplet generator device 30 along with a third oil phase that includes a surfactant to generate a mixed aqueous emulsion in which a uniform fraction of the PEG phase and the dextran phase are present in each droplet suspended in the oil phase. At certain concentration regimes of PEG and dextran, the two phases separate into distinct spatial locations within the droplets with distinct morphologies (FIG. 8). One spatial location includes the enriched phase that includes the PEG component while another spatial location includes the second component of the aqueous two-phase system (e.g., a polymer such as dextran). Cross-linking is then induced in the enriched phase containing the PEG component and the formed droplet carrier particles 12 are washed to remove oil and dextran to produce spherical or elliptical PEG particles having voids or cavities 18 inscribed within the PEG droplet carrier particles 12. The shape of the voids or cavities 18 corresponds to the shape of the previous dextran-rich regions of each droplet.

[0059]

[0096] Crosslinking of the PEG phase can be achieved using a range of techniques. In an exemplary approach, both UV- and pH-initiated crosslinking of the PEG phase has been demonstrated. For example, PEGDA was crosslinked via chain-growth radical polymerization triggered by UV light and a photoinitiator (Irgacure® 2959 (1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one), LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate), etc.). In another embodiment, a thiol-ene reaction between multi-arm PEG-norbornene + dithiol or multi-arm thiol crosslinker is initiated via UV light and a photoinitiator. In a related embodiment, PEG-vinylsulfone / maleimide + dithiol or multi-arm thiol crosslinker is used at a low pH to prevent gelation prior to droplet formation. The pH is increased in the dispersed phase by adding an organic base (e.g., triethylamine) downstream of the oil phase to initiate the crosslinking reaction.

[0060]

[0097] In one exemplary embodiment, droplet carrier particles 12 were produced using the following conditions: a dextran phase comprising 20% ​​w / v 40 kDa dextran and up to 1.3% DTT crosslinker in phosphate buffered saline (PBS) at pH 7 injected at 2.67 μL / min; a PEG phase comprising 17.5% w / v 4-arm PEG-norbornene, 2% w / v LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) and 0.5 mg / ml biotin-PEG-thiol in PBS injected at 8 μL / min; and an oil phase composed of 0.25% v / v Pico-Surf™ in Novec 7500 oil injected at 44 μL / min into a microfluidic droplet generator device 30 with a channel height of 70 microns and a junction width of 60 microns. The droplet carrier particles 12 are illuminated near the exit area of ​​the microfluidic device (height 130 microns) for approximately 1-3 seconds at 50-200 mW / cm through a DAPI filter set and a 4X objective lens. 2The droplet carrier particles 12 were cross-linked by focused UV light at a power of 1000 / s. Using this approach, droplet carrier particles 12 were produced at speeds of up to 1000 / s with high geometric uniformity. To characterize the uniform biotin groups on the particles 12, they were labeled with 8 micrograms / ml of Alexa Flour™ modified streptavidin solution. The droplet carrier particles 12 were then fluorescently imaged in the TRITC channel, as shown in Figures 10C and 10D. Image analysis showed that the droplet carrier particles 12 had an average outer diameter of 82.5 microns and a CV of 1.5%, while the aperture diameter had an average of 51.0 microns and a CV of 2.1%.

[0061]

[0098] The droplet carrier particles 12 are collected off-chip as an emulsion in a continuous phase of oil and surfactant. In embodiments where purified droplet carrier particles 12 are desired in the aqueous phase, or where purified droplet carrier particles 12 in the aqueous phase serve as the starting point for an assay, the droplet carrier particles 12 must undergo several washing steps to remove excess surfactant, unreacted biomolecules, and associated organic phase from the droplet carrier particles 12. In one embodiment, purified droplet carrier particles 12 were isolated through successive washing steps of Novec-7500 oil, hexane, ethanol, and PBS with 0.1% Pluronic F-127. More specifically, the particle emulsion and associated oil phase are collected off-chip and the majority of the oil phase is removed. For collection of 1 mL of droplet carrier particles 12, 3-5 milliliters of PBS and 1 milliliter of 20% perfluoro-octanol in Novec-7500 are added to the particle emulsion. The solution is gently stirred for a few minutes to break the emulsion, and excess oil is again removed. Next, 3 mL of Novec-7500 oil is again added, the solution is stirred and centrifuged at 2000G for 2 minutes, and then excess Novec oil is removed. This process is repeated for a total of three washes. Next, 3 mL of hexane is added to the particle suspension, stirred, centrifuged, and similarly removed. This process is repeated for a total of three additional washes. Next, 3 mL of ethanol is added to the particle suspension, stirred, centrifuged, and similarly removed. This process is repeated for a total of three additional washes. Finally, 3 mL of PBS with 0.1% Pluronic F-127 is added to the particle suspension, stirred, centrifuged, and similarly removed. This process is repeated for a total of three additional washes. After these final washes, the droplet carrier particles 12 are free of remaining organic solvents, surfactants, and unreacted components, and can be used in assays. For applications involving cells, the droplet carrier particles 12 should be stored for an extended period of time in a solution of ethanol for at least 8-10 hours for sterilization, and can be further washed to return to the aqueous phase before cell seeding.

[0062]

[0099] Size and shape changes

[0100] The size of the droplet carrier particles 12 and the volume of the voids or cavities 18 can vary in size and shape. Using the microfluidic droplet generator device 30, the outer diameter of the particles can be adjusted by adjusting the relative flow rate of the oil phase to the water phase and / or by changing the channel geometry. Droplet carrier particles 12 greater than 100 micrometers in diameter and less than 40 micrometers in diameter have been produced using different channel heights / flow rates. For example, droplet carrier particles 12 with a diameter of 100 micrometers were produced using a device with a channel height of 70 micrometers with the following flow rates: oil = 10 microliters / min (in one embodiment), PEG = 4 μL / min, dextran = 1 μL / min. Droplet carrier particles 12 with a diameter of 40 micrometers were produced using a device with a channel height of 18 micrometers with flow rates: oil = 20 μL / min, PEG = 2 μL / min, dextran = 0.5 μL / min (FIG. 9).

[0063]

[0101] The relative cavity volume and morphology can be tuned by adjusting the concentration and therefore the size of the PEG and dextran domains (Figures 8A and 8B). At a critical concentration defined by the binodal curve, PEG and dextran separate into two phases. The interfacial tension between the two phases varies depending on the concentration of PEG and dextran, altering the contact angles between the PEG, dextran and oil phases. For example, increasing the concentration of PEG and dextran increases the interfacial tension between the two phases, resulting in a more protruding shape. By adjusting the relative ratio of PEG and dextran, the relative volumes of the two domains can be manipulated while maintaining consistency in the morphology.

[0064]

[0102] Production uniformity

[0103] The generation of monodisperse droplets using microfluidics (microfluidic droplet generator device 30) allows for high uniformity of the formed droplet carrier particles 12, including void regions. Specifically, in the embodiment described above, the non-crosslinked PEG-dextran emulsion maintains a CV<1% for the outer diameter and a CV<2% for the uniform diameter of the internal dextran phase (FIG. 10A). The formed droplet carrier particles 12, having voids or cavities 18 defined by dextran phase separated regions, maintain high uniformity with a CV<5% (FIG. 10B).

[0065]

[0104] Advantages of crescent-shaped particles for droplet carrier particles

[0105] A distinct advantage of the described geometries over spherical templates is the ability to generate droplets 10 with more uniform reagent volumes outside the material space used for the droplet carrier particles 12. This is particularly important in the encapsulation / quantification of large molecules that cannot freely diffuse within the particle material matrix. Droplets templated by spherical particles contain a small volume fraction outside the particle. This volume fraction is very sensitive to the relative diameter between the dispersed phase of the emulsion and the droplet carrier particles 12. However, generating droplet carrier particles 12 with internal cavities or voids 18 significantly improves the uniformity of the volume outside the droplet carrier particles 12 by having a predefined geometry that contains a large portion of the encapsulated fluid volume. In one embodiment, a three-fold reduction in the volume variation of droplets formed with droplet carrier particles 12 containing cavities or voids 18 is achieved compared to solid spherical particles (see FIG. 11A). The increased uniformity of the volume increases the uniformity of the reaction carried out within the droplets 10. Another advantage of the cavities or voids 18 is that they provide space for encapsulating cells, beads and other microscale objects of interest. Additionally, manipulation of the morphology and size of the voids or cavities 18 can allow for selective capture of objects having a particular size, shape, stiffness, etc.

[0066]

[0106] In one example, the size of the voids or cavities 18 can be adjusted so that there is only space for a single cell, bead, or other microscale object to fit. In this way, a higher fraction of droplets 10 with single cells (or other microscale objects of interest) can be achieved when compared to standard Poisson loading where multiple targets can be encapsulated at a higher concentration. In another example, if multiple cell types are present with different size characteristics (e.g., cell type A with an average diameter of 30 microns and cell type B with an average diameter of 15 microns), the different cell types can be selected using droplet carrier particles 12 with void or cavity 18 sizes adjusted to the cell sizes. For example, cell type B can first be selectively captured by droplet carrier particles 12 with a void size opening of 20 microns and then isolated from the remaining cell population. Optionally, droplet carrier particles 12 with a void or cavity 18 size opening of 40 microns can be used in a second step to capture and encapsulate cell type A.

[0067]

[0107] Other approaches for producing droplet carrier particles

[0108] Cavity-containing particles suitable for acting as droplet carrier particles 12 can be produced through the use of other sacrificial encapsulation materials. In one embodiment, a single sacrificial particle (smaller in size than a droplet) is encapsulated in a droplet containing a crosslinkable material. The crosslinkable material of the droplet is then crosslinked using various methods known in the art (e.g., the methods described above and other crosslinking methods known to those skilled in the art), and the embedded sacrificial particle is washed and / or decomposed. Alternatively, in some embodiments, multiple sacrificial particles / materials are encapsulated in a crosslinkable emulsion to form a micro / nano-porous cavity network containing void volume within each generated droplet carrier particle 12. A single sacrificial particle can be encapsulated in each droplet of a microfluidically generated emulsion, for example, through the use of microfluidic inertial ordering prior to droplet encapsulation. Examples of sacrificial particle materials include alginate, gelatin, MMP cleavable PEG, polystyrene microbeads, and the like. To tailor the nanoporosity, non-crosslinkable PEG is added to the crosslinkable PEG phase prior to crosslinking in one embodiment consisting of porous droplet carrier particles 12. After crosslinking, the gel is washed to remove the non-crosslinked PEG and reveal the void space.

[0068]

[0109] Other embodiments utilize three-phase systems to create voids in the polymerizable fluid. For example, a three immiscible phase emulsion: water, fluorinated oil, trimethylolpropane ethoxylate triacrylate (ETPTA) can be used to create voids within the ETPTA polymerized particles. Other embodiments utilize masking + photoinitiated crosslinking of precursor solutions to create 2D / 3D protrusions in polymerizable materials with contained void spaces (e.g., diffuse liquid casting, stop-flow lithography, continuous-flow lithography).

[0069]

[0110] Droplet carrier particle storage

[0111] In one embodiment, the droplet carrier particles 12 are stored while suspended in the dispersed phase 14. Optionally, glycerol or other cell protectants are used to allow the droplet carrier particles 12 to be stored at low temperatures (-20 or -80°C) without damaging the particles. In other embodiments, the droplet carrier particles 14 may be dried or lyophilized and then redispersed in the dispersed phase 14 or in a sample fluid containing the dispersed phase 14 prior to use. This allows the density of droplet carrier particles 12 for a given dispersed phase volume to be controlled, and allows the droplet carrier particles 12 to be dispersed in the oil continuous phase with highly efficient dispersed phase / sample fluid encapsulation. Lyophilization is one method that allows for long term storage of the droplet carrier particles 12. A method for freeze-drying hydrogel particles that does not damage the structure of the hydrogel particles can be found in Sheikhi et al., “Microengineered emulsion-to-powder (MEtoP) technology for the high-fidelity preservation of molecular, colloidal, and bulk properties of hydrogel suspensions,” ACS Appl. Polym. Mater., 1, 8, 1935-1941 (2019), which is incorporated by reference herein.

[0070]

[0112] Chemical modification of the droplets

[0113] In many embodiments, the droplet carrier particles 12 described herein are fabricated from polymer precursors that are widely available and used in many biotechnology applications. Thus, there are many well-defined methods of chemical modification that can be applied to the droplet carrier particles 12 prior to emulsification to make them compatible or useful in various bioassays. Examples include thiol-ene / thiol-yne click chemistry, Michael addition, NHS ester reactions, azide-alkyne chemistry, biotin-streptavidin binding, antigen-antibody interactions, etc. (FIG. 12). Several embodiments illustrating these modifications and their applications are presented below.

[0071]

[0114] Biotinylation of particles

[0115] Biotin is a small molecule that has found great utility in the biotechnology industry because it binds strongly and specifically to the proteins avidin and streptavidin. Furthermore, biotin is easily conjugated to larger biomolecules without affecting their function, allowing for their rapid and efficient conjugation to streptavidin-coated substrates or other biomolecules of interest. For conjugation to polymers, there are many commercially available biotins attached to short polymer chains that contain reactive groups that are easily crosslinked with a number of hydrogel precursors. One preferred method of biotinylating the droplet carrier particles 12 is by incorporating biotin PEG-thiol into a PEG solution in the microfluidic droplet generating device 30. More specifically, biotin-functionalized hydrogel droplet carrier particles 12 with void structure were achieved by co-flowing 5 mg / mL biotin PEG-thiol with 15 wt% 10 kDa PEG-norbornene in PBS and 20 wt% dextran containing 1.3% DTT in PBS. Conjugation was verified by observing the fluorescent signal after removing the detergent and reconstituting the droplet carrier particles 12 in the aqueous phase by incubating the gel with streptavidin-FITC. Biotinylation of the droplet carrier particles 12 allows for the conjugation of many common reagents such as antibodies, proteins, nucleic acids, cells, nanoparticles and primers, and their use in a wide range of assays including ELISA, PCR, other nucleic acid amplification assays and flow cytometry.

[0072]

[0116] Cell Adhesion Peptides

[0117] Cell adhesion peptides can also be attached to the droplet carrier particles 12 during or after particle production. In one embodiment, RGD peptide (Ac-RGDSPGERCG-NH2) [SEQ ID NO: 1] or another integrin binding peptide or derivative peptide can be incorporated into the precursor solution and covalently crosslinked into the polymer backbone through reaction with cysteine ​​groups present in the peptide. Integrin binding peptides are important for promoting cell adhesion onto substrates and maintaining viability in adherent cell populations. Currently, droplet assays requiring long-term incubation or cell culture are limited to suspension cells due to the absence of a solid surface in standard water-in-oil emulsions to which cells can attach. This platform extends the utility of droplet assays by providing uniformly sized pockets for attachment and quantification of single cell signals (internal void or cavity 18 space) without limiting the total volume of medium available to cells (total droplet volume) since liquids and small molecules can easily diffuse through the solid gel support. Furthermore, a unique capability of the droplets 10 is the ability to control the dimensions of the internal void or cavity 18 both to act as a strainer to limit the size of the encapsulated cells (e.g., trapping white blood cells, red blood cells or bacteria from a background of larger cells) and to control the volume and number of cells that can be encapsulated, or to maintain both adherent and suspended cells simultaneously. This type of co-culture has proven difficult with current techniques, such as probing interactions between pairs of cell populations. For example, it allows for the detection and subsequent expansion of antigen-specific T cells to patient-derived tumor cells.

[0073]

[0118] FSL-modified cell attachment

[0119] Biotinylated droplet carrier particles 12 can be used to similarly bind non-adherent cell types by pretreating the particles with 10-250 μg / mL streptavidin while simultaneously modifying the cells with biotin conjugated to free lipids or cholesterol (commercially available as FSL-biotin, 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-PEG-biotin (DSPE-PEG-biotin), cholesterol-PEG-biotin, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG-biotin (DMPE-PEG-biotin), etc.), or by modifying the cells with biotinylated lipids and streptavidin prior to mixing with the biotinylated particles 12. In a preferred embodiment, 10-100 μg / mL of biotinylated lipid is added to a suspension of one million cells in PBS and incubated at 37° C. for 60-90 minutes. The affinity and binding of non-adherent cells from aqueous solutions allows for an increased concentration of cells encapsulated in droplets 10 rather than satellite droplets 28, which is important for the analysis of highly diluted samples. Furthermore, using standard techniques such as flow cytometry (using a flow cytometer 150), successful sorting of high-performance members of the cell population is more easily achieved in the aqueous phase and is therefore highly dependent on both the ability to retain cells within the droplet carrier particles 12 after breaking the water-in-oil emulsion and the subsequent release of cells from the droplet carrier particles 12 after sorting. Since FSL-biotin is not permanently bound to the cell membrane, it will degrade over time, eventually releasing the cells from their matrix. Degradable peptides can be incorporated into the PEGylated backbone chains during the formation of the droplet carrier particles 12 and can be cleaved upon completion of the assay by exposure to an appropriate protease enzyme. For example, the peptide Ac-GCRDGPQGIWGQDRCG-NH2 [SEQ ID NO:2] can be used as a cross-linking agent during the manufacture of droplet carrier particles 12 because of the presence of two cysteine ​​groups at its termini, and confers degradability due to the peptide sequence being a substrate for matrix metalloproteases.

[0074]

[0120] Cell isolation by selective adhesion

[0121] Conjugation or covalent attachment of modified antibodies to the surface of droplet carrier particles 12 is also easily accomplished through co-incubation in aqueous solution. Surface-conjugated antibodies allow selective binding of subsets of cell populations based on the expression of specific proteins on their surface. This allows capture and enrichment of single cells of interest directly through antibody interactions with surface antigens conserved across all cells in the population, or with rarer surface antigens that enrich for a subset of cells within a large background of exogenous cells in a mixed population. Such enrichment is an important feature in assays of many biological fluid samples, where data from small subpopulations of interest are often confounded by noise resulting from measurements from target cells. In embodiments where capture of all cells through a conserved protein marker is desired, biotinylated antibodies are typically pre-bound to cells to decorate all available surface moieties while conserving reagents. Up to 10 million T cells were modified in 1 mL volume with 10 μg / mL biotin-anti-CD3 antibody and attached to free streptavidin sites on the droplet carrier particles 12. In another embodiment where capture of a selected cell population is required, the droplet carrier particles 12 must be directly modified with antibodies to eliminate adhesion of cells that do not contain the surface marker of interest. Due to the larger total surface area of ​​the droplet carrier particles 12, this requires a higher concentration of antibody, for example a concentration of 10-250 μg / mL per sample, depending on the antibody affinity for the target antigen.

[0075]

[0122] In one embodiment, selective binding and accumulation of cells is achieved by incubating and actively mixing an aqueous solution containing droplet carrier particles 12 conjugated with antibodies with the mixed cell solution. Antibodies targeting cell surface antigens (e.g., CD8, CD4) result in selective enrichment of the cell type of interest (e.g., T cell subpopulation). During mixing, target cells enter into the voids or cavities 18 of the droplet carrier particles 12, bind to the antibodies, and remain attached within the void or cavity regions 18. Non-target cells may enter the void regions but do not selectively attach, and are therefore removed by subsequent mixing of the solutions. Target cells may also bind to the outer surface of the droplet carrier particles 12, but shear during mixing of the solutions may also remove those cells that are not maintained within the void or cavity regions 18. In one embodiment, a concentrated suspension of T lymphocytes (at least 10 million cells / mL) modified as described above with 10 μg / mL biotin-anti-CD3 is added to the concentrated streptavidin-modified droplet carrier particle pellet at a ratio of 5 cells / particle and pipetted using a 100 μL, 200 μL or 1000 μL pipette tip at a rate of 2-4 repetitions per second for 2 minutes. The droplet carrier particles 12 are then filtered and collected using a Fisherbrand 40 μm cell strainer to obtain a population of droplet carrier particles 12 highly enriched by cells attached within the central cavity 18. Adjustment of the average number of cells bound within the "pocket" 18 of the droplet carrier pocket 12 is possible by adjusting the initial cell to droplet carrier particle 12 ratio during seeding. In particular, the cavity or void area 18 may be of a size (less than 10 microns to less than 20 microns) that allows the entry and retention of only a single cell, two cells or a target number of cells on average. The attached cells can then be encapsulated in an oil phase as described herein and in later sections to perform single cell assays. The attached cells can include mammalian cells, but can alternatively include bacteria, algae, fungi or other cells or cell fragments, such as microvesicles or exosomes.

[0076]

[0123] Trapping of micro / nano objects (cells / beads / proteins / etc.) via crosslinkable dispersed phase

[0124] In a separate embodiment, a crosslinkable material can be added to the sample to be encapsulated as the dispersed phase 14 in the droplets 10. The gelation of the dispersed phase can be triggered prior to returning the droplet carrier particles 12 to the aqueous phase to bind or immobilize the cells, beads or other samples in their respective droplet carrier particles 12 for downstream analysis / sorting. In one example, a temperature sensitive gel (agarose, gelatin, etc.) can be used to gel and encapsulate the cells in the gel matrix prior to transition to the aqueous phase (e.g., through cooling to 4° C.). The encapsulated cells can then be flowed through downstream analysis and sorting equipment (e.g., flow cytometry). In one embodiment, a reversible gelation material is used so that the cells can then be released from the droplet carrier particles 12 and gelled regions via a temperature change to melt the agarose (or other gel material) or the use of an enzyme, e.g., agarase, or a combination thereof.

[0077]

[0125] In other embodiments, UV or pH triggerable crosslinking mechanisms are used to initiate gelation and trapping (radical polymerization, thiol-ene, Michael addition, etc.). If desired, reversible crosslinking can be performed using reversible crosslinkers. In certain embodiments, crosslinkers with disulfide bonds can be degraded using reducing agents, using enzymatically cleavable crosslinkers (e.g., through specific peptide sequences), or using crosslinking chemistries that are susceptible to hydrolysis by the addition of base (e.g., molecules conjugated with crosslinkers that include sulfone bonds, acrylamides, esters, thioesters, etc.).

[0078]

[0126] Varying porosity for varying droplet reactivity

[0127] The porosity of each droplet carrier particle 12 can also be manipulated during crosslinking by incorporation of porogens, changes in precursor chain length, or through the addition of secondary chemistries that allow partial degradation as needed. This control provides an additional layer of versatility, allowing droplet carrier particles 12 to be specifically designed based on the requirements of a particular assay. Droplet carrier particles 12 with smaller pores may be impermeable to proteins and other large biomolecules, allowing for more sensitive detection of such species, since the apparent volume of such an assay is the particle void space rather than the entire particle volume. In contrast, larger pore spacing allows diffusion of larger biomolecules, providing more sites for attachment before signal saturation, widening the quantifiable range.

[0079]

[0128] Use of Droplets in Assays

[0129] Droplets 10 can be made compatible with many widely used droplets or other digital assays that rely on compartmentalizing a volume. Through careful selection in material chemistry and particle design, droplets 10 can be directly applied to many assay workflows to enhance collected data through enrichment of desired cell populations or by providing a smaller apparent volume for analyte detection.

[0080]

[0130] Digital Nucleic Acid Amplification Assay

[0131] Nucleic acid amplification is widely used in biology for the identification and characterization of nucleic acid sequences to monitor gene expression, identify inherited diseases, and verify proper gene transfer in genetic modification workflows. The best known of these amplification schemes, polymerase chain reaction (PCR), has been adapted for use in droplets and commercialized by Bio-Rad for use as a digital assay. Droplet digital PCR (ddPCR) uses a microfluidic droplet generator to segment an aqueous volume containing nucleic acids, primers, dNTPs, fluorescent intercalation dyes, and DNA synthesis enzymes into nanoliter volume droplets. When a sample is heat cycled, the target DNA is amplified, resulting in an increase in fluorescent signal (e.g., due to an intercalation fluorescent dye) in droplets containing the target sequence that is easily distinguished from background fluorescence in droplets where amplification did not proceed due to the absence of the target sequence. This technique has the added advantage of providing absolute quantification of the concentration of the target sequence in a biological sample.

[0081]

[0132] Digital nucleic acid amplification approaches (such as digital PCR, digital loop-mediated isothermal amplification (LAMP) or other isothermal amplification approaches) are easily transferred to the droplet 10 system because the hydrogel particles can be easily reconstituted in an aqueous PCR or other amplification cocktail mixed with a sample containing the target nucleic acid sequence ( FIG. 13A ). Subsequent sample emulsification via vortexing or pipetting allows for distribution of the target sequence and cocktail mix, similar to standard ddPCR approaches. The uniform volume within the droplet 10 can then be heat cycled or heated to generate a fluorescent signal if a target is present and analyzed for absolute quantification. Importantly, the presence of droplet carrier particles 12 supporting the droplet 10 can improve the thermal stability of the emulsion and reduce aggregation. Emulsification through this approach can generate multiple satellite droplets 28 as well. However, the relative ratio of these satellite droplets 28 to those filled with particles can be adjusted based on the surfactant used, the dryness (porosity) of the initial microgel sample, and the sample volume compared to the available volume in the droplet carrier particles mixed with the sample. Thus, the total volume of sample encapsulated in the empty satellite droplets 28 can be limited, or the fraction of the total aqueous volume dispensed into the droplets 10 can be calculated and a correction factor applied to quantify the number of target nucleic acid sequences present in the total volume. For example, the target encapsulation efficiency can be approximated for a given initial concentration of droplet carrier particles 12, the droplet carrier particle geometry (particle outer diameter and internal cavity volume), and the expected thickness of the aqueous layer surrounding the droplet carrier particles 12 after the formation of the droplets 10 (see Figures 11A-11C and related text on encapsulation efficiency theory).

[0082]

[0133] In a related embodiment, the droplet carrier particles 12 can be pre-coated with a primer and / or captured nucleic acid sequence specific for the target sequence (or sequences) to promote target nucleic acid binding to the droplet carrier particles 12 in the aqueous phase prior to emulsification. This allows for localized analyte concentration on the droplet carrier particles 12 and eliminates amplification of satellite droplets 28 during heat cycling. By utilizing an excessively large number of droplet carrier particles 12 relative to the expected sample target sequences, these systems operate in a digital counting regime, but the analog fluorescent signal of the higher concentrations of each droplet can also provide information on the nucleic acid concentration. The absolute fluorescence value (not just an on-off threshold) can be used to recalculate the average number of bound sequences per droplet 10 and yield an absolute value for the total number of target sequences in the sample. The ability to concentrate target sequences on the droplet carrier particles 12 and then perform downstream amplification and readout is an inherent advantage of droplet systems. Additionally, the ability to capture nucleic acid sequences from a sample allows for washing and replacement with a different solution (e.g., nucleic acid amplification mixture solution, see washing section above) allowing for the ability to remove background contaminants from the sample solution that may interact with the amplification reaction. Finally, the droplet approach has the added advantage that the digital assays are completely device-free, providing users with no knowledge of microfluidics easy access to these assays that may otherwise be unavailable or require extensive equipment and / or expertise.

[0083]

[0134] Further modifications of the droplets 10 allow the droplets 10 to perform even the most complex embodiments of digital PCR assays, such as BEAM (bead, emulsion, amplified, magnetic) digital PCR. In BEAM, the biofluid sample is again dispensed into droplets with primer-functionalized beads. If a target nucleic acid is present in the droplet with the beads, the nucleic acid is amplified by PCR, leading to the attachment of an amplicon to the primer on the encapsulating bead. The bead-attached amplicon can contain information about a mutation in the target sequence. It is noted that not every droplet contains both the target and one bead in previous implementations of BEAM, because the emulsification of the beads is random, which is a drawback of the technique. Once amplification is complete, the emulsion is broken and each bead is magnetically retrieved and incubated with one or more types of fluorescently labeled nucleic acid reporter probes that are complementary to sequences containing different mutations. The specific mutations of interest that are amplified and attached to the beads give rise to a unique fluorescent signal on that bead. These fluorescently labeled beads can then be passed through a flow cytometer 150 to characterize the prevalence of mutations in the target sequences present in the sample.

[0084]

[0135] However, assays similar to BEAM can be performed using droplets 10 with additional advantages. In one embodiment, the droplet system acts to generate more monodisperse emulsions for BEAM, and the opening size or volume of the cavity or void area 18 in the droplet carrier particle 12 is dimensioned to allow only a single bead to enter into each droplet 10. In another preferred embodiment, the droplet carrier particle 12 itself is conjugated with a primer and acts as the solid phase bead in the BEAM workflow. This has a significant advantage in that each droplet has a single attached solid phase (i.e., the droplet carrier particle 12). The droplet carrier particle 12 can then be resuspended in the aqueous phase 14 for fluorescent probe binding and analysis in a flow cytometer 150. Each droplet carrier particle 12 can also be barcoded in a number of ways to provide a unique index to the droplet carrier particle 12. For example, in one embodiment, the droplet carrier particle may contain separate primer / nucleic acid capture sequences associated with each barcode targeting a different sequence to allow multiplexing of nucleic acid detection assays. Primers on one or both sides of the target sequence may also be immobilized on the droplet carrier particles 12 to produce attached target nucleic acid sequences.

[0085]

[0136] As described herein, the droplets 10 can also be magnetically separated from the background of satellite droplets 28 by covalently attaching or embedding magnetic nanoparticles within the droplet carrier particles 12. In the presence of an externally applied magnetic field, the droplet carrier particles 12 can be accumulated or displaced and washed. By producing magnetic droplet carrier particles 12 below the standard flow cytometry critical size cutoff (<50 micrometers), mutational heterogeneity in a sample can be quantified directly from suprathreshold fluorescent signals associated with each droplet carrier particle 12.

[0086]

[0137] In a related embodiment, the amplified nucleic acid sequences can also be captured on the droplet carrier particles 12 for analysis by including a pre-gel solution of the aqueous phase to gel the aqueous dispersed phase after amplification to prevent loss of the amplicons upon returning the droplet carrier particles 12 to the aqueous phase. This approach is more preferable for long amplicons (e.g., those generated using a LAMP reaction).

[0087]

[0138] In another embodiment, droplet carrier particles 12 are used that have fully enclosed cavities 18. The porosity of the particle matrix can be adjusted to allow the DNA target and reaction mixture to diffuse through the matrix. After droplet 10 formation and amplification, amplification products that are too large to diffuse through the particle matrix can be retained. This is beneficial in that the droplet carrier particles 12 can be returned to the aqueous phase for downstream analysis and quantification (e.g., flow cytometer) without loss of amplification signal that remains retained within the fully enclosed cavities.

[0088]

[0139] Barcoding of droplet carrier particles 12 can be used to simultaneously capture and analyze different target nucleic acid sequences in a mixed sample. A mixture of distinct types of barcoded droplet carrier particles 12 and distinct nucleic acid capture reagents can be introduced to a sample to collect distinct targets. The signal for a particular droplet carrier particle 12 can be linked to the barcode for that droplet carrier particle 12 to identify the target type, and multiplex assays can be performed when multiple droplet carrier particles 12 are analyzed. Approaches for barcoding droplet carrier particles 12 are described further herein.

[0089]

[0140] Digital ELISA

[0141] Digital ELISA is a technology conceptually similar to ddPCR, used to quantify low concentrations of proteins or other analytes in small aqueous distributions using a sandwich immunorecognition reaction. To perform digital ELISA in droplets 10, capture antibodies specific to the target protein or other analyte are first conjugated to the surface of droplet carrier particles 12 (see FIG. 13B). The antibody-coated droplet carrier particles 12 are then placed in a solution containing the target analyte such that the number of droplet carrier particles 12 is in large excess over the number of analytes, and incubated to allow complete binding. After binding, the sample solution is washed and replaced with a washing solution. Once this first binding reaction and washing step is complete, a second antibody that has affinity for a second region of the target analyte and contains a signal amplification component is added to the solution and can bind to the analyte bound to the droplet carrier particles 12. The excess second antibody is washed again from the droplet carrier particles 12 using the techniques described herein, and the solution is replaced with a signal development solution containing a substrate that reacts with the amplification component of the antibody to generate a signal (fluorescence, colorimetric, pH change). Examples of signal amplification components include enzymes such as horseradish peroxidase (HRB), β-galactosidase, esterase, etc. Examples of substrates include fluorescein di-β-D-galactopyranoside (FDG) or other fluorogenic substrates of β-galactosidase, as well as fluorogenic substrates of HRP, such as Amplex Red, QuantaRed, QuantaBlu, etc. Immediately after addition of the signal developer, the droplet carrier particles 12 are emulsified with an oil phase 16 to form droplets 10, and the entire sample is incubated to allow accumulation of fluorescent or other signal by the droplets 10. Following signal creation, the droplets 10 can be placed in a large reservoir where all droplets 10 can be simultaneously imaged and quantified using a microscope or wide-field low-cost imaging device to determine the fraction of droplets 10 with high signal relative to the total number of droplets 10. As previously identified, this allows calculation of the expected concentration of the analyte in the sample.Furthermore, after the analyte is bound to the droplet carrier particles 12, which are washed and resuspended in the ELISA process, and the sample is emulsified, all satellite droplets 28 do not contain signal generating components and can be discarded without issue. In another embodiment, tyramides can be used to link the surface of the droplet carrier particles 12 themselves as a product of HRP enzyme turnover that allows an amplified local signal to be captured on the droplet carrier particles 12. These signals, such as fluorescent signals, can then provide a direct measure of whether analyte has been bound on each droplet carrier particle 12 using downstream techniques, such as flow cytometry (using flow cytometer 150), after the emulsion of the droplets 10 is broken. In one embodiment, Alexa Flour™ 488-tyramide is used as a substrate that is activated by HRP, which acts as an amplification component on the secondary antibody, and the activated fluorescent tyramide conjugate covalently binds to nearby proteins, peptides, or other phenolic groups incorporated on or within the droplet carrier particles 12. In a related embodiment, biotin-tyramide is used as a substrate leading to signal amplification by covalent attachment of multiple biotin molecules to the droplet carrier particles 12. The biotin can then be fluorescently labeled in a number of ways using streptavidin-conjugated fluorophores.

[0090]

[0142] For nucleic acid analysis, barcoding of droplet carrier particles 12 can be used to simultaneously capture and analyze different target analytes in a mixed sample. A mixture of distinct types of barcoded droplet carrier particles 12 and distinct antibody capture reagents can be introduced into the sample to collect distinct targets. The signal for a particular droplet carrier particle 12 can be linked to the barcode for that droplet carrier particle 12 as they coexist. This allows target types for multiple droplet carrier particles 12 to be analyzed to perform multiplex assays for multiple proteins / analytes. Approaches for barcoding droplet carrier particles 12 are further described herein.

[0091]

[0143] Single Cell Sequencing

[0144] The drive to improve the resolution of cellular gene expression measurements, combined with the reduction in cost of the aforementioned sequencing approaches, has prompted interest in droplet-mediated single-cell sequencing of RNA and DNA. In the single-cell RNA sequencing approach, individual cells are co-encapsulated with barcoded beads capable of capturing mRNA with bound poly-T capture nucleic acids that contain a unique cellular barcode and molecular identifier. These barcoded capture nucleic acids also act as primers for reverse transcription. The cells are lysed within the droplet to release the mRNA, which binds to the poly-T capture nucleic acids on the barcoded beads. The emulsion is then broken and the captured RNA on each bead is reverse transcribed. The pooled cDNA contains the barcode from each individual cell to obtain a unique gene expression readout corresponding to that cellular member of the population. With a single barcoded bead and cell present in each droplet, this process attributes a unique molecular signature to the readouts from each individual cell and allows for pooling and bulk gene sequencing and other analysis of the signatures while maintaining sample differentiation. Some common variations of these approaches include Drop-Seq, InDrop, and CytoSeq.

[0092]

[0145] Single cell nucleic acid sequencing is adapted using a droplet system with the distinct advantage of being device-free, again allowing quantification of gene expression from unique cells without the need for complex microfluidic devices or equipment for the end user. In one embodiment using droplets 10 for single cell RNA sequencing, the cavity or void area 18 of the droplet carrier particle 12 is sized to retain a single cell and prevent more than one cell from entering. The droplet carrier particles 12 are further functionalized to include mRNA capture moieties on their surfaces that are uniquely barcoded, as described herein. The droplet carrier particles 12 may optionally be functionalized to include adhesion ligands or antibodies to cells, as described herein, to allow selective cell isolation prior to single cell RNA sequencing. A lysis reagent can also be incorporated into the matrix of the droplet carrier particle 12 prior to cell encapsulation and formation of the droplet 10 emulsion. The lysis reagent can be slowly released to lyse the captured cells, or can be triggered to be released or activated, as discussed further below. Alternatively, the lysis reagent can be added by mixing the droplets 10 with additional droplets containing a lysis agent that interacts with the droplets 10 without removing attached cells. Bulk emulsification of the droplets 10 in the presence of cells can encapsulate millions of single cells in a time scale of minutes, rather than the hours to days required by a single microfluidic device. In another embodiment, a lysis agent that is miscible in both the oil phase 16 and the aqueous phase 14 can be used. In this case, the lysis agent can be added to the oil 16 after the formation of the droplets 10, with some fraction distributing into the droplets 10 to cause cell lysis. In another embodiment, an inactive lysis reagent can be added into the dispersed phase and then activated by an external stimulus after or just before droplet formation. For example, ionic surfactants such as sarkosyl are active over a certain pH range.In this example, the pH of the solution is lowered to leave the Sarkosyl inactivated, and then droplet formation can be activated by increasing the pH through a proton acceptor (e.g., triethylamine) dissolved in the oil phase that partitions into the aqueous phase of the droplet 10.

[0093]

[0146]

[0147] Furthermore, the standard way to barcode nucleic acid capture sequences on microparticles in existing methods is through split-and-pool synthesis, where the microparticles are split into four different solutions, each containing one nucleotide, collected, and randomly split again a total of n times to form random oligomers of length n. This type of addition can be performed directly on the droplet carrier particle 12 used to create the droplet 10, eliminating the need for a second solid phase and restricting DNA amplification to only droplets containing functionalized particles. That is, the use of droplets 10 can provide a significant advantage by overcoming the stochastic process of bead and cell loading, which is a waste of both beads and cells. Although single cells, droplets without beads do not lead to a signal, nor do droplets with beads without cells lead to a signal. The droplet 10 contains a single bead (droplet carrier particle 12) and the void area can be dimensioned to isolate a single cell.

[0094]

[0148] Controlling the release of molecules / reagents from droplet carrier matrices

[0149] In one embodiment, the reagents entrapped within the matrix of the droplet carrier particle 12 are released over time. For example, lytic reagents (i.e., surfactants, detergents and / or enzymes) can be stored within the dried droplet carrier particle 12. Upon hydration, the lytic reagents are slowly released to lyse the cells encapsulated within the droplet 10. In another embodiment, the lytic reagents are incubated within the droplet carrier matrix material until near saturation, and the dispersed phase is exchanged for a non-lytic fluid that contains the cells to be encapsulated, forming the droplet 10. The lytic reagents stored in the droplet carrier matrix are slowly released by diffusion to lyse the encapsulated cells. The porosity of the hydrogel matrix of the droplet carrier particle 12 can be adjusted to modulate the time course of reagent release, with increased porosity resulting in a more rapid release and decreased porosity resulting in release over an extended period of time, as described herein.

[0095]

[0150] In other embodiments, the matrix of the droplet carrier particles 12 is formed from a material that swells in response to changes in environmental conditions (e.g., pH, temperature, light, etc.), allowing for triggered release of encapsulated reagents stored in the matrix. For example, light-triggered degradation of o-nitrophenyl-containing backbone polymers in the droplet carrier particle 12 matrix can result in a decrease in crosslink density, swelling of the droplet carrier matrix, and reagent release. In another embodiment, the matrix of the droplet carrier particles 12 is charged and oppositely charged reagents are bound via charge-to-charge interactions. Release of the charged reagents can be induced by a change in pH, which alters the charge of the reagent, the droplet carrier particles 12, or both. In one embodiment, the pH can be externally adjusted through the addition of organic acids / bases in the oil phase (e.g., acetic acid, triethylamine). In other embodiments, the above-mentioned approach may be used to release drugs / molecules over a period of time to probe cellular responses.

[0096]

[0151] Cell viability during encapsulation

[0152] Some assays require the maintenance of cell viability even after single cell measurements. For example, to screen high performers for the production of useful bioproducts or to select cells with optimal phenotypes for the treatment of certain diseases. These assays can also be performed in a droplet 10 emulsion system by using oils and surfactants known to be highly cytocompatible. In a preferred embodiment, a fluorocarbon continuous phase is used that includes a non-ionic cytocompatible surfactant. To demonstrate this, Jurkat cells stained with both Hoechst and calcein were mixed with dried PEG microgel droplet carrier particles 12 produced as described herein and emulsified by pipette in a Novec™ 7500 with 0.5% Pico-Surf™. Upon emulsification, the droplets 10 were placed on a microscope slide and imaged in bright field, DAPI, and FITC channels via a fluorescent microscope to determine viability (see Figures 14A-C). The majority of cells emulsified within the droplets 10 stained positive for calcein both pre- and post-emulsification, indicating compatibility with the proposed encapsulation approach.

[0097]

[0153] In another example, Chinese Hamster Ovary (CHO) cells were encapsulated in droplets 10 in media using Novec™ 7500 and 2% v / v Pico-Surf™ as the oil phase. Cells were harvested at various time points using Novec™ 20% v / v PFO and stained with calcein AM as a live stain and propidium iodide as a dead stain. It was found that cells maintained high viability throughout this process and for over 24 hours (>80% viability).

[0098]

[0154] Single cell secretion isolation

[0155] Cell populations traditionally thought of as homogenous can in fact exhibit enormous heterogeneity in phenotype that is simply masked by the limited resolution in many common assays. To gather useful indicators of cell population dynamics, these assays must be performed at the single-cell level. For example, in vivo, a large-scale immunological response to perturbations of the local microenvironment is orchestrated through the secretion of signaling molecules between leukocytes. Simply probing bodily fluids in bulk to determine protein composition in this scenario provides total sample concentrations but no information about which subpopulations are secreting, limiting the extent to which such coordinated responses can be understood.

[0099]

[0156] A number of schemes can be applied to capture, quantify and sort cells based on secreted protein signals using droplets 10. Figure 16 shows an example of an exemplary workflow for performing capture and analysis of secretions using droplets 10. Cells can be bound to the surface of droplet carrier particles 12 as described above using either biotinylated lipids, RGD and / or surface marker specific antibodies depending on the desired composition of the encapsulated cell population. To capture secretions, a capture reagent (e.g., an antibody targeting the secreted molecule) can be immobilized on and / or within the matrix of droplet carrier particles 12 as described above for the ELISA system. Once encapsulated, the cells are incubated while the secreted proteins accumulate on the surface of the particles bound to the capture reagent. A secondary reporter antibody, e.g., a secondary reporter antibody with an attached fluorophore that is also present in the emulsified solution or can be introduced upon breaking the emulsion, binds to the captured proteins. Localization of the signal onto the droplet carrier particles 12 can enable sorting through a standard flow cytometry / FACS system 150 after breaking the emulsion and reconstituting the droplet carrier particles 12 into the aqueous phase. Furthermore, reversible binding of cells to the particle surface, for example through biotinylated lipid modification, allows cells to be sorted along with the droplet carrier particles 12, resulting in the selection of specific cells having a desired phenotype as well as quantitative secretion analysis at the single cell level.

[0100]

[0157] General workflow

[0158] (1) Loading of cells onto droplet carrier particles 12 in aqueous solution

[0159] In one embodiment, droplet carrier particles 12 are first loaded, for example by a pipette, into a well plate, well, flask or other container with a flat bottom (see FIG. 16 - Particle Seeding and Cell Loading). In one embodiment, the droplet carrier particles 12 are asymmetric in shape, so that the droplet carrier particles 12 settle with their cavities in an upright orientation (e.g., cavities 18 open on the surface opposite the direction of acceleration due to gravity). This is advantageous in that the open cavities 18 can then be seeded with cells. The seeding volume of droplet carrier particles 12 can be approximated for a given particle size and well surface area by assuming a closed packing of spheres. For example, for 85 micron diameter particles 12 and a 12 well plate (with a surface area of ​​2 cm per well), ... 2 ), it was found that 30 microliters of concentrated particles 12 covered most of the bottom of the well surface.

[0101]

[0160] After the droplet carrier particles 12 have settled (typically 5-10 minutes), cells are carefully seeded (e.g., with a pipette) into the wells and allowed to settle, with a portion of the cells settling into the cavities 18 of the droplet carrier particles 12. The fraction of cells that are outside the cavities 18 relative to the fraction of cells that enter the cavities 18 can be increased by increasing the ratio of the cavity opening width to the droplet carrier particle diameter. In general, the fraction of droplet carrier particles 12 that contain a given number of cells (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cells) can be calculated from Poisson statistics and controlled by adjusting the cell density during the seeding step. In one example experiment, each well of a 24-well plate was seeded with 15 microliters of concentrated droplet carrier particles 12 (85 micron outer diameter, 50 micron inner diameter). Various amounts of cells were added to different wells and allowed to settle into the droplet carrier particles 12. The cells and particles 12 were imaged to determine the number of cells per particle 12. Addition of 10,000 cells per well resulted in a Poisson loading of approximately 0.09 cells per particle 12. More specifically, 93.1% of the particles 12 were empty, 6.65% contained one (1) cell, and 0.25% contained two (2) or more cells. Of the particles 12 with cells, 96% contained only one cell. Addition of 30,000 cells per well resulted in a Poisson loading of approximately 0.2 cells per particle 12. More specifically, 82.2% of the particles 12 were empty, 15.9% contained one (1) cell, and 1.9% contained two (2) or more cells. Of the particles 12 with cells, 89% contained only one cell. Addition of 100,000 cells per well resulted in a Poisson loading of approximately 0.8 cells per particle 12. More specifically, 42.6% of the particles 12 were empty, 32.6% contained one (1) cell, and 24.8% contained two (2) or more cells. Of the particles 12 with cells, 56.8% contained only one cell.

[0102]

[0161] Different cell seeding volumes are ideal for various applications. In embodiments where it is important to capture less than one cell per particle for assay validity, a lower seeding density is ideal (e.g., in the wells of a 24-well plate (surface area 2 cm2)). 2 In contrast, in embodiments where multiple cells per particle 12 are desired, such as for the assessment of cell-cell interactions, a higher seeding density is preferred (e.g., 10,000 cells per well (surface area 2 cm) of a 24-well plate where 15 μL of concentrated droplet carrier particles 12 are seeded in each well). 2 )). In other embodiments, where cell loss is detrimental and should be limited, multiple layers of droplet carrier particles 12 can be arrayed such that cells that do not settle into cavities 18 in a first layer of droplet carrier particles 12 can settle into cavities 18 in a second or subsequent layer of droplet carrier particles 12.

[0103]

[0162] A second method of binding cells to droplet carrier particles 12 utilizes the difference in shear forces experienced by cells bound within the protected cavities 18 of the droplet carrier particles versus the shear forces experienced by cells bound to the outer surface of the droplet carrier particles 12. Briefly, a concentrated suspension of cells with affinity for the surface of the droplet carrier particles 12 (e.g., through adhesive ligands) can be mixed into a concentrated suspension of droplet carrier particles 12 and vigorously agitated using a 100 μL, 200 μL, or 1 mL pipette at 2-4 pipette repetitions per second, 50-100x. The cells distribute throughout the suspension and rapidly attach to the surface of the droplet carrier particles 12. Cells that bind to the outside of the droplet carrier particles 12 are rapidly sheared back into suspension, while cells that are trapped within the voids or cavities 18 are further protected from the majority of the external fluid shear forces and remain attached to the surface of the pocket formed by the voids or cavities 18 of the droplet carrier particles 12. Once the particle suspension has been sufficiently agitated, it can be filtered as described below, which provides a method of rapidly concentrating the fraction of cells found within the cavities 18 of the droplet carrier particles.

[0104]

[0163] Modification of the surface of the droplet carrier particles 12 allows for the attachment and subsequent culture of seeded cells within the cavities 18 of the droplet carrier particles. For example, commonly used integrin-binding peptides such as RGD incorporated into the surface of the droplet carrier particles 12 allow for cell attachment based on the presence of integrins, for example, and maintain cell attachment to the droplet carrier particles 12 even in the presence of vigorous mechanical agitation from a pipette, centrifugation, and flow sorting procedures. In a preferred embodiment, RGD is added at a concentration of at least 4 mg / mL in the dextran phase during the preparation of the droplet carrier particles 12. In this approach, radicals generated from a photoinitiator in the PEG phase induce covalent bonds between free thiols on the peptide cysteine ​​groups and unbound norbornenes on the polymer backbone of the droplet carrier particle 12 precursor. CHO cells seeded on such RGD-modified droplet carrier particles 12 remained associated and spread on the surface of the droplet carrier particles 12 for several days.

[0105]

[0164] Cell lines that are typically non-adherent can also be bound to the surface of droplet carrier particles 12. In one embodiment, biotin-streptavidin interactions are used to link cells to droplet carrier particles 12. More specifically, biotinylated droplet carrier particles 12 are pre-modified with streptavidin and target cells are pre-modified with either biotinylated lipids / cholesterol or biotinylated antibodies that generate affinity between the droplet carrier particles 12 and the cell population or a subset of the cell population. In one preferred workflow, primary T cells can be bound to biotinylated droplet carrier particles 12 by first pre-modifying the biotinylated droplet carrier particles 12 with 10 μg / mL streptavidin in PBS. At the same time, T cells are modified by mixing 10 μg / mL biotin-anti-CD3 antibody to less than 10 million cells and incubated at 37° C. for 20 minutes. Both the droplet carrier particles 12 and the cells are washed several times with PBS to ensure removal of unbound material. The droplet carrier particles 12 are then centrifuged at 2000G for 5 minutes to form a tight pellet to which the concentrated anti-CD3 modified cell suspension is added. The droplet carrier particles 12 cells and suspension are then continuously stirred by manual pipetting for at least 2 minutes. The sample is then filtered using a cell strainer, as described below, to collect only the droplet carrier particles 12 and any cells bound to their surface. An alternative embodiment using biotinylated lipids proceeds in much the same way, with the important caveat that any cells can be modified using this approach, regardless of surface protein composition. Here, the cells are incubated with 10-100 μg / mL of biotinylated lipid at 37° C. for a total of 60-90 minutes, then washed and attached to the droplet carrier particles 12 by pipetting.

[0106]

[0165] (2) Washing away unbound cells and / or background secretions and adding affinity reagents to bind to droplet carrier particles 12 that capture the specific cell secretions of interest.

[0166] In some applications, cells that remain unbound to the droplet carrier particles 12 are undesirable and may even be a source of noise. To reduce background, the droplet carrier particles 12 can be washed prior to the formation of the droplets 10 to eliminate unbound cells from the solution before the assay is performed. In one approach, a suspension of droplet carrier particles 12, droplet carrier particles 12 with attached cells and unbound cells are added to a cell strainer with a mesh size larger than the cell diameter but smaller than the diameter of the droplet carrier particles 12. This allows the droplet carrier particles 12 with attached cells to be retained by the mesh while the unbound cells pass through the mesh. While retained, the droplet carrier particles 12 can be continuously washed by successive additions of buffer to remove cells that are not tightly attached to the surface of the droplet carrier particles 12. The droplet carrier particles 12 and their bound cells are then isolated through simple inversion of the cell strainer, addition of buffer from the underside of the mesh and collection of the resulting solution containing the buffer and the eluted droplet carrier particles 12 with attached cells. One suitable cell strainer for this application is the Ficherbrand 40 μm sterile cell strainer from Fisher Scientific.

[0107]

[0167] In rare cell applications, it may be desirable to collect cells that are not bound to the cavities 18 of the droplet carrier particles. In this case, the strategies described above can be used to use the collected cells that are not bound to the droplet carrier particles 12 for later seeding into a new sample of droplet carrier particles 12.

[0108]

[0168] In some applications, it is desirable to capture secretions on droplet carrier particles 12. In such embodiments, it is beneficial to modify the surface of the droplet carrier particles 12 with affinity reagents, such as antibodies or immunoglobulin binding proteins, to act as molecular capture sites for the secretions (FIG. 16-Capturing and sorting secretions). In an exemplary embodiment where the secretion rate is slow and binding of cells to the droplet carrier particles 12 is rapid, such as in a biotin-streptavidin reaction, the droplet carrier particles 12 can be pre-modified with molecular capture sites within or on the surface of the droplet carrier particles 12 and can be rapidly dispensed into droplets 10 without contaminating the signal resulting from the bound cells. In other embodiments where the secretion rate is rapid and it takes several hours for the cells to strongly attach to the surface of the droplet carrier particles 12, such as when adherent cells (e.g., CHO cells) attach to RGD-linked peptides, the cells are first allowed to attach to the surface of the droplet carrier particles 12, preferably for 2 to 12 hours. Once the cells have attached to the droplet carrier particles 12, the sample can be washed via centrifugation and solution exchange to remove background secretions and unbound cells. The droplet carrier particles 12 can then be modified with affinity reagents such as biotinylated protein A or biotinylated antibodies that bind to the secretions of interest to form molecular capture sites attached to the droplet carrier particles 12, which are then rapidly emulsified into droplets 10. Detailed protocols are disclosed herein. In a preferred embodiment, the droplet carrier particles 12 are functionalized with both RGD peptide and biotin groups by incorporating both 4 mg / mL RGD in the dextran phase of the droplet carrier particle precursor and 0.5-5 mg / mL biotin-PEG-thiol in the PEG phase of the droplet carrier particle precursor during fabrication, as described in the detailed description for FIG. 12. Cells are seeded onto these peptide and biotin modified droplet carrier particles 12. After deposition, each droplet carrier particle 12 sample (30 μL of droplet carrier particles 12 in a 12 well plate) is treated with 0.02 mg / mL streptavidin in PBS, which binds to the biotin groups on the surface of the droplet carrier particles 12. The samples are incubated for 10 minutes and washed several times with PBS with 0.5% BSA.Each concentrated droplet carrier particle 12 sample is then modified with 10 μL of a 0.5 mg / mL stock of biotinylated protein A and incubated for 10 minutes as described above. After appropriate modification, the droplet carrier particles 12 can be directly applied for secretion measurement applications or emulsified as described below.

[0109]

[0169] (3) Emulsification of droplet carrier particles 12 with attached cells and binding affinity reagents into an oil continuous phase to form droplets 10.

[0170] Optimal formation and maintenance of droplets 10 depends on several factors, including the type and concentration of surfactant used and the agitation method. In one exemplary embodiment, 2% Pico-Surf™ (Sphere Fluidics, Inc.) in a Novec™ 7500 operated fluid as the continuous phase was used with droplet carrier particles 12 in PBS or cell culture medium as the dispersed phase at a 2:1 volume ratio, respectively. Under these circumstances, vigorous agitation via manual pipetting (50-100X per second, 2-4 pipetting repetitions per second using a 100 μL, 200 μL or 1 mL pipette) was found to reliably and reproducibly form monodisperse droplets 10 with a high fraction containing only single droplet carrier particles 12.

[0110]

[0171] (4) Incubating the drops 10 for a period of time to accumulate secretions that bind to the affinity reagent.

[0172] To maintain cell viability during incubation, droplets 10 were formed using both cell culture medium (such as RPMI-1640 / DMEM) and PBS enriched with 2% FBS. Since most small molecules can easily diffuse through the pores of the hydrogel droplet carrier particles 12, the total volume of the droplets 10 serves as a nutrient reservoir. Furthermore, the droplets 12 can be maintained at about 37° C. in a standard cell incubator. Adding a layer of light or heavy mineral oil on top of the droplet 10 layer was found to improve stability by reducing evaporation and aggregation.

[0111]

[0173] All analytes produced by single cells encapsulated within the droplets 10 are retained within their associated aqueous phase volume or compartment. These relatively small droplets 10 result in localized accumulation of secretion signals that aid in rapid binding of detectable amounts of secretion on the surface of the droplet carrier particles 12. Depending on the expected secretion levels, incubation times can be adjusted depending on the cell type and secretion target of interest. For example, if low secretion levels are expected, longer incubation times (>12 hours) can be used, whereas if high secretion rates are expected, shorter incubation times (~2-3 hours) can be used.

[0112]

[0174] Depending on the expected secretion level, the number of secretion binding sites and the spatial location of the binding sites can be adjusted. In one example, the available binding sites can be increased by fabricating the droplet carrier particle 12 with a matrix porosity with a pore size that allows the secretion to diffuse freely through the gel matrix. In this embodiment, the complete three-dimensional geometry of the droplet carrier particle 12 can be used to capture the secreted molecules to increase the total number of binding sites, which is beneficial in the case of high secretion levels, since the binding sites are not easily saturated, allowing for a better dynamic range of detection. In some cases, for example, at low secretion levels, it is advantageous to spatially localize the binding sites to generate a more focused signal. For example, by using solid droplet carrier particles 12, or by using particles with a porosity such that the secretion cannot diffuse freely through the matrix of the droplet carrier particle 12, accessible binding sites are localized on the surface of the droplet carrier particle 12. In a further embodiment, the binding sites can be localized on the surface of the internal cavity or cavity 18 to further localize the secretion capture and the obtained signal. In the particular example of CHO cells secreting anti-IL-8 antibodies described herein, the pore size of the droplet carrier particles is small enough to prevent free diffusion of the target secrete, anti-IL-8, such that the secreted anti-IL-8 binds to the outside of the droplet carrier particles 12 (i.e., within the cavities 18 and around the outer periphery), enhancing the visibility of the accumulated fluorescent signal.

[0113]

[0175] (5) Breaking the emulsion of the droplets 10 to return the droplet carrier particles 12 with attached cells to the aqueous solution.

[0176] Once the droplets 10 have been incubated long enough for sufficient secretions to accumulate and bind on the droplet carrier particles 12 for visualization, the emulsion loses stability to recover the droplet carrier particles 12 and their bound cells. This is accomplished by adding a secondary surfactant such as perfluorooctanol (PFO) or Pico-Break™. In one embodiment, all excess oil is removed from the droplet 10 emulsion and replaced with an equal volume of 20% PFO in Novec™-7500 oil. Once the droplets are destabilized, the solution is gently stirred by tapping the tube surface, as a clear boundary between the aqueous and organic phases is visible within 2 minutes. Any remaining small organic satellite droplets 28 can be removed from the aqueous phase through rapid centrifugation at low speed (~100g) for no more than 5 seconds. The aqueous solution and droplet carrier particles 12 are then easily removed (e.g., by pipette) from the dissociated oil phase prior to downstream analysis.

[0114]

[0177] (6) Staining the droplet carrier particles 12 with adherent cells for captured secretions with a second affinity reagent specific for the secretions.

[0178] A number of different reporting schemes can be used to analyze secreted molecules bound to droplet carrier particles 12. In a preferred embodiment, a second antibody conjugated to a fluorophore that is specific for a second epitope on the secreted molecule is added to form a fluorescent sandwich immune complex and report the presence of bound secreted molecules. This method allows for quantification through many commonly used analytical tools such as flow cytometers 150 (illustrated in Figures 13A, 13B, 16 and 18), plate readers and fluorescent microscopes.

[0115]

[0179] For secreted molecules present in particular at low concentrations, an amplification scheme in which a reporter antibody is conjugated to an enzyme such as horseradish peroxidase to cleave a fluorescent dye that binds to a free site on the particle can amplify the signal, as described herein, such as through the use of tyramide chemistry. In a related embodiment, magnetic nanoparticles or microparticles can be used to label the captured secreted molecules of interest. The addition of magnetic properties can be used in many ways, for example, to concentrate droplet carrier particles 12 of interest or to sort samples of interest using magnetic forces. It should be understood that staining is not limited to these two modalities (e.g., fluorescent and magnetic) but can include a combination of multiple modalities. Other modalities can include colorimetric, phosphorescent, light scattering particles, plasmonic nanoparticles, among others known in the art.

[0116]

[0180] (7) Analysis of the stained droplet carrier particles 12 with attached cells (e.g., using a flow cytometer 150) and, optionally, sorting of the cells of interest attached to the droplet carrier particles 12 based on a stain-based intensity threshold corresponding to secretion volume / affinity.

[0181] Once stained, the droplet carrier particles 12 and their bound cells can be analyzed and sorted in a high throughput manner using a commercially available flow sorter 150 (illustrated in Figures 13A, 13B, 16 and 18). In a preferred embodiment, the droplet carrier particles 12 are suspended in nutrient-rich PBS, such as PBS+2% FBS, to maintain cell viability during the sorting process. The relative size of the droplet carrier particles 12 allows clear discrimination of the particles from contaminating dust, cell debris or small oil droplets in solution during sorting, allowing them to be easily identified using forward and side scatter signals. Adherent cells stained with cytoplasmic tracking dyes, nuclear stains, viability stains or reporter antibodies are also easily detected on the droplet carrier particles 12, allowing direct analysis of the cells encapsulating the droplet carrier particles 12. Finally, reporter antibodies added to the surface of the droplet carrier particles 12 upon emulsion breaking allow direct quantification of relative protein production from individual cell clones within the particle cavities 18 from the relative fluorescence intensity on the droplet carrier particle 12 surface. Sorted cells of interest, for example clones secreting high levels of a desired protein, are easily isolated and remain viable for subsequent expansion, allowing enrichment of beneficial cell phenotypes.

[0117]

[0182] In another example, the screening of single cells based on total secretions can be performed over multiple cycles to improve the selection of the desired subpopulation. This general workflow is shown in FIG. 19. According to the approach described above, single cells are isolated in droplet carrier particles 12 and emulsified into droplets 10, where secretions accumulate and are captured on the droplet carrier particles 12 without crosstalk. The droplet carrier particles 12 can then be placed back in water, stained to indicate the amount of secretions, and analyzed / sorted together with the attached cells. Subpopulations of sorted cells can then be expanded to perform iterative selection steps. In one embodiment, the sorted cells attached to the droplet carrier particles 12 can be seeded in well plates or flasks and expanded directly from the droplet carrier particles 12. For example, it was found that after several days of culture, CHO cells grow across the surface of the droplet carrier particles 12 and eventually on the surface of the well plate in which they were seeded. This has the advantage of reducing handling steps and limiting trypsinization of the cells. If desired, the attached cells can be removed from the droplet carrier particles 12 using standard trypsinization and subculture steps. After cell growth, the single cell secretion and sorting cycle can be performed again by seeding the recovered cells back onto the droplet carrier particles 12 and repeating the steps outlined above. In a related embodiment, the single cells seeded onto the droplet carrier particles 12 can be grown to form clonal colonies that are attached to the droplet carrier particles 12 before emulsification. This allows for a combination of clonal growth and secretion-based analysis / sorting. For example, cells can be seeded into the droplet carrier particles 12 at a concentration that predominantly contains only single cells. If desired, the initial population can be screened (e.g., using a flow sorter 150) to remove droplet carrier particles 12 with a large number of cells. After seeding, the cells can be grown directly on the droplet carrier particles 12 so that single cell colonies are formed. This can be done for a variety of times (e.g., <24 hours, 24 hours to 1 week, >1 week) depending on the application.After colony formation, background signal can be washed away and the droplet carrier particles 12 can be modified with secretion binding sites. Secretion screening is then performed as outlined above for single cells. The droplet 10 system is uniquely suited to this workflow because cells can be grown on the droplet carrier particles 12 before encapsulation, providing the opportunity to remove any undesired background signal that accumulates during cell growth. In other approaches where single cells are immediately encapsulated and grown, signal accumulates throughout the growth period which may be undesirable or lead to signal saturation.

[0118]

[0183] Example workflow

[0184] An example of a workflow for selecting high secreting CHO cells is detailed below. The example cell line used is CHO-DP12 clone #1934 (ATCC). Cell culture medium was prepared as specified by ATCC. The CHO-DP12 cell line produces human anti-IL-8 antibody, which is the target secretion for the experiments in this example.

[0119]

[0185] (1) Loading of cells to attach to droplet carrier particles 12 in aqueous solution

[0186] In this example, droplet carrier particles 12 with an outer diameter of 82.5 microns and an inner diameter of 50 microns were used. The droplet carrier particles 12 were modified with 0.5 mg / mL biotin-PEG-thiol (5000 MW, nanocs) and 4 mg / mL RGD (added to the dextran phase during manufacture as described above). 30 μL of concentrated droplet carrier particles 12 were diluted with 1 mL cell culture medium and added into one well of a 12-well plate. The droplet carrier particles 12 were then allowed to settle for 10 minutes. CHO DP-12 cells were concentrated to 4 million cells per ml. For targeted encapsulation of ~0.3 cells per particle, 18 μL of concentrated cell stock was taken and diluted to 50 μL with culture medium and then carefully transferred into the well pre-seeded with droplet carrier particles 12. For targeted encapsulation of ~0.1 cells per particle, 6 μL of concentrated cell stock was diluted to 50 μL and then carefully transferred into wells pre-seeded with droplet carrier particles 12. Cells were allowed to seed for 10 min before moving the well plate to the incubator. A range of 4-12 h was found to be necessary for cells to strongly attach to the droplet carrier particles 12.

[0120]

[0187] (2) Wash away unbound cells and / or background secretions and add affinity reagents that bind to droplet carrier particles 12 that capture the specific cell secretions of interest.

[0188] After the cells were incubated for a sufficient time to attach to the droplet carrier particles 12, the droplet carrier particles 12 were transferred from the well plate to a 15 mL conical tube. This was done by tilting the well plate at an angle of about 30°, pipetting excess medium from the top to shear the droplet carrier particles 12 attached to the surface, and pipetting the removed droplet carrier particles 12 and attached cells into the 15 mL conical tube. To limit the attachment of the droplet carrier particles 12 to the walls of the conical tube, the tube can be pretreated with a solution of PBS with 0.1% Pluronic F-127, PBS with 0.5% BSA (bovine serum albumin), or PBS with 2% FBS (fetal bovine serum). The droplet carrier particles 12 and attached cells were then washed 2-3 times with PBS supplemented with 0.5% BSA (calcium and magnesium ions). This wash removes any biotin that may be present in the medium and any proteins from the cells that may be present in the background medium. For all washing steps, the samples were centrifuged at 300 g for 3 min. The droplet carrier particles 12 were then modified with a 0.02 mg / ml solution of streptavidin, which binds to the available biotin groups on the droplet carrier particles 12. After a 10 min incubation, the droplet carrier particles 12 and bound cells were washed 2-3 times with PBS+0.5% BSA. Next, the droplet carrier particles 12 were modified with biotinylated protein A, which is used as an example of a capture site for secreted anti-IL-8 protein. In this example, 10 μL of a 0.5 mg / ml stock solution of biotinylated protein A (Thermo Fischer Scientific) was added to each sample, which was then incubated for 10 min. Alternatively, biotinylated IgG1, FC mouse anti-human could be used. Finally, the samples were washed 2-3 times with PBS+0.5% BSA. At the final wash, PBS was replaced with cell culture medium.

[0121]

[0189] (3) Emulsifying droplet carrier particles 12 with the attached cells and the binding affinity reagent in the oil continuous phase to form droplets 10.

[0190] The droplet carrier particles 12 and bound cells were concentrated by spinning down the sample. The supernatant was then removed until approximately 50 μL of sample remained. Then 100 μL of Novec™ oil + 2% w / v Pico-Surf™ was added to the sample. The Eppendorf tube was then flicked 2-5x to help break up the sample into larger droplets. Then, using either a 100 μL or 200 μL pipette, the sample was pipetted up and down at a rate of 2-4 pipettes per second, 50-100X, to generate monodisperse droplets 10. Then, after droplets 10 formation, mineral oil was added to the top of the sample (~100-150 μL) to prevent sample evaporation and reduce aggregation while incubating the sample.

[0122]

[0191] (4) Incubating the drops 10 for a period of time to accumulate secretions that bind to the affinity reagent.

[0192] The samples were allowed to incubate for 2-24 hours in a cell incubator (37°C, 5% CO2). During this step, the secreted anti-IL-8 protein attaches to the Protein A binding sites on the droplet carrier particles 12 (see Figure 17A).

[0123]

[0193] (5) Breaking the droplets 10 emulsion to return the droplet carrier particles 12 with attached cells to the aqueous solution.

[0194] To return the droplet carrier particles 12 and cells to the aqueous phase, ∼4-6 mL of PBS + 2% FBS was added to the top of the sample. The added mineral oil was then removed by pipette. 50 μL of 20% v / v PFO in Novec™ oil was then added to the sample to aid droplet aggregation. Aggregation was aided by gently tapping the Eppendorf tube. After 2-5 minutes, the remaining droplets can be aggregated by centrifuging the sample at 100 g for 5-10 seconds. Once phase transfer is complete, the droplet carrier particles 12 and bound cells are transferred to a new 15 ml conical tube (pretreated with 2% FBS or 0.5% BSA or 0.1% Pluronic F-127 in PBS).

[0124]

[0195] (6) Staining the droplet carrier particles 12 with adherent cells for captured secretions using a second affinity reagent specific for the secretions.

[0196] The droplet carrier particle 12 sample in the aqueous phase 14 was washed 2-3 times with PBS+2% FBS. For the final wash, the sample was spun down and the supernatant aspirated until ~100 μL remained. 10 μL of 0.5 mg / ml goat anti-human IgG H&L(Cy5) preadsorbed (Abcam, ab97172) was added to the sample to stain the captured anti-IL-8 protein captured on the droplet carrier particle 12 (Figure 17A). After a 30 minute incubation at 37°C, the sample was washed 2-3X with PBS+2% FBS to remove any unconjugated stain. The sample was then placed on ice during transfer to the flow cytometer 150.

[0125]

[0197] Optionally, during this staining process, the cells bound to the droplet carrier particles 12 can be stained with calcein AM. Figure 17B shows an example of a microscope image taken of a droplet carrier particle 12 with bound cells stained with calcein AM. The fluorescence imaging clearly shows a strong signal in the Cy5 channel (secretion staining channel) on the droplet carrier particles 12 where calcein AM stained live cells are present. Almost no background signal is shown on other droplet carrier particles 12 that do not have bound cells. This indicates successful secretion capture and labeling, as well as limited crosstalk between droplets 10 during the incubation step.

[0126]

[0198] (7) Analyzing the droplet carrier particles 12 stained by the attached cells (e.g., using a flow cytometer 150) based on a threshold of stain-based intensity corresponding to the amount / affinity of secretion, and optionally sorting the cells of interest that are attached to the droplet carrier particles 12.

[0199] Samples were analyzed in high throughput using an On-Chip Sorting Flow Cytometer 150 from On-Chip Biotechnologies, Inc. (Tokyo, Japan). Samples were diluted to 200 μL with PBS+2% FBS and added to the sample inlet. Here, a 150 μm flow chip from On-Chip Biotechnologies was used. PBS+2% FBS was also used as sheath fluid. Forward scatter height (FSC(H)) and side scatter height (SSC(H)) gating was used to select the droplet carrier particles 12 from other background events / noise. Other events may be associated with small amounts of Novec™ 7500 oil droplets still in solution, cells dissociated from the droplet carrier particles 12, or cell debris. An example of a gating plot is shown in FIG. 18. After this initial gating, sorting events were then selected from the relative far-red fluorescence signal. For example, in FIG. 18, the top 10% of the far-red signal was selected as the sorting gate. Samples containing droplet carrier particles 12 with attached cells were then sorted and collected. Using the on-chip sorter flow cytometer 150, the event rate was typically around 50-300 events / sec for a sample. The collected samples were stained by calcein AM and visualized under a fluorescent microscope as shown in Figure 18. After sorting, the majority of cells maintained viability as indicated by the calcein AM live staining signal. Further images in the Cy5 channel (captured secretions staining channel) show a clear accumulation of droplet carrier particles 12, indicating cells associated with high levels of captured secretions after sorting. This demonstrates the ability to sort cells from relative secretions levels using the droplet 10 system.

[0127]

[0200] Barcoding of droplets

[0201] A number of schemes can be used to barcode batches of droplet carrier particles 12 with unique identifiers so that multiple targets (e.g., nucleic acids, proteins, cells, or other analytes) can be probed in the same experimental procedure. These include through split-and-pool synthesis for the generation of DNA oligos as described above, or through fluorescence intensity with various ratios of multiple fluorophores (FIG. 15B), through magnetic affinity via various concentrations of pre-encapsulated magnetic nanoparticles 52 (FIG. 15C), through unique flow cytometry side scattering characteristics from nanoparticles 54 of various sizes and amounts (FIG. 15D), or through changes in particle morphology that allow the diameter or other dimensions of the droplet carrier particles 12 to act as unique identifiers while maintaining uniformly sized cavities or voids (FIG. 15A). Different barcoding approaches can also be combined to further increase the number of different types of droplet carrier particles 12.

[0128]

[0202] Covalent attachment of a small number of differently colored fluorophores provides a vast amount of identifiability simply through variation in the relative concentration of each molecule. For example, attachment or otherwise embedding of only three different fluorophores at eight different concentrations results in over 500 distinct barcodes that can be uniquely detected and assigned to specific droplet carrier particles. Fluorometric barcoding of droplet carrier particles 12 is easily accomplished through attachment of fluorophores conjugated to antibodies, reactive groups, or any other moiety that can be typically attached to the surface of the droplet carrier particle 12 or embedded within the matrix of the droplet carrier particle 12 or linked by covalent bonds. For example, droplet carrier particles 12 manufactured using either PEG-norbornene or PEG-VS can be labeled during or after manufacture by conjugating maleimide-modified fluorescent dyes (e.g., Alexa Flour™ 488 maleimide, Alexa Flour™ 568 maleimide, etc.) to thiolated crosslinkers. Different ratios of these fluorophores can be mixed to create unique barcodes. In another embodiment, biotin-PEG-thiol (Nanocs) can be conjugated to PEG-norbornene, PEG-VS or any other thiol-reactive PEG group polymers before or after crosslinking of droplet carrier particles 12. The biotin groups can then be modified with fluorescent streptavidin molecules (e.g., Alexa Flour™ 488, 568, 647, streptavidin, among others) in various colors, concentrations and ratios. Modification with fluorescent streptavidin after particle manufacture allows for an efficient barcoding process. For example, premixes of different labeled streptavidin molecules can be premixed in different concentrations / ratios and then added to the manufactured droplet carrier particles 12 to effectively form many different barcodes. In another example, fluorescent nanoparticles in different concentrations and ratios can be mixed with hydrogel precursors and embedded within the hydrogel matrix of the droplet carrier particles 12 during manufacture in one embodiment.Fluorescence reading of the bar-coded droplet carrier particles 12 within the aqueous phase or drops 10 can be performed using a standard microscope, flow cytometer 150, plate reader, or any other standard piece of laboratory equipment.

[0129]

[0203] Magnetism can be used to barcode the droplet carrier particles 12 and the resulting drops 10 through the variation of magnetic attractive forces in an applied magnetic field. The droplet carrier particles 12 can be manufactured with different levels of magnetic inclusions to produce different magnetic barcodes. Various levels of magnetic inclusions can be achieved by introducing water-soluble magnetic nanoparticles or microparticles 52 into the droplet carrier particle precursor solution prior to polymerization. One embodiment using magnetic barcoding involves placing the drops 10 or droplet carrier particles 12 on a surface on an array of magnetic pillars in the presence of an oscillating magnetic field. The oscillation results in a net unidirectional movement across the plane of the pillars that are increasingly spaced apart along the plane of movement. The total distance covered by the aqueous phase droplets 10 or droplet carrier particles 12 is then proportional to the degree of magnetic functionalization, as droplet carrier particles 12 with less magnetic inclusions will eventually stop and will not be able to pass the distance to the next pillar array, while those with a greater degree of magnetization will continue to move due to stronger attractive forces as disclosed in U.S. Pat. No. 10,144,911, which is incorporated herein by reference. Magnetic functionalization of the droplet carrier particles 12 also allows rapid movement of the emulsion throughout the continuous phase. The magnetic particles 42 encapsulated within the emulsified microparticles provide sufficient force in the presence of an applied magnetic field to separate the droplet-containing droplet carrier particles 12 from the large background of satellite droplets 28 (see FIG. 5). Thus, application of this barcoding scheme can not only spatially separate subpopulations through variation of the applied force, but also separate the droplet carrier particles 12 from undesired satellite droplets 28 to improve the signal in many assays.

[0130]

[0204] Particle size is a measurement measure commonly used in flow cytometry to separate distinct subsets of cells from each other. The droplet carrier particles 12 can be barcoded by adding light scattering nanoparticles 54 of various sizes and / or concentrations that scatter light at different angles and / or with different intensities. The nanoparticles 54 (e.g., polystyrene nanoparticles) can be loaded into the precursor hydrogel solution in one embodiment prior to polymerization to generate droplet carrier particles 12 with embedded scattering barcodes. This alternative approach allows for analysis of droplet carrier particle 12 types in the flow cytometer 150, for example, by analyzing side scattering signals without the need for extra fluorescent signals that require compensation and that may interfere with the analysis of analytes that are fluorescently labeled and attached to the droplet carrier particles 12.

[0131]

[0205] Another method of barcoding droplet carrier particles includes through the size of the droplet carrier particles 12 themselves. This is shown in FIG. 15A. Different sizes of droplet carrier particles 12 result in different forward scattering signals in the flow cytometer 150. The difference in particle size also allows visual discrimination between particle classes and can be easily classified through wide-field imaging and microscopic observation. Furthermore, microfluidic techniques such as inertial focusing or deterministic lateral displacement can be used to sort droplet carrier particles 12 of different sizes from one another through the variation of the cumulative force applied to each droplet carrier particle 12 in the flow field. As discussed with other non-fluorescent barcodes, such size-based barcoding allows the fluorescent signal across multiple wavelengths to be used for analyte detection without interference from the barcode. Furthermore, for imaging flow cytometer 150, the shape / size of the droplet carrier particles 12 and void or cavity regions 18 can be imaged to provide a method of barcoding as well.

[0132]

[0206] Method for reading and sorting droplets / droplet carrier particles - Patents.com

[0207] In order to convert the signal generated in the droplet assay into a quantifiable measurement, a well-defined method of reading the assay output must be compatible with the droplet system. Furthermore, developing methods to process the signal and sort subsets of droplets 10 into distinct populations is important for the isolation of high performers from single cell studies. In particular, the droplet carrier particles 12 and the droplets 10 formed are compatible with many commonly used laboratory systems, making them relatively easy to both read and sort with appropriate equipment.

[0133]

[0208] In some embodiments, signals corresponding to reactions carried out in the volume encapsulated within the droplet 10 are attached to the surface of the droplet carrier particle 12 or accumulate within the droplet carrier particle 12 and the surrounding aqueous phase 14. This allows detection of fluctuations in fluorescent or colorimetric signals through signal detectors (e.g., photomultiplier tubes (PMTs)) in both custom fabricated microfluidic devices that can be compatible with the droplet 10 in oil continuous phase, and in commercially available flow cytometers 150 and FACS systems that are compatible with the droplet carrier particles 12 once returned to the aqueous phase. Some commercially available flow cytometers 150 are also compatible with oil continuous phase, such as On-Chip Flow and On-Chip Sorting from On-Chip Biotechnologies, Inc., Tokyo, Japan. The diameter of the droplet carrier particle 12 should be adjusted to <~50 micrometers for use with standard commercially available flow cytometry instruments 150, although larger sizes up to ~500 micrometers can be used in other commercially available cytometry systems (e.g., those from On-Chip Biotechnologies, Inc. or the Union Biometrica Biosorter). The barcoding signatures can also be simultaneously read using fluorescence and / or scattering signals as described herein. The number of positive signal droplet carrier particles 12 for each barcode can then be counted from the flow cytometry scattering plot if a threshold or gate is used to discriminate specific subpopulations, leading to multiplex assays by counting the fraction of droplet carrier particles 12 with high vs. low assay signals within a specific barcode gate. Sorting based on these assay signals can also be performed to isolate high vs. low droplet carrier particles 12 for further cell analysis and expansion, or nucleic acid analysis and downstream sequencing. In other embodiments, the droplets 10 containing the captured analytes (e.g., using antibodies) can be further exposed to magnetic particles / microbubble particles that also bind to the captured analytes to form a sandwich. The positive signal particles can then be separated via magnetic fields or buoyancy, respectively.

[0134]

[0209] Standard fluorescence microscopy is also an effective method of probing assay output. Here, wide-field imaging protocols can be applied to measure all droplets 10 within a sample batch to develop a better understanding of the variability within the individual assay measurements. In an embodiment, a portable imaging system can be used to enable on-site / point-of-care analysis (e.g., wide-field fluorescence imaging on a mobile device), similar to that disclosed in U.S. Patent Application Publication No. 2013 / 0157351 or U.S. Patent No. 9,007,433, both of which are incorporated herein by reference. Furthermore, the heterogeneity of the geometry of the droplet carrier particles 12 means that a magnetic field can be utilized to align a population of magnetic droplets 10 in the same direction to obtain a uniform signal from the particle voids or cavities 18 during imaging or flow analysis.

[0135]

[0210] The asymmetry of the crescent-shaped droplet carrier particles 12 can be exploited to align the droplet carrier particles 12 based on buoyancy. For example, it was found that the crescent-shaped droplet carrier particles 12, which are denser than the aqueous phase, tend to settle and orient with their cavities 18 exposed upright. This typically occurred on a time scale of 5-10 minutes. This preferred alignment can be used to facilitate loading of microscale objects (e.g., loading cells) into the exposed cavities 18. The shape of the droplet carrier particles 12 can also be exploited to create a preferred orientation during flow in a confined channel. For example, the shape of the droplet carrier particles 12 can be adjusted so that the particles are oriented in a preferred direction such that the scattering signature is more uniform.

[0136]

[0211] While embodiments of the present invention have been illustrated and described, various changes can be made without departing from the scope of the invention. Accordingly, the invention is not to be limited, except insofar as the following claims and their equivalents are concerned.

Claims

1. A droplet-based system that uses a volume associated with solid phase particles suspended in an immiscible fluid, comprising: a plurality of three dimensional hydrophilic droplet carrier particles formed from a crosslinked hydrogel, each said hydrophilic droplet carrier particle having a void or cavity formed therein; an aqueous fluid bound to the three-dimensional hydrophilic droplet carrier particles and disposed within the voids or cavities of the plurality of three-dimensional hydrophilic droplet carrier particles; The plurality of three-dimensional hydrophilic droplet carrier particles bound to the aqueous fluid are disposed or suspended in an oil phase.

2. The system of claim 1 , wherein the aqueous fluids disposed within the voids or cavities of the three-dimensional hydrophilic droplet carrier particles have substantially the same volume.

3. The system of claim 1 , wherein the voids or cavities open onto the surface of the three-dimensional hydrophilic droplet carrier particle.

4. 4. The system of claim 3, wherein the voids or cavities open onto the surface of the three-dimensional hydrophilic droplet carrier particle with openings having an area less than 33% of the total surface area of ​​the envelope of the three-dimensional hydrophilic droplet carrier particle.

5. 4. The system of claim 3, wherein the voids or cavities open onto the surface of the three-dimensional hydrophilic droplet carrier particle with openings having an area less than 10% of the total surface area of ​​the envelope of the three-dimensional hydrophilic droplet carrier particle.

6. 4. The system of claim 3, wherein the voids or cavities open onto the surface of the three-dimensional hydrophilic droplet carrier particle with openings having an area less than 5% of the total surface area of ​​the envelope of the three-dimensional hydrophilic droplet carrier particle.

7. The system of claim 1 , wherein the voids or cavities are disposed entirely within the three-dimensional hydrophilic droplet carrier particle and do not intersect with a surface of the three-dimensional hydrophilic droplet carrier particle.

8. The system of claim 1 , wherein the plurality of three-dimensional hydrophilic droplet carrier particles are formed from a PEG-based crosslinked hydrogel.

9. The system of claim 1 , wherein the plurality of three-dimensional hydrophilic droplet carrier particles have unique indicia formed thereon or therein.

10. The system of claim 1 , wherein the voids or cavities of at least some of the plurality of three-dimensional hydrophilic droplet carrier particles contain cells or beads therein.

11. The system of claim 1 , wherein the voids or cavities of the plurality of three-dimensional hydrophilic droplet carrier particles have a volume in the range of about 100 fL to about 10 nL.

12. The system of claim 1 , wherein the voids or cavities of the plurality of three-dimensional hydrophilic droplet carrier particles have linear dimensions within the range of about 5 μm and about 250 μm.

13. The system of claim 1 , wherein the plurality of three-dimensional hydrophilic droplet carrier particles further comprises a barcode material incorporated therein or thereon.

14. The system of claim 1 , wherein the plurality of three-dimensional hydrophilic droplet carrier particles further comprises magnetic particles contained therein.

15. The system of claim 1 , wherein the plurality of three-dimensional hydrophilic droplet carrier particles further comprises light scattering particles contained therein.

16. 2. The method of claim 1 , wherein the plurality of three-dimensional hydrophilic droplet carrier particles comprises one or more dyes of varying intensity. The system described in 1.

17. The system of claim 1 , wherein the plurality of three-dimensional hydrophilic droplet carrier particles comprises particles of different sizes.

18. A droplet-based system using a volume associated with solid phase particles suspended in an immiscible fluid. a plurality of three dimensional hydrophilic droplet carrier particles formed from a crosslinked hydrogel, each said hydrophilic droplet carrier particle having a void or cavity formed therein; an aqueous fluid bound to the three-dimensional hydrophilic droplet carrier particles and disposed within the voids or cavities of the plurality of three-dimensional hydrophilic droplet carrier particles; The plurality of three-dimensional hydrophilic droplet carrier particles bound to the aqueous fluid are disposed or suspended in an oil phase to form an emulsion of droplets, substantially all of the droplets comprising a single hydrophilic droplet carrier particle contained therein.

19. 20. The system of claim 18, wherein the aqueous fluids disposed within the voids or cavities of the three-dimensional hydrophilic droplet carrier particles have substantially the same volume.

20. 20. The system of claim 18, wherein the voids or cavities open onto the surface of the three-dimensional hydrophilic droplet carrier particle.

21. 21. The system of claim 20, wherein the voids or cavities open onto the surface of the three-dimensional hydrophilic droplet carrier particle with openings having an area less than 33% of the total surface area of ​​the envelope of the three-dimensional hydrophilic droplet carrier particle.

22. 21. The system of claim 20, wherein the voids or cavities open onto the surface of the three-dimensional hydrophilic droplet carrier particle with openings having an area less than 10% of the total surface area of ​​the envelope of the three-dimensional hydrophilic droplet carrier particle.

23. 21. The system of claim 20, wherein the voids or cavities open onto the surface of the three-dimensional hydrophilic droplet carrier particle with openings having an area less than 5% of the total surface area of ​​the envelope of the three-dimensional hydrophilic droplet carrier particle.

24. 20. The system of claim 18, wherein the voids or cavities are disposed entirely inside the three-dimensional hydrophilic droplet carrier particle and do not intersect with a surface of the three-dimensional hydrophilic droplet carrier particle.

25. The system of claim 18 , wherein the plurality of three-dimensional hydrophilic droplet carrier particles are formed from a PEG-based crosslinked hydrogel.

26. 1. A method for producing hydrophilic droplet carrier particles, comprising: Providing a microfluidic droplet generator device having a plurality of inlets; flowing a solution of a crosslinkable first component of an aqueous two-phase system including a photoinitiator into one of the plurality of inlets, the crosslinkable first component being poly(ethylene glycol) PEG or a PEG derivative; flowing a solution of a second component of the aqueous two-phase system, the second component including a cross-linking agent, into another inlet of the plurality of inlets; flowing an oil phase into another of the plurality of inlets, whereby aqueous droplets are formed within the microfluidic device, each aqueous droplet dividing into distinct regions within each droplet, the distinct regions including a phase rich in a PEG component and a phase rich in a second component of the aqueous two-phase system; and cross-linking said PEG component-rich phase by exposure to light to form hydrophilic droplet carrier particles.

27. 27. The method of claim 26, wherein the crosslinking is initiated by exposure to ultraviolet (UV) light.

28. 27. The method of claim 26, further comprising removing the oil and second component rich phase from the droplet carrier particles.

29. 30. The method of claim 28, further comprising loading said hydrophilic droplet carrier particles into a separate aqueous solution, said hydrophilic droplet carrier particles being bound to droplets of said separate aqueous solution.

30. 30. The method of claim 29, wherein the separate aqueous solution is further disposed within voids or cavities formed within the hydrophilic droplet carrier particles.

31. 27. The method of claim 26, wherein the rich phase of the second component comprises dextran.

32. 1. A method for producing hydrophilic droplet carrier particles, comprising: Providing a microfluidic droplet generator device having a plurality of inlets; flowing a solution of a crosslinkable first component of an aqueous two-phase system including a crosslinker into one of a plurality of inlets, the crosslinkable first component being poly(ethylene glycol) PEG or a PEG derivative; flowing a solution of a second component of the aqueous two-phase system into another inlet of the plurality of inlets; flowing an oil phase into another of the plurality of inlets, whereby droplets are formed in the microfluidic device, each droplet dividing into distinct regions within each droplet that include a phase rich in a PEG component and a phase rich in the second component of the aqueous two-phase system; and cross-linking said rich phase of said PEG component by increasing the pH to form hydrophilic droplet carrier particles.

33. 1. A method for performing a cell secretion assay using droplet carrier particles, comprising: a. providing a plurality of three-dimensional hydrophilic droplet carrier particles, each particle having a void or cavity formed therein; b. loading cells into the voids or cavities of the plurality of three-dimensional hydrophilic droplet carrier particles; c. adding an affinity agent to said plurality of three-dimensional hydrophilic droplet carrier particles specific for a cell secretion of interest; d. emulsifying the plurality of three-dimensional hydrophilic droplet carrier particles containing the cells and the affinity agent to form a plurality of droplets; e. Incubating the plurality of drops; f. breaking the emulsion of said droplets to obtain said plurality of three-dimensional hydrophilic droplet carrier particles in an aqueous solution; g. adding a stain, dye or other secondary affinity reagent specific to the secretion of the subject to one or more of said plurality of three-dimensional hydrophilic droplet carrier particles; h) analyzing said plurality of three-dimensional hydrophilic droplet carrier particles of step (g) for a signal formed or generated by a stain, dye or other secondary affinity reagent specific for a cellular secretion of said subject on one or more of said plurality of three-dimensional hydrophilic droplet carrier particles.

34. 34. The method of claim 33, further comprising washing the plurality of three-dimensional hydrophilic droplet carrier particles containing the loaded cells prior to adding the affinity agent.

35. 34. The method of claim 33, wherein analyzing the plurality of three-dimensional hydrophilic droplet carrier particles in step (g) comprises flowing the plurality of three-dimensional hydrophilic droplet carrier particles through a flow cytometer.

36. 34. The method of claim 33, wherein analyzing the plurality of three-dimensional hydrophilic droplet carrier particles in step (g) comprises viewing or reading the plurality of three-dimensional hydrophilic droplet carrier particles in a well plate or other holder.

37. 36. The method of claim 35, further comprising sorting the plurality of three-dimensional hydrophilic droplet carrier particles based on a threshold fluorescence intensity or color measured by the flow cytometer.

38. 34. The method of claim 33, wherein only the affinity agent is disposed within the voids or cavities of the plurality of three-dimensional hydrophilic droplet carrier particles.

39. 34. The method of claim 33, wherein the affinity agent is disposed on substantially all of the surface of the plurality of three-dimensional hydrophilic droplet carrier particles.

40. 34. The method of claim 33, wherein the steps are performed sequentially.

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