Generating microdroplet emulsions with microfluidic chambers containing pores

Abstract

Provided herein are devices, assemblies, systems, kits, and methods for generating an emulsion with a microfluidic chamber composed of impediments (e.g., posts) regularly or irregularly spaced apart to create pores (e.g., 10-1000 microns in diameter). In certain embodiments, a non-emulsion mixture composed of sample fluid (e.g., containing cells and / or beads) and an immiscible carrier fluid are flown through the microfluidic chamber once or repeatedly such that an emulsion is generated comprising sample microdroplets (e.g., about the same size as the pores, or of irregular shape). In some embodiments, the microfluidic chamber is housed in a pipette tip and a micropipette is used to push or draw the non-emulsion mixture through the microfluidic chamber.

Description

[0001] GENERATING MICRODROPLET EMULSIONS WITH MICROFLUIDIC CHAMBERS CONTAINING PORES

[0002] The present application claims priority to U.S. Provisional application serial number 63 / 489,004, filed March 8, 2023, which is herein incorporated by reference in its entirety.

[0003] FIELD OF THE INVENTION

[0004] Provided herein are devices, assemblies, systems, kits, and methods for generating an emulsion with a microfluidic chamber composed of impediments (e.g., posts) regularly or irregularly spaced apart to create pores (e.g., 10-1000 microns in diameter). In certain embodiments, a non-emulsion mixture composed of sample fluid (e.g., containing cells and / or beads) and an immiscible carrier fluid are flown through the microfluidic chamber once or repeatedly such that an emulsion is generated comprising sample microdroplets (e.g., about the same size as the pores, or of irregular shape). In some embodiments, the microfluidic chamber is housed in a pipette tip and a micropipette is used to push or draw the non-emulsion mixture through the microfluidic chamber.

[0005] BACKGROUND

[0006] Prior described methods of generating microdroplet emulsions are generally dependent on flow rates, and do not allow for aqueous and oil phases to be premixed before the emulsification process. In the case of microfluidic emulsification, precise flow rates and ratios of the aqueous and oil phases need to be injected into the droplet generation junction to produce droplets with desired size and uniformity. What is needed is microdroplet emulsification methods and assemblies that overcome some or all of these problems.

[0007] SUMMARY OF THE INVENTION

[0008] Provided herein are devices, assemblies, systems, kits, and methods for generating an emulsion with a microfluidic chamber composed of impediments (e.g., posts) regularly or irregularly spaced apart to create pores (e.g., 10-1000 microns in diameter). In certain embodiments, a non-emulsion mixture composed of sample fluid (e.g., containing cells and / or beads) and an immiscible carrier fluid are flown through the microfluidic chamber once or repeatedly such that an emulsion is generated comprising sample microdroplets (e.g., about the same size as the pores, or of irregular shape). In some embodiments, the microfluidic chamber is housed in a pipette tip and a micropipette is used to push or draw the non-emulsion mixture through the microfluidic chamber.

[0009] In some embodiments, provided herein are systems, kits, assemblies, and devices for generating an emulsion from a non-emulsion mixture that is composed of sample fluid, an immiscible carrier fluid, and optionally a surfactant comprising: a) a housing component; b) at least one microfluidic chamber housed in, integral with, or configured to be housed in said housing component, wherein said at least one microfluidic chamber comprises: i) at least 2, and optionally at 100, impediments regularly or irregularly spaced apart to create at least 2, and optionally at least 100, pores therebetween, wherein said at least 2, and optionally 100, pores are about 5-2000 microns in diameter, and ii) a first open end and a second open end which are in fluid communication with each other such that, when said non-emulsion mixture is pushed or drawn once or multiple times through said microfluidic chamber from said first open end to said second open end, and / or vice versa, it passes through said at least 2, and optionally said at least 100, pores and generates an emulsion comprising a plurality of sample microdroplets in said immiscible carrier liquid, wherein optionally at least some, or most, of said plurality of microdroplets are about the same size as said at least 2, and optionally said 100 pores, or wherein optionally at least some, or most, of said plurality of microdroplets are of non-uniform size, and / or optionally contain only one cell or only one particle (e.g., bead, core-shell particle, etc.).

[0010] In certain embodiments, the systems, kits, and assemblies further comprise: c) the non- emulsion mixture. In other embodiments, the systems, kits, and assemblies further comprise: c) the sample fluid, and / or the immiscible carrier fluid, and / or the surfactant.

[0011] In particular embodiments, provided herein are methods of generating an emulsion with a microfluidic chamber comprising: a) attaching an emulsion generating assembly to a pressureproviding device, wherein said emulsion generating assembly comprises: i) a housing component; ii) at least one microfluidic chamber housed in, or integral with, said housing component, wherein said at least one microfluidic chamber comprises: A) at least 2, and optionally 100, impediments regularly or irregularly spaced apart to create at least 2, and optionally 100 pores, therebetween, wherein said at least 2, and optionally 100 pores, are about 5-2000 microns in diameter, and B) a first open end and a second open end which are in fluid communication with each other, and iii) wherein optionally an immiscible carrier fluid is present in said microfluidic chamber; and b) activating said pressure-providing device such that a first liquid is pushed or drawn through said microfluidic chamber once or multiple times, from said first open end to said second open end, or from said second open end to said first open end, or both, wherein said first liquid comprises: i) a sample fluid if said immiscible carrier fluid is present in said microfluidic chamber, and / or ii) a non-emulsion mixture comprising: said sample fluid, said immiscible carrier fluid, and optionally a surfactant, wherein when said first liquid is pushed or drawn through said microfluidic chamber once or multiple times, this generates an emulsion comprising a plurality of sample microdroplets in said immiscible carrier liquid, wherein optionally at least some, or most, of said plurality of microdroplets are about the same size as said at least 2 or at least 100 pores, or wherein optionally at least some, or most, of said plurality of microdroplets are of non-uniform size. In certain embodiments, the activating in repeated at least once, or at least 5 times, or at least 10 times or at least 25 times, or at least 50 times. In certain embodiments, the pressure-providing device is configured to provide negative pressure, positive pressure, or negative and positive pressure (e.g., via air flow or liquid flow from pumps).

[0012] In certain embodiments, the surfactant is present in the non-emulsion mixture. In other embodiments, the housing component comprises a micropipette tip, or container (e.g., a plastic container as shown in Figure 8B attached to the micropipette tip) configured to be attached to said micropipette tip, and / or wherein said microfluidic chamber further comprises a semi- permeable membrane. In further embodiments, the housing component comprises at least a portion of a microfluidic device. In certain embodiments, the systems, kits, and assemblies further comprise the sample fluid and / or the emulsion.

[0013] In some embodiments, the sample fluid comprises water and at least one of the following: i) a liquid gel precursor, ii) a plurality of cells, iii) a plurality of capture beads, optionally barcoded and / or composed of hydrogel, and iv) a plurality of core-shell particles. In particular embodiments, the capture beads are configured to hybridize to RNA, DNA, and / or protein targets.

[0014] In further embodiments, the at least one microfluidic chamber comprises at least 2, at least 10, or at least 20 of the microfluidic chambers. In additional embodiments, the at least one microfluidic chamber is located in the housing component. In further embodiments, the about 5- 2000 microns in diameter for the pores is about 10- 1000 microns, or about 20- 100 microns, or about 50-90 microns. In other embodiments, the at least 2 or at least 100 impediments, is at least 1000, at least 10,000, or at least 100,000 impediments. In additional embodiments, the at least 100 pores, is at least 1000, at least 10,000, or at least 100,000 pores.

[0015] In some embodiments, the housing component is configured to mate with a pressureproviding device (e.g., push-fit; releasably attach, etc.) such that the pressure-providing device can apply pressure to the microfluidic chamber. In other embodiments, the pressure -providing device comprises a micro-pipettor or an air-pressure pump or liquid pump. In certain embodiments, the kits, systems, and assemblies further comprise: c) a micro-pipettor. In certain embodiments, the pushed through comprises positive pressure (e.g., air or liquid) and the drawn through comprises negative pressure (e.g., air or liquid).

[0016] In certain embodiments, the emulsion comprises at least 1000, 10,000, 100,000, or 1 million of the sample microdroplets in the immiscible carrier liquid. In other embodiments, the immiscible carrier liquid comprises oil, and / or wherein said immiscible carrier liquid comprises oleogel and / or oleogel. In additional embodiments, the plurality of sample microdroplets are substantially monodisperse in the immiscible carrier fluid.

[0017] In some embodiments, the at least 2 or at least 100 impediments have a shape selected from: square, round, octagon, star shaped, rectangles, rods, posts, and combinations thereof. In particular embodiments, i) wherein the at least 2, and optionally 100, impediments comprises at least 20 impediments (e.g., at least 20 ... 40 ... 60 ... 80, or 100) and at least 20 pores therebetween (e.g., at least 20 ... 40 ... 60 ... 80, or 100), and ii) wherein the at least 20 impediments arc arranged in first and second regularly spaced apart groups that are separate (not intermixed, as shown in Figure 7) from each other, and iii) wherein the first regularly spaced apart group has first impediments that create a first pore size therebetween, and said second regularly spaced apart group has second impediments that create a second pore size therebetween that is different from the first pore size (e.g., the first pore size is 120 um or 150 um or 200 um, and the second pore size is 60 um or 80 um or 100 um), and optionally wherein said first and second impediments have different shapes (e.g., one is posts or rod and one is square or rectangles). In particular embodiments, the sample fluid comprises water and particles, wherein the impediment, the pores, and the particles arc sized such that the plurality of sample droplets on average contain one and only one particle. In other embodiments, the immiscible fluid is present in the microfluidic chamber, and the sample fluid is not present in the microfluidic chamber. In particular embodiments, the microfluidic chamber comprises at least one additional opening configured to introduce the sample fluid, or the immiscible carrier fluid, or the non-emulsion mixture, wherein the configured to introduce is under laminar flow conditions. In other embodiments, the at least one microfluidic chamber and the housing component are integral with each other such that they form a single combined component.

[0018] DEFINITIONS

[0019] As used herein, a bead is the platform upon which nucleic acid and / or protein capture probes and / or primers are attached or linked to. A bead may be any desired shape. The term “beads” include pellets, disks, fibers, gels, particles pads, slides, matrices, etc. A bead state may be solid, porous, rigid, semi-rigid, deformable, hard, etc. Bead material may be any desired composition that maintains the mechanical integrity of a bead, is compatible with the nucleic acid primers and capture sequences or protein capture molecules. A bead surface composition may include, for example, cellulose, glass, poreglass, resin, silica, polystyrene, or other suitable materials.

[0020] DESCRIPTION OF THE FIGURES

[0021] Figure 1 shows an exemplary embodiment where a microchamber (e.g., 1-20) are inside a pipette tip. A close up of the impediments (e.g., posts) and pores therebetween is shown on the right side of this figure.

[0022] Figure 2 shows example emulsions generated by the pipette tip device (left) or, alternatively, by vortexing. The Pipette tip device forms more uniform droplets with fewer small “satellite droplets”.

[0023] Figure 3 shows another emulsion generated by the pipette tip device that, additionally, contains hydrogel particles. By making droplets that are of similar size but slightly larger than the particles, single particles can be encapsulated per droplet at high frequency. Figure 4 shows example images of droplets generated by the pipette tip device that contain particles. By making droplets that arc larger than the particles, but not too large, thick shells of miscible liquid can be encapsulated around the particles. Achieving thick shells with vortexing or particle templated emulsification is challenging due to the proclivity of those methods to form satellites.

[0024] Figure 5 shows other samples of obstacles that can be used to form emulsions with the method.

[0025] Figure 6 shows example post arrays of the pipette tip device that can be used to form emulsions.

[0026] Figure 7A shows a close up of an exemplary mixed array device with 120 um gaps in the square array and 80 um gaps in the brick array. Figure 7B shows a zoomed-out version of this exemplary mixed array device, which could, for example, be fitted in, or to, a pipette tip.

[0027] Figure 8A shows an exemplary housing attached to, or integral with, the end of a pipette tip configured for housing an exemplary microfluidic chamber herein, wherein the housing is in a microtube. Figure 8B shows this same exemplary housing outside of the microtube. Figure 8C shows an exemplary microfluidic chamber, that is configured to be in the housing shown in Figures 8 A and 8B.

[0028] Figure 9 shows exemplary core- shell- shell beads (which are polyacrylamide-agarose- agarose) that encapsulate cell nuclei stained with Hoechst dye, and which are generated via a mixed array device, such as shown in Figure 7.

[0029] DETAILED DESCRIPTION

[0030] Provided herein are devices, assemblies, systems, kits, and methods for generating an emulsion with a microfluidic chamber composed of impediments (e.g., posts) regularly or irregularly spaced apart to create pores (e.g., 10-1000 microns in diameter). In certain embodiments, a non-emulsion mixture composed of sample fluid (e.g., containing cells and / or beads) and an immiscible carrier fluid are flown through the microfluidic chamber once or repeatedly such that an emulsion is generated comprising sample microdroplets (e.g., about the same size as the pores, or of irregular shape). In some embodiments, the microfluidic chamber is housed in a pipette tip and a micropipette is used to push or draw the non-emulsion mixture through the microfluidic chamber. In some embodiments, the particles (e.g., beads and / or core-shell particles) or cells or liquid gel precursor may reside in the sample to be encapsulated such that they end up in the microdroplets that are generated. In certain embodiments, the impediments or obstacles are posts in a microfluidic chamber with dimensions from 10-1000 microns, and wherein the spacing between the posts are between 10-1000 microns. In further embodiments, the microchamber may have thousands or hundreds of thousands (or more) of impediments (e.g., posts) in a 2D-like pattern. In other embodiments, the microchamber has a 3D-like pattern composed of membranes with pore dimensions 10-1000 microns, and wherein these membranes may be stacked in a tube. In certain embodiments, the non-emulsion mixture is driven through the microchamber via application of a pressure, such as via a pipette in the form of a pipette tip. In particular embodiments, the non-emulsion mixture comprises hydrogel particles with a selected size, and wherein the particle and impediments sizes are selected such that droplets with the desired number of particles (e.g., 1, 2, 3, or more) are generated, such as containing a single particle.

[0031] In some embodiments, the encapsulating phase resides within the device or assembly prior to injection of the encapsulation phase. In further embodiments, additional inlets (for the microchamber) from the X, Y, and Z direction are used to introduce additional encapsulated and encapsulating phases under laminar flow conditions. In certain embodiments, the encapsulated and encapsulating phases are pre-mixed before being introduced into the device. In particular embodiments, device or assembly is built in the form factor of a pipette tip and may be operated by a pipette and may be disposable. In further embodiments, the device or assembly is used to generate an emulsion for digital PCR, single cell genomics, etc.

[0032] The methods, assemblies, kits and systems herein provide, in certain embodiments, for the formation of microdroplets that are generally monodispersed. Compared to other microfluidic processes, this approach is generally robust against clogging, scalable for forming large amounts of monodispersed emulsions, and largely independent of the flow rates used. This is due to the structure of the devices and assemblies and emulsification mechanism. Conventional microfluidic emulsification methods are dependent on flow rates, but the methods described herein generally have a broader range of flow rates over which the droplet size is independent. In certain embodiments, the devices and assemblies comprise a microfabricated porous material with a pore size determined by the distance and geometry of impediments. In general, over a range of flow rates, the droplet size converges close to the pore size, making the process scalable by parallelizing the pores. Unlike membrane emulsification, this method can form generally monodispersed emulsions with varying flow rates and fluid viscosities. Geometrically-mediated breakup in a T-junction also depends on flow rates, but the method described herein, in general, forms monodispersed emulsions even if flow rates are not well controlled.

[0033] Another advantage compared to previously described methods such as membrane emulsification, microfluidic droplet formation, and geometrically-mediated droplet formation is that the present assemblies and methods allow the aqueous and oil phases to be premixed before the emulsification process. In the case of prior microfluidic emulsification, precise flow rates and ratios of the aqueous and oil phases need to be injected into the droplet generation junction to produce droplets with desired size and uniformity. Geometrically-mediated breakup also results in droplets that are flow-rate dependent and with sizes determined by the flow rates and sizes of the device.

[0034] The ability to form droplets with desired size based generally on device geometry as described herein is a major advantage for creating a simple device without flow rate controls. This is important in many applications, particularly when the device is intended to be disposable, such as in digital PCR in a clinical setting. The devices, assemblies, and methods herein are a significant improvement as it can be used, for example, to directly operate on well plates, for example, by fabricating it as a pipette tip that can be used with a pipettor. In certain embodiments, to emulsify a sample, oil and surfactant can be added on top of the sample fluid by pipetting, then the tip immersed in the sample to repeatedly draw it through the microchamber. The resulting emulsion size is generally determined by the emulsifier dimensions, which is, in certain embodiments, composed of plastic and pore features of comparable size to the desired droplets (e.g. 10, 100, or 1000 microns). In particular embodiments, these features are simple and inexpensive to produce through scalable microfabrication and injection molding techniques, leading to a low-cost, disposable pipette-operated device. In a clinical setting, this would allow for pipette tips to be disposed of between each emulsification process, avoiding crosscontamination between samples.

[0035] In certain embodiments, the microdroplet emulsions generated by the assemblies and methods here are applied to single cell genomics. In certain embodiments, an important step in the workflow is encapsulating single cells with barcode beads (e.g., in the form of compressible hydrogels). In the art, hydrogel bead suspensions are often stored at high particle concentration, making them difficult to encapsulate with microfluidics due to their non-Newtonian fluid properties with strange and challenging flow behavior. The assemblies and methods herein simplify this process as they are, in certain embodiments, largely flow-rate independent, resulting in droplets that generally depend only on the relative sizes of the pores. In some embodiments, to ensure high encapsulation efficiency, the pore size can be set to produce a droplet of the required size to encapsulate the particles. For example, if a single particle is desired in each droplet, the droplet can be made slightly larger than the particle to hold only one particle. This enables rapid and controlled emulsification of emulsions that can be directly used for single cell genomics applications.

[0036] In particular embodiments, another advantage of the assemblies and methods herein are its scalability. In certain embodiments, the porous network in the microchamber is composed of a simple array of posts and pores, which can be replicated in a massively parallel format in two and three dimensions. This allows the device to be scaled up to accommodate large volumes of sample, resulting in a faster emulsification process than with traditional microfluidic methods. For example, emulsifying 1 milliliter of sample using microfluidics typically takes an hour, but with the emulsification devices, assemblies, and methods herein, the process can be completed in seconds. In particular embodiments, the parallel nature of the assemblies and devices herein, along with the ability to quickly draw and eject samples without affecting droplet size, allows for quick emulsification of large amounts of reagents.

[0037] In some embodiments, the devices and assemblies herein can be augmented in various ways to suit different applications and use cases. In certain embodiments, all reagents including aqueous, particles, and encapsulation oil can be premixed and drawn repeatedly through the microchamber. Alternatively, in particular embodiments, the microfluidic nature of the devices and assemblies herein can be leveraged for more complex operations. For instance, aqueous reagents can be injected into the microchamber by utilizing a Y-junction upstream, adding aqueous phases that can be mixed or not mixed, depending on the flow. Oil can be added from a separate channel or already reside in the device, and a small amount of surfactant, for example, is sufficient to encapsulate the sample in the desired droplets. The geometry under which the reagents enter the microchamber can be optimized, for example, by merging from different directions, for example, a planar array of impediments in a quasi-2D rectangle with a certain thickness and chamber height. Reagents can be added from channels in a single plane or from the Z direction, uniformly distributing the fluid and suspending particle input. Under laminar flow conditions, this can result in rapid encapsulation of the sample without mixing prior to encapsulation, which is important for single-cell genomics applications where cells should generally not mix until fully encapsulated.

[0038] A porous network is a three-dimensional arrangement of obstacles and interstitial openings in a solid structure. Porous networks can be found both in nature, such as rocks and fiber-based systems like paper, and in engineered applications. They are useful for various purposes, including filtering and emulsifying fluids. Provided herein are methods to use a porous network to encapsulate fluids. The size of the droplets that form generally depends on the size of the pores in the network. If the network has uniform pore sizes, the droplets will generally be uniform in size as well. However, if the fluid passes through the porous network for a long time, the droplets will tend to a size that is comparable to the smallest pore size in the network, which may have a relatively uniform size distribution.

[0039] Porous networks of the devices, systems, and assemblies herein can be created through various processes, including methods that imitate natural processes. For example, aggregating particles together can result in a porous network. If the particles are uniform, the pores in the network will also be somewhat uniform, although not perfectly so if the stacking is random. Porous networks can also be created by stacking particles in a regular lattice, such as a crystal lattice, which results in substantially uniform pores and, therefore, uniform droplets. The lattice geometry can also influence the pore size and shape and, thus, the emulsion properties.

[0040] Another process for creating a porous network is using fibers. Fibers can be stacked either randomly or in a more controlled manner, such as through weaving, to produce porous networks that can be used for filtering or, in this case, emulsifying fluids. The aqueous and immiscible fluids can be passed through the porous network multiple times to produce the emulsion, the size of which depends on the pore size and geometry. Fibers have the advantage of being able to be woven together, which allows for greater control over the microscale geometry and makes the process simple and scalable.

[0041] In some embodiments, for generally greater control over the porous network, microfabrication techniques, such as lithography, can be used. Lithography can precisely fabricate a porous network in a planar or three-dimensional device. For a simple planar device, microfabrication can be used to create a chamber with obstacles placed on a lattice, where the fluid flows laterally through the chamber and passes the obstacles to form an emulsion. To create a more three-dimensional device, the chamber geometry can be replicated in the Z dimension by stacking chambers together. This allows for more fluid to pass through the device at a lower pressure and, therefore, increases the throughput and capacity for generating an emulsion. Other fabrication processes, such as 3D printing or 3D lithography, can be used to create 3D porous networks with interconnections between different Z stacks. These networks more closely resemble natural porous media, such as fiber or particle stacks, while allowing for precision control over the porous network geometry.

[0042] In general, the use of a porous network for emulsifying fluids offers several advantages, including the ability to form relatively uniform droplets without precision control overflow properties and pressures, the ability to operate at high flow rates and pressures for quick emulsification, and robustness to clogging. Thus, in certain embodiments, this approach allows for the formation of uniform emulsions using simple pipetting and well plates, and can emulsify large volumes of reagents in a matter of seconds or minutes, compared to minutes, hours, or even days with a microfluidic device. Thus, it has significant advantages for settings that require simplicity, speed, and reliability, such as in the clinical or research setting.

[0043] The assemblies, devices, and methods described herein have a wide range of applications and are valuable for various applications that already use microfluidic emulsification. For example, in certain embodiments, they are a substantially simple, and are resistant to crosscontamination, clogging, and reliable. For example, existing digital PCR instruments that use droplet generators can be upgraded with this devices and assemblies herein, generally eliminating the need for microfluidic instrumentation. Single cell sequencing instruments that rely on microfluidics for encapsulating particles and cells in droplets can also be replaced with the devices and assemblies herein (e.g., making them simpler). This includes, for example, the 10X genomics Chromium instrument, Mission Bios Tapestri instrument, Sphere Fluidics, and Dolomites Fluidics, which all use microfluidic emulsification. Finally, industrial processes that use various types of emulsification can be upgraded with the approaches herein to yield more monodispersed emulsions that are also scalable and cost-effective. These processes often rely on membrane emulsification or high pressure injection through a nozzle, which can be difficult to scale to large volumes and are susceptible to clogging. In certain embodiments, the devices, assemblies and methods herein avoid these issues. In some embodiments, the methods herein allow for capturing RNA (e.g., mRNA), from lysed cells in the microdroplcts, that can be combined with DNA and protein sequencing approaches as desired to perform multi-omics. Multiple approaches may be employed to allow capture of mRNA with this method. For example, in one embodiment, a process is described in which mRNA hybridizes to a bead (in the sample fluid) and is reverse transcribed onto the bead to generate the barcoded cDNA products (“drop seq”). Alternatively, another approach is to release Poly T capture probes upon lysis of the cell (in the microdroplets) such that the Poly T capture probes can bind to the mRNA of the lysed cell in solution, rather than on the surface of the bead (InDrops). In certain embodiments, if the reverse transcriptase enzyme and necessary reagents are present in the sample fluid and microdroplets, this results in cDNA synthesis of the mRNA while it is tethered to the bead, thereby barcoding the mRNA.

[0044] In some embodiments, the microdroplets formed herein are flowed in a microfluidic device, which may be used to combine multiple microdrops such that all the reagents are combined into a single aqueous droplet. In certain embodiments, the microfluidic devices employed herein comprise a microenvironment on Demand (MOD) device, described in PCT application W02020232072A1 and Cole et al., Proc. Natl. Acad. Sci., 114(33): 8728-8733, 2017, which are both incorporated by reference herein in their entireties. In certain embodiments, the MOD platform is composed of a combination of deterministic single-cell droplet sorter and droplet-assembler that can selectively assemble cells and reagents. MOD performs a cyclic buildup and release of designer droplets through the merging of select droplets on a defined dielectrophoretic trapping position inside the microfluidic device. This approach is advantageous because it is less prone to contamination, higher throughput, and requires fewer moving parts than other devices. The flexible nature of the MOD platform makes it a well-suited technology to perform integrated and functional cell-cell, cell-ECM interaction analysis and link any perturbations to select expressed gene sequences or transcriptome profiles at a single cell level. Essentially, MOD allows for precise, flexible, scalable liquid handling that can build a large number of predetermined reaction conditions. MOD not only allows for the soiling and combination of particulates (e.g., cells, capture beads, liquid gelling precursor, etc.), but also sorts and assembles diverse droplet contents. Furthermore, droplets constructed with MOD are compartmentalized and miniaturized (e.g., -100 pL) providing contained reactions in concentrated volumes. These two aspects of MOD, reagent selection and reaction miniaturization, provide a powerful approach to phenotypically screen large numbers of single cells.

[0045] In certain embodiments, droplet-based cell culture or RNA and DNA sequencing library prep is performed with the microdroplets as prepare herein. The duration of cell culture in sub- nanoliter droplets is limited by a finite amount of encapsulated media and localized buildup of metabolic waste products. In cases where longer duration incubations are desired or required, it may be appropriate to convert a microdroplet to a media-permeable format while keeping encapsulated objects in place. This can be achieved by flowing hydrogel (liquid gel) precursors into droplets along with cells, then triggering gelation to form either gel beads or permeable capsules. After gelation, in certain embodiments, the emulsion is broken, the emulsion oil is removed, and the cell-laden capture- shells arc suspended in media and cultured for a time. Examples of the hydrogel bead approach are given in Wan et al., (Polymers (Basel)., vol. 4, no. 2, pp. 1084-1108, 2012), Utech et al., (Adv. Healthc. Mater., 2015), and Dolega et al. (Biomaterials, vol. 52, no. 1, pp. 347-357, 2015.), all of which are herein incorporated by reference in their entireties. Examples of permeable capsules are given by Yu et al, (Biomed. Microdevices, vol. 17, no. 2, 2015.), van Loo et al (Mater. Today Bio, vol. 6, no. February, p. 100047, 2020.), and Leonaviciene et al. (Lab Chip, no. Advanced Article, 2020), all of which are herein incorporated by reference in their entireties. Extended cell culture is especially useful in cases where cell proliferation is important, such as clonal expansion of single cells and cell-cell interaction assays where proliferation is a readout. In some cases, it may be desired to break down a gel bead or capsule via chemical, enzymatic, or thermal means in order to access the contents for further processing.

[0046] Microdroplets as generated in connection with the subject methods, devices, and / or systems may be sphere shaped or they may have any other suitable shape, e.g., an ovular or oblong shape. Microdroplets as described herein may include a liquid phase and / or a solid phase material. In some embodiments, aqueous droplets according to the present disclosure include a gel material. In certain embodiments, the aqueous droplets comprise double emulsions (or multiple emulsion) or are treated to generate hydrogel shells.

[0047] In certain embodiments, the microdroplets as generated herein may be treated to comprise a hydrogel shell or microcapsule, such as exemplified in U.S. Pat. 10,710,045 and U.S. Pat. Pub. 20140127290, both of which are herein incorporated by reference in their entireties, particularly for such hydrogel shells or microcapsules. In certain embodiments, the hydrogel shells for the microdroplcts comprise a liquid core, and at least one external envelope totally encapsulating the liquid core at its periphery, said external envelope being able to retain the liquid core when the capsule is immersed into a gas and comprising at least one gelled polyelectrolyte and / or a stiffened biopolymer. In certain embodiments, such microdroplets contain a cell and / or other reagents discussed herein.

[0048] In some embodiments, the subject microdroplets have a dimension, e.g., a diameter, of or about 1.0 pm to 1000 pm, inclusive, such as 1.0 pm to 750 pm, 1.0 pm to 500 pm, 1.0 pm to 100 pm, 1.0 pm to 10 pm, or 1.0 pm to 5 pm, inclusive. In some embodiments, microdroplets as described herein have a dimension, e.g., diameter, of or about 1.0 pm to 5 pm, 5 pm to 10 pm, 10 pm to 100 pm, 100 pm to 500 pm, 500 pm to 750 pm, or 750 pm to 1000 pm, inclusive. Furthermore, in some embodiments, microdroplets as described herein have a volume ranging from about 1 fL to 1 nL, inclusive, such as from 1 fL to 100 pL, 1 fL to 10 pL, 1 fL to 1 pL, 1 fL to 100 IL, or 1 IL to 10 fL, inclusive. In some embodiments, aqueous droplets as described herein have a volume of 1 fL to 10 fL, 10 IL to 100 fL, 100 fL to 1 pL, 1 pL to 10 pL, 10 pL to 100 pL or 100 pL to 1 nL, inclusive.

[0049] In some embodiments, the microdroplets as described herein refer to small, generally spherically structures, containing at least a first fluid phase, such as an aqueous phase (e.g., water), bounded by a second fluid phase (e.g., oil) which is immiscible with the first fluid phase. In some embodiments, microdroplets according to the present disclosure may contain a first fluid phase (e.g., oil) bounded by a second immiscible fluid phase (e.g., an aqueous phase fluid, such as water). In some embodiments, the second fluid phase is an immiscible phase carrier fluid. Thus, droplets according to the present disclosure may be provided as aqueous-in-oil emulsions or oil in aqueous emulsions. Droplets may be sized and / or shaped as described herein for aqueous droplets. For example, droplets according to the present disclosure generally range from 1 pm to 1000 pm, inclusive, in diameter. Droplets according to the present disclosure may be used to encapsulate cells, nucleic acids (e.g., DNA and / or RNA), enzymes, reporter dyes, reagents, and a variety of other components. The term microdroplet may be used to refer to a droplet produced in, on, or by a microfluidic device and / or flowed from or applied by a microfluidic device. Aspects of the disclosed methods may include making microdroplets using one or more cells from a biological sample. In such cases, each microdroplct may contain zero, one, or more than one cell. In some cases, such microdroplets can be made by incorporating the biological sample, cells from the biological sample, lysate from cells of the biological sample, or any other sample derived from the biological sample into a mixed emulsion. In some cases, the method further includes separating one or more components of the biological sample or otherwise processing the biological sample (e.g. via centrifugation, filtration, and the like), before making the microdroplets.

[0050] One or more lysing agents may be added to the microdroplets (as generated herein) containing a cell, under conditions in which the cell(s) may be caused to burst, thereby releasing their genomes and target proteins. The lysing agents may be in buffer containing detergents such as Triton X100 and / or proteinase K. One or more primers may be introduced into microdroplets or core-shell particles for each of the genes to be detected. Hence, in certain aspects, primers for all target genes (e.g., antibody genes) may be present in the aqueous droplet at the same time, thereby providing a multiplexed assay. The microdroplets or core-shell particles may be temperature-cycled so that they will undergo PCR. hi certain embodiments, rolling circle amplification (RCA)-based proximity ligation is employed.

[0051] In some embodiments, a surfactant may be used to stabilize the microdroplets. Accordingly, a microdroplet may involve a surfactant stabilized emulsion. Any convenient surfactant that allows for the desired reactions to be performed in the microdroplets, may be used. In other aspects, microdroplet is not stabilized by surfactants or particles. The surfactant used depends on a number of factors such as the oil and aqueous phases (or other suitable immiscible phases (e.g., any suitable hydrophobic and hydrophilic phases)) used for the emulsions. For example, when using aqueous droplets in a fluorocarbon oil, the surfactant may have a hydrophilic block (PEG-PPO) and a hydrophobic fluorinated block (Krytox® FSH). If, however, the oil was switched to be a hydrocarbon oil, for example, the surfactant would instead be chosen so that it had a hydrophobic hydrocarbon block, like the surfactant AB IL EM90. In selecting a surfactant, desirable properties that may be considered in choosing the surfactant may include one or more of the following: (1) the surfactant has low viscosity; (2) the surfactant is immiscible with the polymer used to construct the device, and thus it doesn’t swell the device; (3) biocompatibility; (4) the assay reagents are not soluble in the surfactant; (5) the surfactant exhibits favorable gas solubility, in that it allows gases to come in and out; (6) the surfactant has a boiling point higher than the temperature used for PCR (c.g., 95°C); (7) the emulsion stability; (8) that the surfactant stabilizes drops of the desired size; (9) that the surfactant is soluble in the carrier phase and not in the droplet phase; (10) that the surfactant has limited fluorescence properties; and (11) that the surfactant remains soluble in the carrier phase over a range of temperatures.

[0052] In some cases, microdroplets herein a capture bead (e.g., present in original sample fluid). In some cases, at least one dimension of the capture bead (e.g., diameter, is between about 0.5 pm and about 500 pm). In some cases, the bead is made of a polymeric material, such as polystyrene. In some cases, the bead is magnetic or contains a magnetic component. In some cases, the bead has a biomolecule attached to its surface, such as poly-TTT oligos, an antibody, a protein, an antigen, DNA, RNA, streptavidin, or a combination thereof.

[0053] In some embodiments, the methods, assemblies, devices, and / or systems described herein can be used to sequence nucleic acid (e.g., separated mRNA / cDNA and genomic DNA molecules) derived from single cells. For example, individual cells can be encapsulated in the microdroplets formed herein which include the assay reagents. The cells can then be lysed and subjected to molecular biological processing to amplify and / or tag their nucleic acids with barcodes. The material from all the droplets can then be pooled for all cells and sequenced and the barcodes used to sort the sequences according to single droplets or cells. These methods can be used, for example, to sequence the genomes or transcriptomes of single cells in a massively parallel format.

[0054] In certain embodiments, nucleic acid sequence assay components that employ barcoding for labelling individual RNA (e.g., mRNA), cDNA, and DNA molecules, and / or for labeling for cell / well source (e.g., if wells pooled before sequencing analysis), and / or for labeling particular affixed entities (e.g., if droplet from two or more affixed entities are pooled prior to sequencing) are employed. Examples of such barcoding methodologies and reagents are found in Pat. Pub. US2007 / 0020640, Pat. Pub. 2012 / 0010091, U.S. Pat. 8,835,358, U.S. Pat. 8,481,292, Qiu et al. (Plant. Physiol., 133, 475-481, 2003), Parameswaran et al. (Nucleic Acids Res. 2007 Oct; 35(19): el30), Craig et al. reference (Nat. Methods, 2008, October, 5( 10): 887-893), Bontoux et al. (Lab Chip, 2008, 8:443-450), Esumi et al. (Neuro. Res., 2008, 60:439-451), Hug et al., J. Theor., Biol., 2003, 221:615-624), Sutcliffe et al. (PNAS, 97(5): 1976- 1981 ; 2000), Hollas and Schuler (Lecture Notes in Computer Science Volume 2812, 2003, pp 55-62), and W0201420127; all of which arc herein incorporated by reference in their entireties, including for reaction conditions and reagents related to barcoding and sequencing of nucleic acids.

[0055] In certain embodiments, the DropSeq method employing beads with primers attached to them are employed to sequence nucleic acids from microdroplets. An example of such a method is described in Macosko et al., Cell, 161(5): 1202- 1214 (see, e.g., Figure 1 therein), which is herein incorporated by reference in its entirety. In certain embodiments employing DropSeq, barcoded template switch oligos are bound to beads and oligo dT is supplied in solution along with RT PCR reagents. Reverse transcription (RT) can, for example, be performed as described in Kim et al., Anal Chem. 2018 Jan 16;90(2): 1273-1279, herein incorporated by reference. In other embodiments, barcoded oligo-dT beads are provided, the cells are lysed, mRNAs is captured on the beads, the emulsion is broken, and the drop is re-emulsified to capture mRNA beads with barcoded TSO beads where the TSO can be released by UV. Solution phase TSO can then be used for performing RT-PCR. Primers specific to the variable regions displayed on the surface of the SD cells can be employed to amplify such variable regions prior to sequencing.

[0056] In certain embodiments, unique oligo drops are provided to the fixed entities, and allow a link between imaging and genomics. For example, the unique oligos can contain two part 8-mer barcodes linked to polyA or TSO followed by 8-mer barcodes. In this regard, if one employs 96 barcoded oligos, selecting any three can generate 142,880 combinations. It is known what combination of three oligos are printed at each well position to identify that particular well. These oligos will also be sequenced and so when one sees a particular 3-oligo combination in the sequencing readouts, one knows the fixed entity and the image for that fixed entity.

[0057] In certain embodiments, the barcode tagging and sequencing methods of WO2014201273 (“SCRB-seq” method, herein incorporated by reference) are employed. The necessary reagents for the SCRB-seq method (e.g., modified as necessary for small volumes) are added to the microdroplets, each containing a lysed cell. Briefly, the SCRB-seq method amplifies an initial mRNA sample from cells from a single fixed entity. Initial cDNA synthesis uses a first primer with: i) N6 for cell / well identification, ii) N10 for particular molecule identification, iii) a poly T stretch to bind mRNA, and iv) a region that creates a region where a second template- switching primer will hybridize. The second primer is a template switching primer with a poly G 3’ end, and 5’ end that has iso-bases. After cDNA amplification, the tagged cDNA single fixed entity samples are pooled. Then full-length cDNA synthesis occurs with two different primers, and full-length cDNA is purified. Next, a NEXTERA sequencing library is prepared using an i7 primer (adds one of 12 i7 tags to identify particular multi-well plates) and P5NEXTPT5 to add P5 tag for NEXTERA sequencing (P7 tag added to the other end for NEXTERA). The library is purified on a gel, and then NEXTERA sequencing occurs. As a non-liming example, with twelve i7 plate tags, and 384 cell / well- specific barcodes, this allows total of 4,608 single cell transciptomes to be done at once. This method allows for quantification of mRNA transcripts in single fixed entity.

[0058] In other embodiments, the barcode tagging and sequencing methods employ concepts from the Multi- seq method. For example, cells are incubated with anchor and co-anchor lipid modified oligonucleotides (LMO) and encapsulated in droplets. Individual barcodes in droplets can hybridize to exposed regions of the LMOs and these barcodes can be used instead of Drop- seq beads. Anchor-coanchor LMOs remain bound to individual cells at 4°C but can freely equilibrate between cells in a droplet at 37°C. Thus, a specific LMO-barcode combination can be used to link two cells in that droplet that can be tracked after emulsion breaking. In one example, a unique LMO-barcode combination can be randomly assembled in every microfluidic droplet. Barcodes may also be deterministically pre-printed to a microwell array, and additionally provide linkage to imaging data recoded at specific microwell positions. In another embodiment, one cell in each combination may be LMO-barcoded before the combination in microdroplets. During incubation at 37°C, the LMO-barcodes will re-equilibrate to the initially non-barcoded cell and provide lasting information about co-encapsulation. If a unbarcoded B-cell is combined with an LMO-barcoded antigen presenting cell (APC), this process will allow the type of APC to be read out by sequencing only the B-cell.

[0059] In practicing the methods of the present disclosure, one or more sorting steps may be employed. A sorting step sorts a microdroplet into one of two or more locations (e.g. into one of two or more fluid channels). In some cases, the sorting is into one of two fluid channels. In addition, such sorting may either be passive sorting or active sorting. Active sorting includes the detection of one or more properties of a microdroplet, or a component within the aqueous droplet, and sorting based on the detected property. Passive sorting involves sorting a microdroplet without the active detection of a property. Sorting approaches of interest include, by are not necessarily limited to, approaches that involve the use of one or more sorting channels and one or more sorting elements.

[0060] A variety of different components can be included in the microdroplcts to facilitate detection, including one or more fluorescent dyes (e.g., as part of oligonucleotide probe and / or to stain cell(s)). Such fluorescent dyes may be divided into families, such as fluorescein and its derivatives; rhodamine and its derivatives; cyanine and its derivatives; coumarin and its derivatives; Cascade Blue and its derivatives; Lucifer Yellow and its derivatives; BODIPY and its derivatives; and the like. Exemplary fluorophores include indocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa fluor-355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, JOE, Lissamine, Rhodamine Green, BODIPY, fluorescein isothiocyanate (FITC), carboxy-fluorescein (FAM), phycoerythrin, rhodamine, dichlororhodamine (dRhodamine), carboxy tetramethylrhodamine (TAMRA), carboxy-X-rhodamine (ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen, RiboGreen, and the like. Descriptions of fluorophores and their use, can be found in, among other places, R. Haugland, Handbook of Fluorescent Probes and Research Products, 9th ed. (2002), Molecular Probes, Eugene, Oreg.; M. Schena, Microarray Analysis (2003), John Wiley & Sons, Hoboken, N.J.; Synthetic Medicinal Chemistry 2003 / 2004 Catalog, Berry and Associates, Ann Arbor, Mich.; G. Hermanson, Bioconjugate Techniques, Academic Press (1996); and Glen Research 2002 Catalog, Sterling, VA.

[0061] REFERENCES

[0062] 1. U.S. Patent 11,130,120

[0063] 2. Datta et al., Fluid breakup during simultaneous two-phase flow through a three- dimensional porous medium, Physics of Fluids 26, 062004 (2014).

[0064] 3. Amstad et al., The micro fluidic post-array device: high throughput production of single emulsion drops, Lab Chip, 2014,14, 705-709.

[0065] 4. Kim et al., Repeated geometrical T-junction breakup microfluidic filter device by injection of premixed emulsion for microdroplet production, Journal of Industrial and Engineering Chemistry 81, 81-87. All publications and patents mentioned in the present application are herein incorporated by reference. Various modification and variation of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMSI claim:

1. A system, kit, or device for generating an emulsion from a non-emulsion mixture that is composed of sample fluid, an immiscible carrier fluid, and optionally a surfactant comprising: a) a housing component; b) at least one microfluidic chamber housed in, integral with, or configured to be housed in said housing component, wherein said at least one microfluidic chamber comprises: i) at least 2, and optionally at 100, impediments regularly or irregularly spaced apart to create at least 2, and optionally at least 100, pores therebetween, wherein said at least 2, and optionally 100, pores are about 5-2000 microns in diameter, and ii) a first open end and a second open end which are in fluid communication with each other such that, when said non-emulsion mixture is pushed or drawn once or multiple times through said microfluidic chamber from said first open end to said second open end, and / or vice versa, it passes through said at least 2, and optionally said at least 100 pores, and generates an emulsion comprising a plurality of sample microdroplets in said immiscible carrier liquid, wherein optionally at least some, or most, of said plurality of microdroplets are about the same size as said at least 2, and optionally said 100 pores, or wherein optionally at least some, or most, of said plurality of microdroplets are of non-uniform size.

2. The system or kit of claim 1, further comprising: c) said non-emulsion mixture.

3. The system or kit of claim 2, wherein said surfactant is present in said non-emulsion mixture.

4. The system or kit of claim 1, further comprising: c) said sample fluid, and / or said immiscible carrier fluid, and / or said surfactant.

5. The system, kit, or device of claim 1 , wherein housing component comprises a micropipcttc tip or container configured to be attached to said micropipcttc tip, and / or wherein said microfluidic chamber further comprises a semi-permeable membrane.

6. The system, kit, or device of claim 1, wherein housing component comprises at least a portion of a microfluidic device.

7. The system or kit of claim 1, further comprising; said sample fluid, and wherein said sample fluid comprises water and at least one of the following: i) a liquid gel precursor, ii) a plurality of cells, iii) a plurality of capture beads, optionally barcoded and / or composed of hydrogel, and iv) a plurality of core-shell particles.

8. The system or kit of claim 7, wherein said capture beads are configured to hybridize to RNA, DNA, and / or protein targets.

9. The system or kit of claim 1, further comprising: c) said emulsion.

10. The system, kit, or device of claim 1, wherein said at least one microfluidic chamber comprises at least 2, at least 10, or at least 20 of said microfluidic chambers.

11. The system, kit, or device of claim 1, wherein said at least one microfluidic chamber is located in said housing component.

12. The system, kit, or device of claim 1, wherein said about 5-2000 microns in diameter for said pores is about 10-1000 microns, or about 20-100 microns, or about 50-90 microns.

13. The system, kit, or device of claim 1, wherein said at least 2 or at least 100 impediments, is at least 1000, at least 10,000, or at least 100,000 impediments.

14. The system, kit, or device of claim 1, wherein said at least 2 or at least 100 pores, is at least 1000, at least 10,000, or at least 100,000 pores.

15. The system, kit, or device of claim 1, wherein said housing component is configured to mate with a pressure-providing device such that said pressure -providing device can apply pressure to said microfluidic chamber.

16. The system, kit, or device of claim 15, wherein said pressure-providing device comprises a micro-pipettor or an air-pressure pump.

17. The system, kit, or device of claim 1, further comprising: c) a micro-pipettor.

18. The system, kit, or device of claim 1, wherein said pushed through comprises positive pressure and said drawn through comprises negative pressure.

19. The system, kit, or device of claim 1, wherein said emulsion comprises at least 1000, 10,000, 100,000, or 1 million of said sample microdroplets in said immiscible carrier liquid.

20. The system, kit, or device of claim 1, wherein said immiscible carrier liquid comprises oil, and / or wherein said immiscible carrier liquid comprises oleogel and / or oleogel.

21. The system, kit, or device of claim 1, wherein said plurality of sample microdroplets are substantially monodisperse in said immiscible carrier fluid.

22. The system, kit, or device of claim 1, wherein said at least 2 or at least 100 impediments have a shape selected from: square, round, rectangle, posts, octagon, star shaped, and combinations thereof.

23. The system, kit, or device of claim 1, i) wherein said at least 2, and optionally 100, impediments comprises at least 20 impediments and at least 20 pores therebetween, and ii) wherein said at least 20 impediments are arranged in first and second regularly spaced apart groups that are separate from each other, andiii) wherein said first regularly spaced apart group has first impediments that create a first pore size therebetween, and said second regularly spaced apart group has second impediments that create a second pore size therebetween that is different from said first pore size, and optionally wherein said first and second impediments have different shapes.

23. The system or kit of claim 1, further comprising; c) said sample fluid, and wherein said sample fluid comprises water and particles, wherein said impediment, said pores, and said particles are sized such that said plurality of sample droplets on average contain one and only one particle.

24. The system or kit of claim 1, further comprising: c) said immiscible fluid, wherein said immiscible fluid is present in said microfluidic chamber, and said sample fluid is not present in said microfluidic chamber.

25. The system, kit, or device of claim 1, wherein said microfluidic chamber comprises at least one additional opening configured to introduce said sample fluid, or said immiscible carrier fluid, or said non-emulsion mixture, wherein said configured to introduce is under laminar flow conditions.

26. The system, kit, or device of claim 1, wherein said at least one microfluidic chamber and said housing component are integral with each other such that they form a single combined component.

27. A method of generating an emulsion with a microfluidic chamber comprising: a) attaching an emulsion generating assembly to a pressure-providing device, wherein said emulsion generating assembly comprises: i) a housing component; ii) at least one microfluidic chamber housed in, or integral with, said housing component, wherein said at least one microfluidic chamber comprises:A) at least 2, and optionally 100, impediments regularly or irregularly spaced apart to create at least 2, and optionally 100 pores, therebetween, wherein said at least 2, and optionally 100 pores, are about 5-2000 microns in diameter, andB) a first open end and a second open end which are in fluid communication with each other, and iii) wherein optionally an immiscible carrier fluid is present in said microfluidic chamber; and b) activating said pressure-providing device such that a first liquid is pushed or drawn through said microfluidic chamber once or multiple times, from said first open end to said second open end, or from said second open end to said first open end, or both, wherein said first liquid comprises: i) a sample fluid if said immiscible carrier fluid is present in said microfluidic chamber, and / or ii) a non-emulsion mixture comprising: said sample fluid, said immiscible carrier fluid, and optionally a surfactant, wherein when said first liquid is pushed or drawn through said microfluidic chamber once or multiple times, this generates an emulsion comprising a plurality of sample microdroplets in said immiscible carrier liquid, wherein optionally at least some, or most, of said plurality of microdroplets are about the same size as said at least 2 or at least 100 pores, or wherein optionally at least some, or most, of said plurality of microdroplets are of non- uniform size.

28. The method of claim 27, wherein said activating in repeated at least once.

29. The method of claim 27, wherein said activating is repeated at least five times.

30. The method of claim 27, wherein said pressure-providing device is configured to provide negative pressure, positive pressure, or negative and positive pressure.

31. The method of claim 27, wherein said surfactant is present in said non-emulsion mixture.

32. The method of claim 27, wherein housing component comprises a micropipette tip or container configured to be attached to said micropipcttc tip, and / or wherein said microfluidic chamber further comprises a semi-permeable membrane.

33. The method of claim 27, wherein housing component comprises at least a portion of a microfluidic device.

34. The method of claim 27, wherein said sample fluid comprises water and at least one of the following: i) a liquid gel precursor, ii) a plurality of cells, iii) a plurality of capture beads, optionally barcoded and / or composed of hydrogel, and iv) a plurality of core-shell particles.

35. The method of claim 34, wherein said capture beads are configured to hybridize to RNA, DNA, and / or protein targets.

36. The method of claim 27, wherein said plurality of microdroplets comprise a plurality of nuclei acid molecules, and the method further comprises: c) processing said plurality of microdroplets such that said plurality of nucleic acid molecules are sequenced.

37. The method of claim 27, wherein said at least one microfluidic chamber comprises at least 2, at least 10, or at least 20 of said microfluidic chambers.

38. The method of claim 27, wherein said at least one microfluidic chamber is integral with said housing component.

39. The method of claim 27, wherein said about 5-2000 microns in diameter for said pores is about 10-1000 microns, or about 20-100 microns, or about 50-90 microns.

40. The method of claim 27, wherein said at least 2 or at least 100 impediments, is at least 1000, at least 10,000, or at least 100,000 impediments.41 . The method of claim 27, wherein said at least 2 or at least 100 pores, is at least 1000, at least 10,000, or at least 100,000 pores.

42. The method of claim 27, wherein said attaching comprises push-fitting said emulsion generating assembly to said pressure -providing device.

43. The method of claim 27, wherein said pressure-providing device comprises a micropipettor or an air-pressure pump.

44. The method of claim 27, wherein said activating said pressure -providing device pushes said first liquid through said microfluidic chamber with positive air pressure.

45. The method of claim 27, wherein said activating said pressure -providing device draws said first liquid through said microfluidic chamber with negative air pressure.

46. The method of claim 27, wherein said emulsion comprises at least 1000, 10,000, 100,000, or 1 million of said sample microdroplets in said immiscible carrier liquid.

47. The method of claim 27, wherein said immiscible carrier liquid comprises oil, and / or wherein said immiscible carrier liquid comprises oleogel and / or oleogel.

48. The method of claim 27, wherein said plurality of sample microdroplets are substantially monodisperse in said immiscible carrier fluid.

49. The method of claim 27, wherein said at least 100 impediments have a shape selected from: square, round, octagon, star shaped, rectangle, posts, and combinations thereof.

50. The method of claim 27, i) wherein said at least 2, and optionally 100, impediments comprises at least 20 impediments and at least 20 pores therebetween, and ii) wherein said at least 20 impediments are arranged in first and second regularly spaced apart groups that are separate from each other, andiii) wherein said first regularly spaced apart group has first impediments that create a first pore size therebetween, and said second regularly spaced apart group has second impediments that create a second pore size therebetween that is different from said first pore size, and optionally wherein said first and second impediments have different shapes.

51. The method of claim 27, wherein said sample fluid comprises water and particles, wherein said impediments, said pores, and said particles are sized such that said plurality of sample microdroplets on average contain one and only one particle.

52. The method of claim 27, wherein said immiscible fluid is present in said microfluidic chamber and said first liquid is said sample fluid.

53. The method of claim 27, wherein said microfluidic chamber further comprises a third and / or fourth opening configured to introduce said sample fluid, or said immiscible carrier fluid, or said non-emulsion mixture into said microfluidic chamber.

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