A platform for deterministic assembly of microfluidic droplets

Abstract

Methods are provided for selectively combining individual entities, including, for example, cells, reagents, drugs, hydrogels, extracellular matrices, beads, particles, biological materials, media, or combinations thereof. In certain embodiments, the methods include sorting a plurality of individual entities and capturing the two or more individual entities for a time sufficient to combine two or more individual entities to form a combined individual entity. In certain embodiments, the methods include creating a plurality of individual entities. In certain embodiments, the methods include detecting or analyzing the individual entities, e.g., via optical detection. In certain embodiments, the methods include manipulating or analyzing the combined individual entities or components therein, e.g., imaging, sequencing, culturing, e.g., three-dimensional culturing, and measuring cell-cell interactions. Systems and devices for practicing the subject methods are also provided.

Description

[Technical Field]

[0001] Reference to priority document This application claims the benefit of priority under 35 U.S.C. §119(e) of co-pending U.S. provisional patent application Serial No. 62 / 847,791, filed May 14, 2019. The disclosure of the provisional patent application is incorporated herein by reference in its entirety.

[0002] Government support This invention was made with government support under Grant No. R43 HG010128 awarded by the National Institutes of Health and Grant Nos. D17PC00405 and 140D6318C0010 awarded by the Defense Advanced Research Projects Agency. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Studying interactions between biomolecules, reagents, cells, or combinations thereof involves bringing the components together under specific conditions. The development of individual-entity microfluidics has provided tools for manipulating single cells and small amounts of reagents or biomolecules. However, such microfluidic manipulation generally only involves sorting each individual entity into a specific microfluidic channel. Thus, such microfluidic devices lack the ability to selectively combine individual entities.

[0004] overview Methods are provided for selectively combining individual entities, including, for example, cells, reagents, drugs, hydrogels, extracellular matrices, beads, particles, biological materials, media, or combinations thereof. In certain embodiments, the methods include sorting a plurality of individual entities and capturing the two or more individual entities for a time sufficient to combine two or more individual entities to form a combined individual entity. In certain embodiments, the methods include creating a plurality of individual entities. In certain embodiments, the methods include detecting or analyzing the individual entities, e.g., via optical detection. In certain embodiments, the methods include manipulating or analyzing the combined individual entities or components therein, e.g., imaging, sequencing, culturing, e.g., three-dimensional culturing, and measuring cell-cell interactions. Systems and devices for practicing the subject methods are also provided.

[0005] The present disclosure provides a method for selectively combining individual entities, for example, by flowing a plurality of individual entities in a carrier fluid through an inlet channel, wherein the plurality of individual entities are insoluble, immiscible, or a combination thereof in the fluid; selectively sorting at least two of the individual entities to a first outlet channel; and temporarily capturing at least two individual entities in an individual entity merging region of the first outlet channel such that the at least two individual entities combine to form a combined individual entity, wherein the inlet channel, the first outlet channel, and the individual entity merging region are each part of a single microfluidic device.

[0006] The present disclosure also provides microfluidic devices that can be utilized in practicing the methods described herein. For example, the present disclosure provides a microfluidic device that includes: a) an inlet channel; b) a sorting channel in fluid communication with the inlet channel; c) a first outlet channel and a second outlet channel in fluid communication with the sorting channel, where the first outlet channel includes an individual entity merging region; d) a sorting element disposed proximate to the sorting channel, where the sorting element is configured to sort individual entities in the sorting channel relative to the first outlet channel; and e) a capture element disposed proximate to the individual entity merging region, where the capture element and the individual entity merging region are configured to capture multiple individual entities in the multiple individual entity merging region for a time sufficient to combine the multiple individual entities to form a combined individual entity.

[0007] The present disclosure also provides systems including a subject microfluidic device, for example as described herein, and one or more or all of the following: i) an individual entity fabrication apparatus configured to fabricate a plurality of individual entities, wherein the individual entity fabrication apparatus is located within the microfluidic device or separate from the microfluidic device; ii) an individual entity library including two or more types of individual entities; iii) a detector configured to detect individual entities in the input flow path, wherein the microfluidic device is configured to sort individual entities in the sort flow path based on the detection by the detector; iv) a temperature control module operably connected to the microfluidic device; v) an incubator operably connected to the microfluidic device; vi) an imaging apparatus configured to image the combined individual entities; and vii) a sequencer operably connected to the microfluidic device or the incubator. [Brief explanation of the drawings]

[0008] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which: [Figure 1] 1 provides a block schematic diagram of an exemplary microfluidic device having an inlet channel, a sorting channel, a sorting element, first and second outlet channels, a capture element, a separate entity fusion region, and upstream and downstream regions. [Figure 2] 1 provides an image of a microfluidic device having a spacer fluid channel, a bias fluid channel, a stacked oil inlet channel, a concentric sorting channel, a flow divider, and a recess according to an embodiment of the present disclosure. [Figure 3] 1 provides an image of a microfluidic device having concentric sorting channels, recesses, and a roughly triangular downstream region according to an embodiment of the present disclosure. [Figure 4] 1 provides images illustrating an example of combining four individual entities using a microfluidic device, where each individual entity contains a different reagent or single cell, according to an embodiment of the present disclosure. [Figure 5] 1 provides a schematic flow diagram of a method for selectively combining individual entities using a microfluidic device, according to an embodiment of the present disclosure. [Figure 6] 1 shows an annotated image of combined individual entities formed using a microfluidic device, where the enclosed combined individual entities each contain three different cells, according to an embodiment of the present disclosure. [Figure 7] 1 shows the percentage of combined individual entities containing exactly three cells using a microfluidic device according to an embodiment of the present disclosure compared to the expected percentage based on a random combination of the individual entities. [Figure 8] We provide graphs showing the efficiency of loading unique bead and / or cell combinations into microfluidic droplets. Random combinations are calculated from Poisson statistics with an average occupancy of 10% for each unique object. Deterministic combinations are calculated from a 98% combinatorial efficiency from published results of similar techniques. [Figure 9] Schematics are provided showing exemplary configurations for capturing individual entities. Panel i) shows a bipolar electrode pair embedded in the same sidewall of a channel. Panel ii) shows a bipolar electrode pair embedded on opposite sides of the channel. Panel iii) shows a bipolar electrode pair embedded in the floor or ceiling of a channel. [Figure 10] Schematics are provided showing exemplary configurations for directing individual entities to the individual entity fusion region. Panel i) shows the application of laminar flow to confine droplet-containing laminar flow to the sidewalls of a channel. Panel ii) shows a partial-height flow distributor that allows fluid, but not droplets, to enter the central portion of the channel. Panel iii) shows a configuration in which grooves of similar height to droplet dimensions are patterned near the sidewalls of the channel, while the remainder of the channel is configured with a reduced height to exclude droplets. Panel iv) shows a porous flow distributor that allows fluid, but not droplets, to enter the central portion of the channel. Panel v) shows a partial-height flow distributor that directs droplets to a trap in the center of a microfluidic channel. [Figure 11]

[0023] Figure 1 provides a schematic diagram illustrating an exemplary embodiment in which trapping is facilitated by a mechanical valve. Panel i) shows an initial stage in which the individual entities are trapped by the valve. Panel ii) shows a second stage in which the individual entities are combined, e.g., by electrical, chemical, or other means. Panel iii) shows a third stage in which the combined individual entities are released by opening the valve and carried downstream. [Figure 12] Schematics are provided illustrating exemplary embodiments with different channel geometries proximate to an electromagnetic capture element. Panel i) shows an individual entity merging region upstream of a bend in the channel wall. Panel ii) shows an individual entity merging region at a lateral facet within the channel wall. Panel iii) shows individual entities being captured in a region vertically higher than the main channel. [Figure 13] 1 provides a flowchart illustrating an example sequence of operations for selectively combining individual entities and releasing the combined individual entities. [Figure 14]10 provides images showing exemplary combinations of individual entities containing beads and cells. Panel i) shows the capture of an individual entity containing a single cell. Panel ii) shows the combined individual entities after the addition of a single rigid bead. Panel iii) shows the downstream release of the combined individual entities. [Figure 15] 1 provides images showing exemplary combinations of cell-containing individual entities. Panel i) shows delivery of a first cell in a first individual entity to the individual entity fusion region. Panel ii) shows the region after addition of a second cell. Panel iii) shows the region after addition of a third cell. Panel iv) shows downstream release of the combined individual entities. [Figure 16] Images and graphs showing the analysis of combined individual entities are provided. Panel i) shows an image of combined individual entities with three types of cells. Panel ii) shows cytokine (IL-2) secretion from immune cells stimulated using a bead-based immunoassay. [Figure 17] Provided are images and graphs showing the results of cell-cell interaction experiments using chimeric antigen receptor (CAR) T cells, target cells (RAJI), and cell death readouts. [Figure 18] 1 provides an image showing the results of a combined cell death and cytokine (IFg) assay on combined CAR-T and RAJI cells after incubation. [Figure 19] A flowchart is provided showing an exemplary multi-omics workflow for creating, selecting, and combining individual entities followed by incubation, analysis, and sequencing. [Figure 20] 1 provides a flowchart illustrating an example of an integrated imaging and real-time barcoding workflow. [Figure 21A]We demonstrate precision in droplets assembled with two beads of two colors. A) Assay droplets are constructed accurately with high throughput, as demonstrated by the assembly of 20,000 droplets with exactly one red and one blue bead. A.1) Representative composite fluorescence image of input droplets shows sparse loading of blue and red fluorescent beads. [Figure 21B] A representative composite fluorescent image of assembled "assay" droplets is provided, showing a uniform content of one red bead and one blue bead per droplet. [Figure 21C] Provided that the assembled droplets are twice the volume of the input droplets. A.4) 90% of the assembled droplets contain exactly one blue and one red bead. B) Exact assembly of one red, one blue, and one green cell in the droplet. [Figure 22A] Providing CAR-T cytokine detection after stimulation with RAJI cells in assembled droplets. A) Input droplets contain a single CAR-T cell, a single RAJI cell, or detection reagents. [Figure 22B] Provide that CAR-T, RAJI, and reagents are sorted, fused into assay droplets, and incubated for 12 hours. [Figure 22C] It is provided that the assay droplets are sorted according to the peak intensity of the cytokine-detecting antibody. DETAILED DESCRIPTION OF THE INVENTION

[0009] Methods are provided for selectively combining individual entities, including, for example, cells, reagents, drugs, hydrogels, extracellular matrices, beads, particles, biological materials, media, or combinations thereof. In certain embodiments, the methods include sorting a plurality of individual entities and capturing the two or more individual entities for a time sufficient to combine two or more individual entities to form a combined individual entity. In certain embodiments, the methods include creating a plurality of individual entities. In certain embodiments, the methods include detecting or analyzing the individual entities, e.g., via optical detection. In certain embodiments, the methods include manipulating or analyzing the combined individual entities or components therein, e.g., imaging, sequencing, culturing, e.g., three-dimensional culturing, and measuring cell-cell interactions. Systems and devices for practicing the subject methods are also provided.

[0010] Before the present invention is described in detail, it is to be understood that the invention is not limited to the particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0011] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, is also specifically disclosed. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Any method and material similar or equivalent to the method and material described herein can be used to implement or test this invention, but some potential and exemplary methods and materials can be described here.Any and all publications mentioned herein are incorporated by reference into this specification to disclose and describe the method and / or material that the publication is cited in.In the event of any discrepancy, it should be understood that this disclosure shall prevail over any disclosure of the incorporated publication.

[0013] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a droplet" includes a plurality of such droplets, a reference to "the individual entity" includes a reference to one or more individual entities, and so forth.

[0014] It is further noted that the claims may be drafted to exclude any element, including any optional element. Accordingly, this specification is intended to serve as a predicate basis for using exclusive language such as "solely," "only," and the like, or for using "negative" limitations in connection with the recitation of claim elements.

[0015] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. To the extent that a definition or use of any term herein conflicts with a definition or use of that term in an application or reference incorporated herein by reference, the present application shall control.

[0016] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0017] definition The individual entities used or produced in connection with the subject methods, devices, and / or systems may be spherical, or they may have any other suitable shape, e.g., oval or elliptical. The individual entities described herein may comprise liquid and / or solid phase materials. In some embodiments, individual entities according to the present disclosure comprise gel materials. In some embodiments, the subject individual entities have dimensions (e.g., diameters) of, for example, 1.0 μm to 1,000 μm, such as 1.0 μm to 750 μm, 1.0 μm to 500 μm, 1.0 μm to 100 μm, 1.0 μm to 10 μm, or 1.0 μm to 5 μm, inclusive. In some embodiments, the discrete entities described herein have dimensions (e.g., diameters) of about 1.0 μm to 5 μm, 5 μm to 10 μm, 10 μm to 100 μm, 100 μm to 500 μm, 500 μm to 750 μm, or 750 μm to 1,000 μm, inclusive. Further, in some embodiments, the discrete entities described herein have volumes in the range of about 1 fL to 1 nL, such as about 1 fL to 100 pL, 1 fL to 10 pL, 1 fL to 1 pL, 1 fL to 100 fL, or 1 fL to 10 fL, inclusive. In some embodiments, the discrete entities described herein have volumes of about 1 fL to 10 fL, 10 fL 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. Additionally, the individual entities described herein may have a size and / or shape such that they can be generated within, on, or by a microfluidic device and / or flow from or applied by a microfluidic device.

[0018] In some embodiments, the discrete entities described herein are droplets. The terms "drop," "droplet," and "microdroplet" are used interchangeably herein to refer to small, generally spherical structures containing at least a first fluid phase, e.g., an aqueous phase (e.g., water), bounded by a second fluid phase (e.g., oil) that is immiscible with the first fluid phase. In some embodiments, droplets 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 (e.g., 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 water-in-oil emulsions or oil-in-water emulsions. The droplets may be of any size and / or shape described herein for the discrete entities. For example, droplets according to the present disclosure generally range in diameter from 1 μm to 1,000 μm, inclusive. Droplets according to the present disclosure can be used to encapsulate cells, nucleic acids (e.g., DNA), enzymes, reagents, and various other components. The term droplet can be used to refer to droplets generated within, on, or by a microfluidic device and / or flowing from or applied by a microfluidic device.

[0019] As used herein, the term "dielectrophoretic force" refers to the force exerted on an uncharged particle caused by the polarization and interaction of the particle with a non-uniform electric field. The dielectrophoretic force can be directed toward an "attractive dielectrophoretic force," i.e., away from a "repulsive dielectrophoretic force," or in any direction relative to the electric field source. Before being contacted by the electric field, the particle can be positively charged, negatively charged, or neutral.

[0020] As used herein, the term "electrophoretic force" refers to the force exerted on a charged particle caused by its interaction with an electric field. The electrophoretic force can be directed toward an "attractive electrophoretic force," i.e., away from a "repulsive electrophoretic force," or in any direction relative to the source of the electric field. Before being contacted by the electric field, the particle can be positively charged, negatively charged, or neutral.

[0021] As used herein, the term "carrier fluid" refers to a fluid configured or selected to contain one or more discrete entities (e.g., droplets) described herein. The carrier fluid may include one or more substances and may have one or more properties, e.g., viscosity, that allow it to flow through a microfluidic device or portion thereof. In some embodiments, the carrier fluid includes, for example, oil or water, and may be in a liquid or gas phase. Suitable carrier fluids are described in more detail herein.

[0022] As used herein, the term "biological sample" encompasses various sample types obtained from various sources, including biological materials. For example, the term includes biological samples obtained from mammalian subjects, e.g., human subjects, as well as biological samples obtained from food, water, or other environmental sources. This definition encompasses blood and other liquid samples of biological origin, as well as solid tissue samples, such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. This definition also includes samples that have been manipulated in any way after procurement, such as by treatment with reagents, solubilization, or enrichment for specific components, such as polynucleotides. The term "biological sample" encompasses clinical samples, including cells in culture, cell supernatants, cell lysates, cells, serum, plasma, biological fluids, and tissue samples. "Biological samples" include cells; biological fluids, such as blood, cerebrospinal fluid, semen, and saliva; bile; bone marrow; skin (e.g., skin biopsy); and antibodies obtained from an individual.

[0023] As used herein, "polynucleotide" or "oligonucleotide" refers to a linear polymer of nucleotide monomers and may be used interchangeably. Polynucleotides and oligonucleotides can have a variety of structural configurations, e.g., single-stranded, double-stranded, or any combination thereof, as well as higher-order intramolecular or intermolecular secondary / tertiary structures, e.g., hairpins, loops, triple-stranded regions, etc. Polynucleotides, when generally referred to as "oligonucleotides," typically range in size from a few monomer units, e.g., 5-40, to several thousand monomer units. Unless otherwise specified or clear from the context, when a polynucleotide or oligonucleotide is represented by a series of letters (upper or lower case), such as "ATGCCTG," it is understood that the nucleotides are in 5'-3' order from left to right, with "A" representing deoxyadenosine, "C" representing deoxycytidine, "G" representing deoxyguanosine, "T" representing thymidine, "I" representing deoxyinosine, and "U" representing uridine. Unless otherwise noted, terminology and atomic numbering conventions follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999).

[0024] As used interchangeably herein, the terms "polypeptide," "peptide," and "protein" refer to polymeric forms of amino acids of any length. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide. Standard polypeptide nomenclature, J. Biol. Chem., 243 (1969), 3552-3559, is used.

[0025] As used herein, "operably connected" and "operably coupled" mean that the disclosed system or device and its various components are connected in a particular manner (e.g., a manner that allows fluid (e.g., water) and / or power to be transferred) that allows them to operate effectively in the manner described herein.

[0026] In certain embodiments, the flow channel is one or more "micro" channels. The channel can have at least one cross-sectional dimension on the order of a millimeter or less (e.g., about 1 millimeter or less). For specific applications, this dimension can be adjusted, and in some embodiments, at least one cross-sectional dimension is about 500 micrometers or less. In some embodiments, the cross-sectional dimension is about 100 micrometers or less, or about 10 micrometers or less, and in some cases about 1 micrometer or less. While the cross-sectional dimension is generally perpendicular to the centerline flow direction, it should be understood that when encountering flow through an elbow or other feature that tends to change flow direction, the cross-sectional dimension need not be strictly perpendicular to the flow. It should also be understood that in some embodiments, a microchannel can have two or more cross-sectional dimensions, such as the height and width of a rectangular cross-section, or the major and minor axes of an elliptical cross-section. Any of these dimensions can be compared to the sizes presented herein. It should be noted that microchannels used in this disclosure may have rectangular cross sections that are highly disproportionate in two dimensions, e.g., heights of about 100-200 micrometers and widths on the order of one centimeter or more. Of course, a particular device may employ channels that are very similar or identical in size in two or more axes (e.g., channels with square or circular cross sections).

[0027] method As summarized above, the disclosed subject matter includes methods for selectively combining individual entities. The present disclosure provides methods for selectively combining individual entities, for example, by flowing a plurality of individual entities in a carrier fluid through an inlet channel, where the plurality of individual entities are insoluble, immiscible, or a combination thereof in the fluid; selectively sorting at least two of the individual entities to a first outlet channel; and temporarily capturing at least two individual entities in the individual entity merging region of the first outlet channel to combine the at least two individual entities to form a combined individual entity, where the inlet channel, the first outlet channel, and the individual entity merging region are each part of a single microfluidic device.

[0028] In some cases, the first outlet flow path further comprises an upstream region positioned between and in fluid communication with the sorting flow path and the individual entity merging region, hi some cases, the first outlet flow path further comprises a downstream region positioned adjacent and in fluid communication with the individual entity merging region.

[0029] In some cases, the method includes releasing the combined individual entities from the individual entity fusion region.

[0030] FIG. 1 presents a non-limiting, simplified schematic diagram of one type of device and method according to the present disclosure. The microfluidic device of FIG. 1 is labeled as microfluidic device 100. FIG. 1 shows a diagram of an inlet channel 101 through which individual entities that are insoluble and / or immiscible in the carrier fluid can flow to a sorting channel 102 that is in direct fluid communication with the inlet channel 101. The individual entities can then be sorted by a sorting element 103 to a first outlet channel 104 or a second outlet channel 105, both of which are in direct fluid communication with the sorting channel. The sorting element 103, in some cases, can be an electrode, e.g., an electrode configured to exert a dielectrophoretic force on the individual entities. The sorting element 103 of FIG. 1 is configured to sort the individual entities in the sorting channel 102 to either the first outlet channel 104 or the second outlet channel 105. In some cases, when the individual entities are sorted into the second outlet flow path 105, they are sorted into a waste container or recycled to the inlet flow path 101. Figure 1 shows an embodiment in which the first outlet flow path 104 includes an upstream region 106, an individual entity merging region 107, and a downstream region 108. In some cases, the individual entity merging region contains a change in the dimensions of the first outlet flow path, for example, the individual entity merging region 107 has a larger cross-sectional area than the upstream region 106.

[0031] 1 device includes a capture element 109. In some cases, the capture element 109 includes a capture electrode, which is configured to exert a force (e.g., a dielectrophoretic force) that captures the individual entities within the individual entity fusion region 107. Furthermore, the individual entity fusion region 107 and the capture element 109 are configured such that the force applied by the capture electrode is sufficient to capture the multiple individual entities within the individual entity fusion region for a time sufficient to cause the multiple individual entities to combine in the individual entity fusion region to form a combined individual entity. In some cases, the capture electrode is configured to provide an electric field that affects the surfaces of the individual entities so that the individual entities can more easily fuse, for example, so that the individual entities spontaneously fuse. In some cases, the effect is destabilizing.

[0032] 1 includes flowing a plurality of individual entities through inlet channel 101 into sorting channel 102, sorting the plurality of individual entities with sorting element 103 into first outlet channel 104 or second outlet channel 105, and capturing at least two individual entities with capture element 109 in individual entity fusion region 107 for a time sufficient to combine at least two individual entities to form a combined individual entity. Figure 5 shows a schematic diagram of an exemplary method in which individual entities containing cells are selectively combined.

[0033] FIG. 2 provides an additional non-limiting, simplified schematic diagram of one type of device and method according to the present disclosure.

[0034] In some cases, the individual entity merging region includes a recess, for example, as shown as recess 107 in FIG. 2. In some cases, the individual entity merging region includes a flow divider, for example, as shown as flow divider 113 in FIG. 2. In some cases, the device further includes a stacked oil inlet, for example, as shown as stacked oil inlet 112 in FIG. 2. In some cases, the capture element includes two electrodes having significantly different shapes from each other, for example, as shown as electrode 109 in FIG. 2. In some cases, the capture element includes two electrodes that generate a region of high electric field gradient extending within the microfluidic channel. In some cases, the individual entity merging region includes a change in the angle of flow between the individual entity merging region and an adjacent upstream region, for example, as shown in FIG. 3.

[0035] In some cases, the device further includes a spacer fluid inlet. As an example, the device of FIG. 2 includes a spacer fluid channel 110 in fluid communication with the inlet channel 101. The spacer fluid channel can be configured such that flowing the spacer fluid through the spacer fluid channel positions the spacer fluid between two individual entities flowing through the inlet channel, thereby maintaining or increasing the distance between the two individual entities, thereby allowing each of the two individual entities to be sorted or not sorted independently.

[0036] In some cases, the device further includes a bias fluid inlet. As an example, the device of FIG. 2 includes a bias fluid channel 111 in fluid communication with the sorting channel 102. The bias fluid channel can be configured such that bias fluid flowing through the bias fluid channel moves individual entities closer to the second sidewall of the sorting channel and away from the first sidewall of the sorting channel. Thus, as an example, the spacer fluid inlet 111 moves individual entities closer to the wall of the inlet channel near the bottom of the figure and further away from the wall near the top of the figure. Thus, one or more bias fluid channels can be configured such that individual entities preferentially flow to the first exit position or the second exit position in the absence of a force from the sorting element. In some cases, the bias fluid inlet channel can be configured such that individual entities preferentially flow to the second exit channel in the absence of a dielectrophoretic force from the sorting electrode. As an example, bias fluid inlet 111 of FIG. 2 preferentially directs individual entities into second outlet channel 105 in the absence of a force exerted on the individual entities by sorting electrode 103 .

[0037] In some cases, the device includes a detector configured to detect individual entities in the input channel, and the microfluidic device is configured to sort the individual entities in the sorting channel based on detection by the detector. As an example, Figure 2 shows an embodiment in which individual entities in the detection region 114 of the input channel 101 can be detected by the detector, and then the sorting electrode 103 can sort the individual entities into the first outlet channel 104 or the second outlet channel 105.

[0038] 2 also includes shielding electrodes 115a, 115b, 115c, and 115d. As used herein, the term "shielding electrode" is used synonymously with "moat electrode." Each shielding electrode may be configured to perform one or more functions, including at least partially shielding individual entities from unwanted electromagnetic fields, assisting in sorting individual entities, and assisting in capturing individual entities.

[0039] Thus, as used herein, a shielding electrode may also be referred to as a screening electrode or a capture electrode when the electrode is configured to participate in the screening or capture of an individual entity. Thus, shielding electrode 115a may be referred to as a screening electrode when configured to form a bipolar electrode pair with screening electrode 103 to facilitate screening of an individual entity. Similarly, shielding electrode 115d may be referred to as a capture electrode when configured to form a bipolar electrode pair with capture electrode 109 to facilitate capture of an individual entity.

[0040] In some cases, the shielding electrodes can generate electromagnetic fields such that individual entities within the device are at least partially shielded from undesired electromagnetic fields. The undesired electromagnetic fields can originate from outside the microfluidic device or from within the microfluidic device. In some cases, the undesired electromagnetic fields are electric fields that are not generated by the sorting or capture electrodes. By at least partially shielding individual entities within the microfluidic device, the shielding electrodes can inhibit unintended fusion of individual entities, i.e., fusion of individual entities outside of the individual entity fusion region. In some cases, the shielding electrodes 115a, 115b, and 115c can be used to at least partially shield individual entities from electromagnetic fields that are not generated by the sorting or capture electrodes.

[0041] In some cases, the shielding electrode can assist in sorting of individual entities. As an example, the shielding electrode 115a may interact with the sorting electrode 103 to facilitate sorting, for example, by forming a bipolar electrode pair with the sorting electrode 103. In some cases, the sorting electrode 103 may be a charged electrode (e.g., positively charged) and the shielding electrode 115a may be grounded. Stated another way, the shielding electrode 115a may be configured to affect the shape of the electromagnetic field generated by the sorting electrode 103 to facilitate sorting.

[0042] In some cases, the shielding electrode can assist in the capture of individual entities. As an example, the shielding electrode 115d may interact with the capture electrode 109 to facilitate capture, for example, by forming a bipolar electrode pair with the capture electrode 109. In some cases, the sorting electrode 109 may be a charged electrode (e.g., positively charged), and the shielding electrode 115d may be grounded. Stated another way, the shielding electrode 115d may be configured to affect the shape of the electromagnetic field generated by the capture electrode 109 to facilitate sorting.

[0043] In some cases, one or more of the shielding electrodes are separate elements, e.g., all of the shielding electrodes are separate elements. In some cases, one or more of the shielding electrodes are directly electrically connected. In some cases, one or more of the shielding electrodes are different regions of a single electrode, e.g., part of a single metal piece. In some cases, one or more of the shielding elements are attached to ground.

[0044] In some cases, the device includes one or more shielding electrodes, as shown in Figure 2. In some cases, the device includes zero shielding electrodes, e.g., individual entities are sorted using a single sorting electrode and individual entities are captured using a single capture electrode.

[0045] Thus, individual entities are sorted and selectively combined within the microfluidic device, i.e., without leaving the microfluidic device, or stated another way, individual entities are sorted and combined without leaving the microfluidic sized channels and regions.

[0046] In addition, the present disclosure provides examples of specific elements and steps that can be used in the described devices, systems, and methods. As outlined above, the capture and sorting elements can be electrodes that exert dielectrophoretic forces on individual entities. In some cases, the electrodes are microfluidic channels containing conductive materials, such as salt water, liquid metal, molten solder, or conductive inks that are subsequently annealed. In some cases, the electrodes are patterned on a substrate of the microfluidic device, such as a patterned indium tin oxide (ITO) glass slide. In some cases, the capture element includes two electrodes. In some cases, the capture element is a selectively actuable bipolar droplet capture electrode. In some cases, the sorting element includes two electrodes. In some cases, the sorting element includes a selectively actuable bipolar droplet sorting electrode.

[0047] In some cases, the sorting flow path includes a partial-height flow divider, as described further below. In some cases, the sorting flow path has a concentric or essentially concentric flow path, and a portion of the sorting electrode is positioned at the center of an arc of the concentric or essentially concentric flow path, as described further below.

[0048] In some cases, the discrete entity includes a particle, e.g., a cell. In some cases, the discrete entity includes a chemical reagent, e.g., a lysis agent or a PCR reagent. In some cases, the discrete entity includes both a cell and a chemical reagent. In some cases, the discrete entity includes a fluorescently tagged cell.

[0049] In some cases, the sorting is passive sorting. In some cases, the sorting is active sorting, i.e., the sorting element sorts individual entities into one of at least two locations based on a detected property of the individual entities or components within the individual entities. In some cases, the detected property is an optical property, and the device further includes an optical detector, e.g., an optical detector configured to detect an optical property of the individual entities or components within the inlet channel. In some cases, the optical property is fluorescence, and the device further includes an excitation light source. In some cases, the sorting is based on detected fluorescence of fluorescent tags on cells within the individual entities.

[0050] In some cases, the individual entity fusion region can include structural elements configured to assist in the capture and combination of the individual entities therein, in some cases, the structural elements are configured to assist in the capture and combination by altering the speed or direction of fluid flow through the region of the individual entity fusion region.

[0051] The present disclosure also provides methods of using a system including, for example, a microfluidic device as described above, and one or more additional components, such as (a) a temperature control module operably connected to the microfluidic device; (b) a detector configured to detect individual entities in an input flow path, wherein the microfluidic device is configured to sort individual entities in a sorting flow path based on detection by the detector; (c) an incubator operably connected to the microfluidic device or an individual entity fabrication apparatus; (d) a sequencer operably connected to the microfluidic device; (e) a device configured to fabricate a plurality of individual entities, i.e., an individual entity fabrication apparatus, disposed within the microfluidic device or disposed separately from the microfluidic device; and (f) one or more conveyors configured to transport particles (e.g., cells) or individual entities, wherein the individual entities can, in some cases, include particles between any combination of the incubator, the device configured to fabricate a plurality of individual entities, the microfluidic device, and the sequencer.

[0052] In some cases, the method includes controlling the temperature of the microfluidic device using a temperature control module operably connected to the microfluidic device. In some cases, the method includes detecting individual entities in an input channel of the microfluidic device, e.g., detecting optical properties of the individual entities or components therein, and sorting the individual entities based on the detection. In some cases, the method includes incubating cells in an incubator operably connected to an individual entity fabrication device or the microfluidic device. In some cases, the method includes fabricating individual entities using an individual entity fabrication device, the individual entity fabrication device being located within the microfluidic device or separate from the microfluidic device. In some cases, the method includes moving the individual entities between components of the system, e.g., with one or more conveyors.

[0053] The present disclosure also provides steps that can be performed after release of the combined microfluidic droplet from the individual entity fusion region. In some cases, the method includes recovering components, such as cells, compounds, or combinations thereof, from the combined individual entities. If the combined individual entities include one or more cells, the one or more cells can be analyzed, for example, genetic information therein can be sequenced using a sequencer. Genetic information can include, for example, DNA and RNA. In some cases, sequencing can include PCR. In some cases, analyzing the individual entities can include mass spectrometry. In some cases, the method includes printing the combined individual entities onto a substrate, for example, as described in U.S. Patent No. 2018 / 0056288, which is incorporated herein by reference for its disclosure of printing individual entities onto a substrate.

[0054] The present disclosure also provides a method for selectively performing a reaction by combining two or more individual entities, as described above, where the reaction occurs between one or more components from each individual entity. The components can be one or more cells, one or more products derived from cells, one or more reagents, or a combination thereof. In some cases, the one or more products derived from cells include cell lysate, DNA, RNA, or a combination thereof. As an example, FIG. 4 illustrates the combination of four individual entities, three of which each contain a different reagent, and the fourth individual entity contains a single cell. Thus, FIG. 4 illustrates that the microfluidic devices described herein can be used to selectively combine different individual entities, resulting in the formation of a combined individual entity containing, for example, three reagents and a cell.

[0055] Thus, the method for selectively performing a reaction can include a combination of two or more individual entities, for example, three or more and four or more entities. In some cases, the number of individual entities that include at least one cell is zero individual entities, one individual entity, two individual entities, or three or more individual entities. In some cases, the number of cells in the individual entities is one.

[0056] In some cases, the method includes repeating the selective combination of the individual entities, for example, performing the selective combination two or more times, three or more times, or four or more times.

[0057] The method allows for the selective combination of two or more individual entities without requiring precise timing of the release or sorting of the two or more individual entities. Thus, in some cases, a first individual entity is captured in the individual entity fusion region after the second individual entity with which it is to be combined has been sorted and before it enters the outlet flow path. In some cases, the second individual entity has not entered the sorting flow path, has not entered the inlet flow path, or has not even been created when the first individual entity is captured in the individual entity fusion region.

[0058] The method also allows for more efficient use of raw materials when constructing a desired combined individual entity. As an example, a desired combined individual entity may include one cancer cell and one immune cell. Thus, an individual entity creation device can be used to create a first group of individual entities, each containing a cancer cell. However, only about 10% of the first individual entities may contain cancer cells. Similarly, only about 10% of a second group of individual entities may contain immune cells. Thus, random combination of one first individual entity with one second individual entity will result in only about 1% of the combined individual entities having the desired cancer cell and immune cell.

[0059] In contrast, the present method allows for sorting of individual entities based on, for example, whether they contain cancer cells or immune cells, or neither cell type, and allows for the selective combination of only those individual entities containing the desired components. Thus, the present method can generate combined individual entities such that the fraction of combined individual entities containing cancer cells and immune cells is higher than 1%, i.e., the value expected based on random combination. If the desired combined entity contains three components, the expected value of random combination may be even lower than 1%.

[0060] As an example, Figure 8 shows the efficiency of loading unique bead and / or cell combinations into microfluidic droplets. Random combinations are calculated from Poisson statistics with an average occupancy of 10% for each unique object. Deterministic combinations are calculated from a 98% combinatorial efficiency from published results of similar techniques.

[0061] Thus, in some cases, the fraction of combined individual entities having the desired content is 1% or more, e.g., 2% or more, 5% or more, 10% or more, 25% or more, 50% or more, 75% or more, or 90% or more.

[0062] In some cases, the methods involve producing 5 or more combined individual entities per minute, including 10 or more, 25 or more, 50 or more, 75 or more, 100 or more, 150 or more, 200 or more, or 300 or more. In some cases, the methods involve producing 300 or more combined individual entities per hour, including 1,500 or more, 3,000 or more, 4,500 or more, 6,000 or more, 9,000 or more, 12,000 or more, or 21,000 or more.

[0063] In some cases, the sorting step is performed such that individual entities are sorted at a rate of 0.01 Hz or greater, e.g., 0.1 Hz or greater, 1 Hz or greater, 10 Hz or greater, 100 Hz or greater, 1 kHz or greater, 10 kHz or greater, or 30 kHz or greater. Sorting with a dielectrophoretic sorter as described herein can achieve sorting at rates of at least up to 30 kHz. In some cases, an electromagnetic sorter is used instead of a mechanical sorter, e.g., a valve, to enable faster sorting speeds.

[0064] In some cases, the capturing and combining steps are carried out to form or release combined individual entities at a rate of 0.1 Hz or greater, such as, for example, 1 Hz or greater, 10 Hz or greater, 100 Hz or greater, or 1,000 Hz or greater. The methods described herein can achieve the formation of combined individual entities at a rate of at least up to 1,000 Hz. In Example 10 discussed below, 17,500 droplets were assembled in 8.5 minutes, resulting in a rate of approximately 3.4 Hz. In some cases, the methods can continuously assemble 1,000 or more droplets without stopping, such as 10,000 or more or 100,000 or more.

[0065] In some cases, the individual entities are flowed in such a way that they reach the individual entity fusion region between 0.1 ms and 1,000 ms after sorting, e.g., between 1 ms and 100 ms, between 2 ms and 50 ms, and between 5 ms and 25 ms. In some cases, the length of the first outlet channel is between 0.2 mm and 5 mm. In some cases, the first outlet channel has dimensions, i.e., width, height, or diameter, between 5 μm and 500 μm, e.g., between 10 μm and 100 μm.

[0066] In some cases, the carrier fluid containing the individual entities is flowed into the inlet channel at a rate between 1 μl and 10,000 μl per hour, e.g., between 10 μl and 1,000 μl per hour, between 25 μl and 500 μl per hour, and between 50 μl and 250 μl per hour.

[0067] In some cases, the spacer fluid is infused at a rate between 100 μl and 20,000 μl per hour, e.g., between 500 μl and 5,000 μl per hour. In some cases, the bias fluid is infused at a rate between 100 μl and 20,000 μl per hour, e.g., between 500 μl and 5,000 μl per hour.

[0068] In some cases, the fluid used to create the cell-containing individual entities has a concentration between 1,000 cells and 10,000,000 cells per mL, e.g., between 10,000 cells and 1,000,000 cells per mL, and between 50,000 cells and 200,000 cells per mL.

[0069] In some cases, the individual entities have a volume between 1 pl and 10,000 pl, such as between 10 pl and 1,000 pl, or between 50 pl and 500 pl.

[0070] In some cases, one or more cells from the combined individual entities are cultured for at least 30 minutes or more, e.g., 1 hour or more, 6 hours or more, 12 hours or more, 24 hours or more, 3 days or more, or 7 days or more.

[0071] In some cases, the device can operate continuously by selectively combining individual entities for 10 minutes or more, e.g., 30 minutes or more, 45 minutes or more, 90 minutes or more, or 180 minutes or more. In some cases, the device can produce at least 100 combined individual entities, e.g., 1,000 or more combined individual entities, 10,000 or more combined individual entities, or 100,000 or more combined individual entities while operating continuously.

[0072] Creating Individual Entities As outlined above, in some cases, the method includes fabricating one or more individual entities, e.g., with an individual entity fabrication device. In such cases, the individual entity fabrication device can be part of a microfluidic device or separate from the microfluidic device, as described elsewhere herein. When the individual entity fabrication device is separate from the microfluidic device, the individual entity fabrication device can be operably connected to the microfluidic device, e.g., such that the individual entities can flow from the fabrication device to the microfluidic device, or the individual entity fabrication device and the microfluidic device can be operably connected to move the individual entities to the microfluidic device.

[0073] The systems and devices can include one or more individual entity creating devices configured to form individual entities from the fluid stream. Suitable individual entity creating devices include selectively activatable droplet creating devices, and the method can include forming one or more individual entities via selective activation of the droplet creating devices. The method can also include forming the individual entities using the droplet creating devices, the individual entities including one or more entities that differ in composition.

[0074] In some cases, the individual entity creating device contains a T-junction and the method includes T-junction drop making. In some cases, creating the individual entities includes step emulsification. In some cases, the individual entity creating device is partially or entirely made of a polymer. In some cases, one or more surfaces of the individual entity creating device are coated with fluorosilane, e.g., the individual entity creating device can be used when a fluorinated fluid is passed through the individual entity creating device.

[0075] In some cases, when multiple types of individual entities are created (e.g., individual entities with different contents), the contents may affect the ability of the individual entity creation device to successfully create the individual entities. Thus, in some cases, different conditions for the individual entity creation device are used to create a first group of individual entities having a first content than those used to create a second group of individual entities having a second content.

[0076] Embodiments of the disclosed methods may include creating individual entities using one or more cells from a biological sample. In such cases, each individual entity may include zero, one, or more cells. In some cases, the individual entities may be created by incorporating the biological sample, cells from the biological sample, a lysate from cells of the biological sample, or any other sample derived from the biological sample into a mixed emulsion. In some cases, the biological sample may be whole blood. In some cases, the method further includes separating one or more components of the biological sample or processing the biological sample, for example, via centrifugation, filtration, etc., prior to creating the individual entities.

[0077] In some cases, after creation of the individual entities, but before introducing the individual entities into the inlet channels of the microfluidic devices described herein, the individual entities may be further modified, for example, by adding cells, reagents, drugs, hydrogels, extracellular matrices, beads, particles, biological materials, media, or combinations thereof. In some cases, the reagents are primers, probes, lysing agents, surfactants, detergents, barcodes, or fluorescent tags. In some cases, the beads are RNA capture beads. In some cases, the beads are immunoassay beads. In some cases, the barcodes are oligonucleotides. In some cases, different types of individual entities are labeled with different types of barcodes, fluorescent tags, or combinations thereof.

[0078] Fluorescent tags can be used to image individual entities within an individual entity fusion region or combined individual entities. Fluorescent tags can also be used to identify the specific types of individual entities combined to create a given combined individual entity. Thus, the properties of a combined individual entity or its components can be correlated with the contents used to create the original individual entity. For example, different types of immune cells can be labeled with different fluorescent tags and incorporated into the individual entity. After combining the immune cell-containing individual entity with other individual entities, such as those containing cancer cells, the outcome of the combined individual entity can be observed, for example, whether the immune cells kill the cancer cells or whether cytokines are secreted. Additionally, fluorescent tags can be measured to correlate outcomes with immune cell types. In other cases, outcomes correlated with fluorescent tags are the results of sequencing, e.g., single-cell sequencing. Because some of all the original individual entities can be labeled with fluorescent tags, the resulting combined individual entity can have multiple fluorescent tags. In other cases, the combined individual entity has only one fluorescent tag.

[0079] Oligonucleotide barcodes can be used in a similar manner to fluorescent tags, however, instead of detecting optical fluorescence, the oligonucleotide barcodes can be sequenced to identify the original individual entities that formed the combined individual entity.

[0080] Methods and devices that can be used to encapsulate components from biological samples are described in PCT International Publication No. WO2014 / 028378, the disclosure of which is incorporated herein by reference in its entirety and for all purposes. Encapsulation approaches of interest include, but are not limited to, hydrodynamically induced drop formation and those described in Link, et al., Phys. Rev. Lett. 92, 054503 (2004), the disclosure of which is incorporated herein by reference. Other methods of encapsulating cells in droplets can also be applied. If desired, the cells may be stained with one or more antibodies and / or probes before encapsulation within the droplets.

[0081] One or more lysis agents may also be added to individual entities (e.g., droplets) containing cells under conditions that can rupture the cell(s), thereby releasing their genomes. The lysis agent may be added after the cells are encapsulated in the individual entities, e.g., microdrops. Any convenient lysis agent, such as proteinase K or a cytotoxin, may be used. In certain embodiments, cells may be co-encapsulated in droplets with a lysis buffer containing a detergent, such as Triton X100 and / or proteinase K. The specific conditions that can rupture the cell(s) will vary depending on the specific lysis agent used. For example, if proteinase K is incorporated as a lysis agent, the individual entities (e.g., droplets) may be heated to about 37-60°C for about 20 minutes to lyse the cells and allow the proteinase K to digest cellular proteins. They may then be heated to about 95°C for about 5-10 minutes to inactivate the proteinase K.

[0082] In certain embodiments, cell lysis may also, or alternatively, rely on techniques that do not involve the addition of a lysing agent. For example, lysis may be achieved by mechanical techniques that may use various geometric features to puncture, shear, polish, etc., cells. Other types of mechanical disruption, such as acoustic techniques, may also be used. Furthermore, thermal energy may be used to lyse cells. Any convenient method for cell lysis may be used in the methods described herein, as needed.

[0083] One or more primers can be introduced into an individual entity (e.g., a droplet) for each gene (e.g., an oncogene) to be detected. Thus, in certain embodiments, primers for all target genes (e.g., an oncogene) can be simultaneously present in an individual entity (e.g., a droplet), thereby providing a multiplexed assay. The individual entities (e.g., droplets) can be temperature cycled, for example, so that individual entities (e.g., droplets) containing cancer cells undergo PCR. During this time, only primers corresponding to genes (e.g., oncogenes) present in the genome induce amplification, creating many copies of these genes (e.g., oncogenes) in the individual entity (e.g., droplet). Detecting the presence of these PCR products can be achieved by various methods, such as using FRET, staining with an intercalating dye, or attaching them to beads. Further information regarding different options for such detection is also provided herein. The individual entities (e.g., droplets) can be optically probed, for example, using a laser to detect PCR products. Optically probing individual entities (e.g., droplets) may include counting the number of target cells (e.g., tumor cells) present in the initial population and / or allowing identification of targets (e.g., cancer genes) present in each cell (e.g., tumor cell).

[0084] Embodiments of the subject methods can be used to determine whether a biological sample contains particular cells of interest, e.g., tumor cells. In certain embodiments, the subject methods may include quantifying the number of cells of interest, e.g., tumor cells, present in the biological sample. Quantifying the number of cells of interest, e.g., tumor cells, present in the biological sample may be based, at least in part, on the number of distinct entities (e.g., droplets) in which PCR amplification products are detected. For example, the distinct entities (e.g., droplets) may be generated under conditions in which a majority of the distinct entities (e.g., droplets) are expected to contain zero or one cell. Techniques described more fully herein may be used to remove these distinct entities (e.g., droplets) that do not contain cells. After performing the PCR steps described above, the total number of distinct entities (e.g., droplets) detected to contain PCR products may be counted to quantify the number of cells of interest, e.g., tumor cells, in the biological sample. In certain embodiments, the methods may also include counting the total number of distinct entities, e.g., droplets, to determine the fraction or proportion of cells from the biological sample that are cells of interest, e.g., tumor cells.

[0085] Method embodiments may include modulating the environment of the individual entities, for example, by adding and / or removing contents of a droplet, thereby modulating the contents of the individual entities. The modulation may include modulating the temperature, pH, pressure, chemical composition, and / or radiation level of the environment of one or more individual entities. The modulation may also be the immediate environment of one or more individual entities, such as an emulsion in which the individual entities are provided, and / or one or more spaces, such as a conduit, channel, or vessel within a microfluidic device. The immediate environment of an individual entity that may be modulated may also include a fluid volume, such as a fluid stream in which the individual entities are provided. One or more individual entities may also be stored within the modulated environment.

[0086] The composition and properties of the discrete entities (e.g., microdroplets) prepared and / or utilized in connection with the disclosed methods can vary. For example, in some embodiments, a discrete entity can include one cell, and can include one or fewer cells. In other embodiments, a discrete entity can include multiple cells, i.e., two or more cells. In some aspects, a discrete entity according to the present disclosure can include a nucleic acid or multiple nucleic acids. In some cases, a discrete entity including a nucleic acid or multiple nucleic acids can be devoid of cells. In some embodiments, as described above, a discrete entity can include one or more solid and / or gel materials, such as one or more polymers.

[0087] In some embodiments, surfactants can be used to stabilize the individual entities (e.g., microdroplets). In some cases, the individual entities or the associated emulsion lack surfactants. Thus, the microdroplets may comprise surfactant-stabilized emulsions. Any convenient surfactant that allows the desired reaction to occur within the individual entities (e.g., microdroplets) may be used. In other aspects, the individual entities (e.g., microdroplets) are not stabilized by surfactants or particles.

[0088] The surfactant used depends on several factors, such as the oil and water phases (or other suitable immiscible phases, e.g., any suitable hydrophobic and hydrophilic phases) used in the emulsion. For example, when using aqueous droplets in fluorocarbon oil, the surfactant may have a hydrophilic block (PEG-PPO) and a hydrophobic fluorinated block (Krytox® FSH). However, if the oil is switched to a hydrocarbon oil, for example, the surfactant is instead selected to have a hydrophobic hydrocarbon block, such as the surfactant ABIL EM90. Desired properties that may be considered in selecting a 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 therefore does not swell the device; (3) biocompatibility; (4) the assay reagents are insoluble in the surfactant; (5) the surfactant exhibits good gas solubility, in that it allows gas in and out; (6) the surfactant has a boiling point higher than the temperatures used for PCR (e.g., 95 °C); (7) emulsion stability; (8) the surfactant stabilizes drops of the desired size; (9) the surfactant is soluble in the carrier phase but not in the droplet phase; (10) the surfactant has limited fluorescent properties; and (11) the surfactant remains soluble in the carrier phase over a range of temperatures.

[0089] Other surfactants, including ionic surfactants, are also contemplated. Other additives may also be included in the oil to stabilize the individual entities (e.g., droplets), including polymers that increase the stability of the individual entities (e.g., droplets) at temperatures above 35°C.

[0090] The discrete entities (e.g., microdroplets) described herein may be prepared as emulsions, e.g., aqueous phase fluids dispersed in immiscible phase carrier fluids (e.g., fluorocarbon or hydrocarbon oils), or vice versa. In some cases, the carrier fluid contains a fluorinated compound. In some cases, the carrier fluid is an aqueous fluid. For example, the hydrophilic or hydrophobic nature of the microfluidic channels (or coatings thereon) may be selected to match the type of emulsion utilized at a particular point in the microfluidic workflow.

[0091] Emulsions can be generated using microfluidic devices, as described in more detail below. Microfluidic devices can form emulsions composed of droplets with extremely uniform sizes. The microdroplet generation process can be achieved by pumping two immiscible fluids, such as oil and water, into a junction. The junction geometry, fluid properties (e.g., viscosity, interfacial tension), and flow rate affect the properties of the generated microdroplets. For a relatively wide range of properties, methods such as T-junctions and flow-focusing can be used to generate microdroplets of controlled, uniform size. To vary the microdroplet size, the flow rate of the immiscible liquid can be varied. This is because, for T-junctions and flow-focusing methods over a certain range of properties, the microdroplet size depends on the total flow rate and the ratio of the two fluid flow rates. To generate emulsions using microfluidic methods, the two fluids are typically loaded into two inlet reservoirs (syringes, pressure tubing) and then pressurized as needed to generate the desired flow rate (using a syringe pump, pressure regulator, gravity, etc.). This allows the fluid to be pumped through the device at a desired flow rate, producing microdroplets of a desired size and velocity.

[0092] In some cases, the cells of the individual entities may be labeled, for example, with a fluorescent label, a barcode, or a combination thereof.

[0093] In practicing the subject methods, several reagents may be added to, i.e., incorporated into, and / or encapsulated by, the discrete entities (e.g., microdroplets) in one or more steps (e.g., about 2, about 3, about 4, or about 5 or more steps). Such reagents may include, for example, amplification reagents such as polymerase chain reaction (PCR) reagents. The manner in which reagents are added to the discrete entities (e.g., microdroplets) may vary in several ways. Approaches of interest include, but are not limited to, those described by Ahn, et al., Appl. Phys. Lett. 88, 264105 (2006); Priest, et al., Appl. Phys. Lett. 89, 134101 (2006); Abate, et al., PNAS, November 9, 2010 vol. 107 no. 45 19163-19166; and Song, et al., Anal. Chem., 2006, 78(14), pp. 4839-4849, the disclosures of which are incorporated herein by reference.

[0094] For example, a reagent may be added to an individual entity (e.g., a microdroplet) by a method including fusing the individual entity (e.g., a microdroplet) with a second individual entity (e.g., a microdroplet) containing the reagent, for example, in an individual entity fusion region of a microfluidic device described herein. The reagent(s) contained in the second individual entity may be added by any convenient method, including those specifically described herein. This second individual entity may be fused with the first individual entity to create a combined individual entity (e.g., a microdroplet) containing the contents of both the first and second individual entities.

[0095] One or more reagents can also, or alternatively, be added using techniques such as droplet aggregation or pico-injection. In droplet aggregation, a target droplet (i.e., a microdroplet) may flow with a microdroplet containing a reagent(s) to be added to the microdroplet. The two microdroplets may flow so that they contact each other but not the other microdroplet. The droplets may then pass through electrodes or other means to apply an electric field, which can destabilize the microdroplets so that they fuse together.

[0096] Reagents may also, or alternatively, be added using pico-injection. In this approach, target droplets (i.e., microdroplets) can flow through a channel containing the reagent(s) to be added, where the reagent(s) are at high pressure. However, in the absence of an electric field, the microdroplets pass through without being injected because the presence of an electric field can prevent the microdroplets from entering due to the presence of surfactants that coat the microdroplets. However, when an electric field is applied as the microdroplets pass through the injector, fluid containing the reagent(s) is injected into the microdroplets. The amount of reagent added to the microdroplets may be controlled by several different parameters, such as adjusting the injection pressure and velocity of the flowing droplets, or by switching the electric field on and off.

[0097] In various embodiments, one or more reagents can also, or alternatively, be added to a microdroplet by fusing two droplets together or by methods that do not rely on injecting a liquid into the droplet. Rather, one or more reagents can be added to a microdroplet by a method that involves emulsifying the reagent into a stream of very small droplets and fusing these small droplets with a target microdroplet. Such methods are referred to herein as "reagent addition via coalescence of multiple droplets." These methods take advantage of the fact that, due to the small size of the added droplets compared to the target droplets, the small droplets flow faster than the target droplets and are collected behind the target droplets. The collection can then be fused, for example, by applying an electric field. Using this approach, or alternatively, multiple reagents can be added to a microdroplet by using several co-flowing streams of small droplets of different fluids. To enable effective fusion of microdroplets with target droplets, it is important that the microdroplets be smaller than the channel containing the target droplets and that the distance between the electrode applying the electric field and the channel into which the target droplets are injected be long enough to allow the microdroplets time to "follow" the target droplets. If the channel is too short, not all microdroplets will fuse with the target droplets, resulting in less reagent being added than desired. This can be compensated for to some extent by increasing the magnitude of the electric field, but this tends to allow droplets further away to fuse. In addition to generating microdroplets on the same microfluidic device, they can also be generated offline or homogenized using a separate microfluidic droplet generator before being injected into the device containing the target droplets.

[0098] Thus, in some embodiments, a reagent can be added to a microdroplet by a method that includes emulsifying the reagent into a droplet stream, where the droplets are smaller than the size of the microdroplet, flowing the droplets together with the microdroplet, and fusing the droplets with the microdroplet. The diameter of the droplets contained in the droplet stream can vary within a range of about 75% or less of the diameter of the microdroplet. For example, the diameter of the flowing droplets can be within a range of about 75% or less of the diameter of the microdroplet, about 50% or less of the diameter of the microdroplet, about 25% or less of the diameter of the microdroplet, about 15% or less of the diameter of the microdroplet, about 10% or less of the diameter of the microdroplet, about 5% or less of the diameter of the microdroplet, or about 2% or less of the diameter of the microdroplet. In certain embodiments, multiple flowing droplets, such as two or more droplets, three or more droplets, four or more droplets, or five or more droplets, can be fused with the microdroplet. Such merging can be achieved in a variety of ways, including, but not limited to, applying an electric field, where the electric field is effective to cause the fluidized droplets to merge with the microdroplets.

[0099] In another aspect, a reagent is added to an earlier-formed droplet (e.g., a microdroplet) by encapsulating the droplet to which the reagent is to be added (i.e., the "target droplet") within a droplet containing the reagent to be added (the "target reagent"). In certain embodiments, the method is carried out by first encapsulating the target droplet in a shell of a suitable hydrophobic phase, such as an oil, to form a double emulsion. The double emulsion is then encapsulated by droplets containing the target reagent to form a triple emulsion. To combine the target droplet with the droplet containing the target reagent, the double emulsion is then ruptured using any suitable method, including, but not limited to, applying an electric field, adding a chemical that destabilizes the droplet interface, flowing the triple emulsion through constrictions and other microfluidic geometries, applying mechanical agitation or ultrasound, increasing or decreasing the temperature, or encapsulating magnetic particles within the droplets that can rupture the double emulsion interface when attracted by a magnetic field.

[0100] In some cases, the discrete entities include beads. In some cases, at least one dimension (e.g., diameter) of the beads is between about 0.5 μm and about 500 μm. In some cases, the beads are made of a polymeric material (e.g., polystyrene). In some cases, the beads are magnetic or include a magnetic component. In some cases, the beads have biomolecules bound to their surfaces, such as antibodies, proteins, antigens, DNA, RNA, streptavidin, or combinations thereof. In some cases, the beads are immunoassay beads. In some cases, the beads are RNA capture beads.

[0101] Thus, the present disclosure provides a method for selectively combining a biomolecule with another compound or cell, the method comprising: selectively isolating a biomolecule from a composition using beads; creating a separate entity comprising the beads and the biomolecule; and selectively combining the separate entity containing the beads and the biomolecule with one or more separate entities containing one or more other compounds or cells using a microfluidic device described herein. Methods for selectively isolating biomolecules using beads are known in the art (e.g., U.S. Patent No. 2010 / 0009383), which is incorporated herein by reference for its disclosure of methods for separating biomolecules or cells using beads.

[0102] Individual entity selection In practicing the methods of the present disclosure, one or more sorting steps can be used. The sorting step sorts individual entities into one of two or more locations, for example, into one of two or more fluid flow paths. In some cases, the sorting is into one of two fluid flow paths.

[0103] Individual entities are sorted based on one or more characteristics of the individual entities or components within the individual entities. Additionally, the sorting can be either passive or active. Active sorting involves detecting one or more characteristics of the individual entities or components within the individual entities and sorting based on the detected characteristics. Passive sorting involves sorting individual entities without actively detecting characteristics. Sorting approaches of interest include, but are not necessarily limited to, approaches involving the use of one or more sorting channels and one or more sorting elements.

[0104] Sorting approaches that can be utilized in connection with the disclosed methods, systems, and devices include those described herein, as well as those described in Agresti, et al. PNAS, vol. 107, No. 9, 4004-4009.

[0105] Active Sorting Structure For active sorting, the device includes one or more sorting elements and one or more detectors, each configured to detect one or more characteristics of individual entities or components within the individual entities, and each sorting element configured to sort the individual entities to one of two or more locations based on detection by the detection element. In some cases, the sorting element is positioned proximate to the sorting flow path, e.g., an electrode is positioned proximate to the sorting flow path. In some cases, the sorting element is positioned within the sorting flow path, e.g., a partial-height splitter within the sorting flow path. In some cases, the device includes a sorting element positioned within the sorting flow path and one or more, e.g., two, sorting elements positioned proximate to the sorting flow path.

[0106] Exemplary structures and methods for actively sorting individual entities are described in Cole et al., PNAS, 2017, 114, 33, 8728-8733, doi:10.1073 / pnas.1704020114; Clark et al., Lab Chip, 2018, 5, 18, 710-713, doi:10.1039 / C7LC01242J, and Sciambi et al., Lab on a Chip, 2015, 15, 47-51, doi:10.1039 / C4LC01194E, the disclosures of which are incorporated herein by reference for purposes of sorting elements.

[0107] In some cases, the sorting element includes an electrode configured to exert a dielectrophoretic force, an electrode configured to exert an electrophoretic force, an element configured to exert an acoustic force, a valve, or a combination thereof.

[0108] In some cases, the sorting element contains an electrode disposed proximate the sorting flow path, e.g., an electrode configured to exert a dielectrophoretic force on the individual entities, or an electrode configured to exert an electrophoretic force on the individual entities. In some cases, the electrode is configured to exert an electrophoretic force on the individual entities.

[0109] The dielectrophoretic force on the individual entities can be directed toward the electrode (i.e., attractive force), away from the electrode (i.e., repulsive force), or in any other direction. In some cases, the sorting electrode is a liquid electrode, e.g., a microfluidic channel containing a conductive material such as salt water, liquid metal, molten solder, or a conductive ink that is subsequently annealed. In some cases, the electrodes are micropatterned on a planar surface and the microfluidic device is bonded to the surface. In some cases, the electrodes are patterned on a substrate of the microfluidic device, e.g., a patterned indium tin oxide (ITO) glass slide. In some cases, the sorting element comprises a selectively actuable dipole sorting electrode. In some cases, the sorting element comprises two electrodes. In some cases, the sorting element comprises a selectively actuable bipolar droplet sorting electrode. In some cases, the electrodes are solid electrodes prepared from any suitable conductive material and may be utilized.

[0110] In some cases, the electrodes are connected to an AC power source at a frequency between about 0.1 kHz and about 100 kHz, e.g., between about 1 kHz and about 50 kHz. In some cases, the electrodes are connected to a voltage between about 10 V and about 10,000 V (e.g., about).

[0111] In some cases, the capture element includes two electrodes, e.g., two electrodes that exert a dielectrophoretic force. In some cases, the distance between the first and second capture electrodes is between about 25 μm and about 500 μm, e.g., between about 50 μm and about 200 μm, or between about 75 μm and about 150 μm.

[0112] In some cases, the distance between the electrode and the interior of the sorting channel is between about 1 μm and about 100 μm, for example, between about 5 μm and about 50 μm, or between about 10 μm and about 25 μm.

[0113] The distance between capture electrodes and the distance between the capture electrodes and the interior of the individual entity fusion region can be varied to improve capture. Placing the electrodes closer to the interior of the individual entity fusion region increases the electromagnetic force exerted on the individual entities. Bringing the electrodes closer together increases the strength of the electric field and therefore the electromagnetic force exerted on the individual entities. On the other hand, capture electrodes can be positioned farther from other electrodes in the individual entity fusion region to, for example, reduce the electromagnetic force to capture the individual entities with less force. The location of the sorting electrodes can be varied for similar reasons, or to provide a larger individual entity fusion region, for example, to combine or allow for a greater number or size of combined individual entities.

[0114] In some cases, the sorting element includes three or more sorting electrodes, e.g., four or more, five or more, ten or more, or twenty or more sorting electrodes. In such cases, the sorting electrodes may be configured to form one or more bipolar electrode pairs, e.g., two or more pairs, three or more pairs, or five or more pairs.

[0115] In certain embodiments, the liquid electrode is energized using a power supply or a high-voltage amplifier. In some embodiments, the liquid electrode flow channel includes an inlet port so that a conductive liquid can be added to the liquid electrode flow channel. The conductive liquid can be added to the liquid electrode flow channel, for example, by connecting a liquid-filled tube to the inlet port and applying pressure. In certain embodiments, the liquid electrode flow channel also includes an outlet port for expelling the conductive liquid from the flow channel.

[0116] In some cases, the sorting element includes two sorting electrodes. In some cases, the two sorting electrodes have substantially different shapes, for example, as shown in FIG. 2. In some cases, the two sorting electrodes generate electric field lines having substantially different shapes. In some cases, the shapes are such that the pair of electrodes provides a constant electric field gradient. Thus, individual entities can be exposed to the sorting force for a longer time and over a longer distance, thereby allowing for the use of lower voltages. In some cases, the electric field is directed radially inward.

[0117] In some cases, a portion of the first sort electrode is positioned at the center of the arc of a concentric or essentially concentric sort channel, and the second sort electrode is positioned on the side of the sort channel opposite the first sort electrode, for example, as shown in Figure 2. In some cases, the sort channel defines a concentric or nearly concentric channel, and a portion of the sort electrode is located at the center of the concentric or nearly concentric channel.

[0118] In some cases, the two sorting electrodes are positioned on the same side of the sorting channel, and the shortest distance between the two sorting electrodes is between about 20 μm and about 500 μm, e.g., between about 50 μm and about 200 μm, between about 75 μm and about 150 μm, between about 100 μm and about 150 μm, or between about 120 μm and about 140 μm.

[0119] In some cases, the shortest distance between the sorting electrode and the interior of the sorting channel is between about 5 μm and about 100 μm, e.g., between about 10 μm and about 50 μm, between about 20 μm and about 40 μm, between about 25 μm and about 35 μm, or between about 28 μm and about 32 μm.

[0120] In some cases, the sorting element includes an element configured to exert an acoustic force. In some cases, the acoustic force is created by acoustic streaming. In some cases, the acoustic force is generated by surface acoustic wave sorting. Various acoustic sorting methods known in the art can be used in the methods of the present invention, including those described below. Junru Wu, Acoustic Streaming and its Applications, Fluids, 2018, 3, 108, doi:10.3390 / fluids3040108; Schmid et al. Sorting dropsand cells with acoustics: acoustic microfluidic fluorescence-activated cell sorter, Lab on a Chip, 2014, 14, 3710, doi:10.1039 / c4lc00588k; Franke et al, Surface acoustic wave actuated cell sorting (SAWACS), Lab on a Chip, 2010, 6, 789-794, doi:10.1039 / B915522H. Each of these is incorporated by reference for its method of acoustic sorting.

[0121] In some cases, the sorting element comprises a valve. In some cases, the valve is disposed within the sorting channel. In some cases, the valve is a microfluidic valve, a membrane valve, a branching channel, a surface acoustic wave generator. In some cases, the sorting channel comprises a partial-height wall divider, a concentric or essentially concentric sorting channel, or a combination thereof. Various valves useful in the present method are known in the art, including those described in Abate et al., Microfluidic Sorting with high-speed single-layer membrane valves, Applied Physics Letters, 2010, 96, 203509, doi:10.1063 / 1.3431281, which is incorporated herein by reference for methods of valve sorting.

[0122] In some cases, valves can be used to at least partially block or allow certain paths of individual entities, thereby filtering out the individual entities.

[0123] In other cases, valves may be used to control the flow of fluids into a sorting channel, thus allowing for indirect control of the fluid dynamics within the sorting channel, thereby sorting individual entities.

[0124] Thus, if the sorting element includes a valve, the sorting step includes mechanical movement of a portion of the device. However, in other cases, sorting is performed by non-mechanical means, such as dielectrophoretic sorting, as described elsewhere herein. Thus, in some cases, the sorting step does not include mechanical movement of a portion of the device. In some cases, the entire method is devoid of movement of a portion of the device. That is, individual entities are sorted, captured, bound, and released without movement of a portion of the device.

[0125] In some cases, the sorting flow path includes a partial height shunt, e.g., as described in Sciambi et al., Lab Chip, 2015, 15, 47-51, doi:10.1039 / C4LC01194E, or includes a concentric or essentially concentric region, e.g., as described in Clark et al., Lab on a Chip, 2018, 5, 18, 710-731, doi:10.1039 / C7LC01242J, where a portion of the sorting electrode is positioned at the center of an arc of the concentric or essentially concentric region.

[0126] In some embodiments, the present disclosure provides microfluidic devices with improved sorting architectures that facilitate high-speed sorting of individual entities (e.g., microdroplets). This sorting architecture may be used in conjunction with other embodiments described herein or in any other suitable application where high-speed sorting of microdroplets is desired. Related methods and systems are also described. For example, in some embodiments, a microfluidic device may include a sorting channel, a first outlet channel in fluid communication with the sorting channel, a second outlet channel in fluid communication with the sorting channel, and a dividing wall separating the first and second outlet channels, wherein the dividing wall contains a first proximal portion having a height less than that of the inlet channel and a second distal portion having a height equal to or greater than that of the inlet channel.

[0127] In some embodiments, the height of the first proximal portion of the dividing wall is about 10% to about 90% of the height of the inlet channel, e.g., about 20% to about 80%, about 30% to about 70%, about 40% to about 60%, or about 50% of the height of the inlet channel.

[0128] In some embodiments, the height of the first proximal portion of the dividing wall is about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, or about 80% to about 90% of the height of the inlet channel.

[0129] In some embodiments, the length of the proximal portion of the dividing wall is equal to or greater than the diameter of a microdroplet described herein, e.g., a microdroplet to be sorted using a microfluidic device described herein. For example, in some embodiments, the length of the proximal portion of the dividing wall is about 1 to about 100 times the diameter of a microdroplet described herein, e.g., about 1 to about 10 times, about 10 to about 20 times, about 20 to about 30 times, about 30 to about 40 times, about 40 to about 50 times, about 50 to about 60 times, about 60 to about 70 times, about 70 to about 80 times, about 80 to about 90 times, or about 90 to about 100 times the diameter of a microdroplet described herein.

[0130] Active Selection Detection As described above, active sorting involves one or more sorting elements that sort individual entities based on detection of one or more characteristics of the individual entities or components therein by one or more detectors. Characteristics of interest include, but are not limited to, optical properties, size, viscosity, mass, buoyancy, surface tension, conductivity, charge, magnetic force, and type. In some cases, the characteristics are characteristics of the individual entities. In some cases, the characteristics are characteristics of the individual entities, such as particles, cells, fluorescent tags on cells, and barcoded components on cells. Sorting can be based on the presence, absence, or type of components detected within the individual entities. In some cases, sorting is based on whether the cells are normal or cancerous. In some cases, the individual entities are detected while the individual entities are in the inlet flow path.

[0131] In some cases, the optical property is fluorescence. Thus, in some cases, the detector includes an excitation light source and a fluorescence detector. In some cases, the excitation light includes visible light, ultraviolet light, or a combination thereof. In some cases, the detector is an optical scanner. In some cases, the detector includes an optical fiber for directing the excitation light to the individual entities to direct the fluorescence light to the fluorescence detector, or a combination thereof. In some cases, a suitable optical scanner utilizes a laser light source directed toward the back of the object and focused onto a microfluidic channel (e.g., an inlet channel) through which the droplets flow, for example, to excite fluorescent dyes within one or more individual entities. Scanning one or more individual entities can enable one or more properties of the scanned entities, such as size, shape, or composition, to be determined.

[0132] A variety of different components can be included in individual entities to facilitate detection, including one or more fluorescent dyes, which may be categorized 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, etc. 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, and Rhodamine. Green, BODIPY, fluorescein isothiocyanate (FITC), carboxyfluorescein (FAM), phycoerythrin, rhodamine, dichlorodamine (dRhodamine), carboxytetramethylrhodamine (TAMRA), carboxy-X-rhodamine (ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen, RiboGreen, etc.Descriptions of fluorophores and their uses can be found, inter alia, in 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, NJ; 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.

[0133] Thus, in practicing the subject method, the component can be detected, for example, based on a change in fluorescence. In certain embodiments, the change in fluorescence is due to fluorescence resonance energy transfer (FRET). This approach may use a special primer set in which the 5' primer has a quencher dye and the 3' primer has a fluorescent dye. These dyes can be located anywhere on the primers, either at the ends or in the middle. Because the primers are complementary, they exist as double strands in solution. Because they are close to each other, the emission of the fluorescent dye is quenched by the quencher dye, making the solution appear dark. After PCR, these primers become farther apart as they are incorporated into long PCR products. This causes the fluorescent dye to emit light, making the solution fluorescent. Therefore, the intensity of individual entities (e.g., droplets) at the wavelength of the fluorescent dye can be measured to detect the presence of a specific target gene (e.g., an oncogene). To detect the presence of different target genes (e.g., an oncogene), this is done with different colored dyes for different primers. This causes the individual entities (e.g., droplets) to fluoresce at all wavelengths corresponding to the primers for the target gene (e.g., an oncogene) present in the cell.

[0134] In some embodiments, the disclosed methods may include encapsulating or incorporating unique identifier molecules, e.g., nucleic acid barcodes, into a plurality of individual entities (e.g., droplets) such that each individual entity of the plurality of individual entities contains a different set of unique identifier molecules. Alternatively or additionally, the disclosed methods may include incorporating a unique identifier molecule into each molecule within a particular individual entity (e.g., droplet).

[0135] In various aspects of the subject methods, multiple biomarkers can be detected and analyzed for a particular individual entity or one or more components thereof, such as a cell(s) encapsulated therein. The detected biomarkers may include, but are not limited to, one or more proteins, transcripts, and / or gene signatures in the cell's genome or a combination thereof. With standard fluorescence-based detection, the number of biomarkers that can be simultaneously interrogated may be limited to the number of fluorescent dyes that can be independently visualized within each individual entity (e.g., microdroplet). In certain embodiments, the number of biomarkers that can be individually detected within a particular individual entity (e.g., microdroplet) can be increased. For example, this may be achieved by separation of dyes into different portions of the individual entity (e.g., microdroplet). In certain embodiments, beads (e.g., LUMINEX® beads) conjugated with dyes and probes (e.g., nucleic acid or antibody probes) may be encapsulated within the individual entity (e.g., microdroplet) to increase the number of biomarkers analyzed. In another embodiment, fluorescence polarization may be used to achieve more detectable signals for different biomarkers for a single cell. For example, fluorescent dyes may be attached to various probes, and the individual entities (e.g., microdroplets) may be visualized under different polarization conditions. In this way, the same colored dye can be utilized to provide signals for different probe targets in a single cell. The use of fixed and / or permeabilized cells may also allow for increased levels of multiplexing. For example, labeled antibodies may be used to target protein targets localized to cellular components, while labeled PCR and / or RT-PCR products are released into individual entities (e.g., microdroplets). This allows the same colored dye to be used for the antibody and the amplicon produced by RT-PCR.

[0136] Passive Screening Passive sorters of interest include hydrodynamic sorters, which sort individual entities (e.g., microdroplets) into different channels according to size based on the different ways in which large and small droplets move through microfluidic channels. Also of interest are bulk sorters, a simple example of which is a tube containing droplets of different masses in a gravitational field. By centrifuging, stirring, and / or vibrating the tube, lighter, buoyant droplets spontaneously move to the top of the container. Droplets with magnetic properties can be sorted using a similar process, but by applying a magnetic field to the container, the droplets spontaneously move according to the magnitude of their properties. Passive sorters used in the subject methods can also include relatively large channels that simultaneously sort a large number of droplets based on their flow characteristics. Furthermore, in some embodiments, sorting is performed via activation of one or more valves, e.g., microfluidic valves.

[0137] Pico-injection can also be used to modify the electrical properties of droplets. This can be used, for example, to change the conductivity of the droplets by adding ions, which can then be used to sort them using, for example, dielectrophoresis. Alternatively, pico-injection can be used to charge droplets. This can be achieved by injecting a fluid into charged droplets so that the droplets are charged after injection. This creates a collection of droplets, some of which are charged and others are uncharged. The charged drops are extracted by flowing them through an electric field region, which allows them to be deflected based on their charge amount. By modulating the pico-injection to inject different amounts of liquid, or by modulating the voltage to inject different charges for a fixed injection volume, the final charge on the droplets can be adjusted to create droplets with different charges. These can then be deflected by different amounts in the electric field region, allowing them to be sorted into different containers.

[0138] Enrichment by selection A population (e.g., a population of individual entities) can be enriched by sorting, in that a population containing a mixture of members that may or may not have a desired property can be enriched by removing members that do not have the desired property, thereby producing an enriched population that has the desired property.

[0139] In some cases, more than one sorting step may be applied to individual entities or populations of types thereof (e.g., microdroplets), e.g., about two or more sorting steps, about three or more, about four or more, or about five or more, etc. When multiple sorting steps are applied, the steps may be substantially identical or different in one or more ways (e.g., sorting based on different characteristics, sorting using different techniques, etc.).

[0140] In some cases, droplets can be purified, for example, as follows: Most of the fluid in the droplet is replaced with a purified solution without removing any individual reagents, such as cells or beads, that may be encapsulated in the droplet. The microdroplets are first injected with a solution to dilute impurities therein. The diluted microdroplets are then flowed through a microfluidic channel to which an electric field is applied using electrodes. The dielectric forming forces generated by the electric field cause cells or other individual reagents to be displaced within the flow as they pass through the field. The droplets are then split so that all objects are in a single microdroplet. Thus, the initial microdroplets are purified in that contaminants may be removed while maintaining the presence and / or concentration of any individual reagents, such as beads or cells, that may be encapsulated within the resulting microdroplets.

[0141] Sorting may be used, for example, to remove individual entities (e.g., microdroplets) that do not contain cells. Encapsulation may result in one or more individual entities (e.g., microdroplets) that contain a large proportion of individual entities (e.g., microdroplets) that do not contain cells. If such empty droplets are left in the system, they will be processed as other droplets, wasting reagents and time. To achieve maximum speed and efficiency, these empty droplets can be removed by droplet sorting. For example, a droplet generator may operate close to the drip-to-jet transition in the absence of cells, such that droplets of a first size, e.g., 8 μm, are formed. In contrast, when cells are present, disturbances created in the flow cause the jet to break up, forming droplets of a second size, e.g., 25 μm in diameter. Thus, the device can generate a bidisperse population of single-cell-containing droplets of a first size (e.g., 8 μm empty droplets) and a bidisperse population of single-cell-containing droplets of a second size (e.g., 25 μm), which can then be sorted by size, e.g., using a hydrodynamic sorter, to recover only the single-cell-containing droplets of the second size (e.g., the larger size).

[0142] Recovery and / or recycling of individual entities In some cases, flow path entities sorted to a particular location (e.g., the second outlet flow path) are recovered and / or recycled, e.g., by reinjection into the carrier fluid upstream of the sorting flow path. Various embodiments of the methods disclosed herein include repeated recycling of individual entities not selected for direction to the first outlet flow path in a particular pass through the sorting flow path. In accordance with subject embodiments, sorting is described in further detail below. Also, in various embodiments, one or more individual entities, e.g., all individual entities present in the mixed emulsion, remain, e.g., encapsulated in a carrier fluid, e.g., a hydrophobic solution (e.g., oil) or a hydrophilic solution (e.g., aqueous solution), prior to and / or throughout the sorting process performed by the sorter and / or throughout the process of directing one or more individual entities to the individual entity merging region of the first outlet flow path.

[0143] Sorter function In some embodiments, a microfluidic device according to the present disclosure includes an electrode, e.g., a liquid electrode, configured to selectively apply an electric field within an inlet channel of the microfluidic device upstream of the dividing wall to effect sorting of one or more microdroplets.

[0144] In some cases, the microfluidic device includes concentric or nearly concentric sorting channels, i.e., a portion of the sorter electrodes is located at the center of a concentric or nearly concentric arc of the sorting channel. Some examples of such sorting architectures are described in Clark et al., Lab Chip, 2018, 5, 18, 710-713, doi:10.1039 / C7LC01242J.

[0145] As described herein, microfluidic devices according to the present disclosure may include a molybdenum salt solution provided in the appropriate flow channels (to generate the electric field gradient used for dielectrophoretic deflection and to limit stray electric fields that may cause unintended droplet fusion).

[0146] Thus, as described in more detail in the Experimental Section, microfluidic devices are provided having gapped dividing walls that facilitate high-speed sorting. The gapped dividing walls of the present disclosure, in combination with one or more detectors described herein and one or more electrodes described herein, facilitate high-speed sorting of microdroplets.

[0147] Capturing and Combining Individual Entities As noted above, after the individual entities have been sorted, the methods described herein can include directing the individual entities to an individual entity fusion region. Accordingly, the devices described herein can include an individual entity fusion region and a capture element positioned proximate the individual entity fusion region.

[0148] The capture element can capture the multiple individual entities within the individual entity fusion region for a time sufficient to combine the multiple individual entities to form a combined individual entity by exerting an electromagnetic force, exerting a mechanical force, applying heat, applying light, exerting an electrical force, providing a reagent, or a combination thereof. In some cases, the electromagnetic force is a dielectrophoretic force. In some cases, the electromagnetic force is an electrophoretic force.

[0149] In some cases, the individual entity merging region includes a feature selected from a geometric change in the dimensions of the first outlet flow path, a flow obstacle, a flow divider, a stacked fluid inlet, a valve, or a combination thereof. In some cases, the geometric change is a change in the cross-sectional area of ​​the first outlet flow path, e.g., the individual entity merging region has a larger cross-sectional area than the upstream region. In some cases, the geometric change is a change in one dimension of the first outlet flow path, e.g., the individual entity merging region is narrower than the downstream region. In some cases, the geometric change includes a recess in the flow path wall. In some cases, the recess includes a region that is not collinear with the fluid flow from the upstream region, as shown, for example, in item 107 of FIG. 2. In some cases, when a valve is utilized, the valve is configured to switch between at least two states. In some cases, in a first state, the valve obstructs the flow of the individual entities through the individual entity merging region while allowing the flow of carrier fluid through the individual entity merging region. In some cases, in a second state, the valve is configured to allow combined individual entities to flow unimpeded through the region of the individual entities. In some cases, the method includes placing the valve in a first state to allow the individual entities to be captured and combined into a combined individual entity, and then placing the valve in a second state to release the individual entities from the individual entity fusion region. In some cases, the valve is a membrane valve.

[0150] The stacking fluid inlet functions in a manner similar to certain embodiments of the spacer fluid inlet described above, i.e., the stacking fluid inlet is configured so that fluid flowing through it moves further away from the first side of the flow channel and closer to the second side of the flow channel. Stated another way, fluid flowing through the stacking fluid inlet comes into contact with fluid moving into the individual entity merging region from an upstream region of the first outlet flow channel, thereby influencing the flow of fluid coming from the upstream region. In some cases, the fluid is oil or otherwise a fluid that is immiscible with the fluid of the individual entities.

[0151] FIG. 2 illustrates an embodiment in which the individual entity fusion region includes a recess 107, a flow diverter 113, and a stacked fluid inlet 112. In FIG. 2, the stacked fluid provides a force that pushes the individual entities into the recess 107 and toward the capture electrode 109. Additionally, the flow diverter 113 in FIG. 2 further influences the interaction of the stacked fluid with fluid from the upstream region, thereby increasing the force that pushes the individual entities into the recess 107. Thus, an individual entity fusion region according to the present disclosure can include a stacked oil inlet and / or a flow diverter, one or more of which are configured such that oil flowing through the stacked oil inlet channel generates a force that pushes the individual entities in the individual entity fusion region toward the capture electrode, the recess, or a combination thereof. In some embodiments, the device can include a flow diverter without a stacked fluid inlet.

[0152] In some cases, the downstream region of the first outlet flow path is configured to assist in the capture of individual entities within the individual entity fusion region. In some cases, the downstream region has a larger cross-sectional area than the individual entity fusion region, which is an example of a geometric change in the first outlet flow path. In some cases, the downstream region has a triangular or approximately triangular shape. In some cases, the downstream region has a triangular or approximately triangular shape, with the individual entity fusion region located at or near the apex of the triangle. As an example, the system of FIG. 3 has downstream region 208 and individual entity fusion region 207.

[0153] In some cases, the longitudinal axis of the downstream region is parallel to the longitudinal axis of the separate entity fusion region, while in other cases, the longitudinal axes are not parallel. In some cases, the axes are parallel but not collinear. In some cases, the axes are parallel and collinear. In some cases, the angle between the axes is greater than 0°, e.g., 5° or more, 10° or more, 15° or more, 30° or more, 45° or more, 60° or more, 75° or more, 90° or more, 135° or more, or 175° or more. In some cases, the angle is between about 15° and about 135°. In some cases, the angle is between about 60° and about 120°, as shown, for example, in FIG. 3 .

[0154] In some embodiments, the capture element includes one or more electrodes, e.g., an electrode configured to exert a dielectrophoretic force on the individual entities. In some cases, the electrode is configured to exert an electrophoretic force. The dielectrophoretic force on the individual entities can be directed toward the electrode (i.e., attractive force), away from the electrode (i.e., repulsive force), or in any other direction. In some cases, the capture electrode is a liquid electrode, i.e., a microfluidic channel containing a conductive material, e.g., salt water, liquid metal, molten solder, or a conductive ink that is subsequently annealed. In some cases, the electrode is patterned on a substrate of the microfluidic device, e.g., a patterned indium tin oxide (ITO) glass slide. In some cases, the capture element includes a selectively actuable bipolar capture electrode. In some cases, the capture element includes two electrodes. In some cases, the capture element includes a selectively actuable bipolar droplet capture electrode. In some cases, the electrode is a solid electrode prepared from any suitable conductive material and may be utilized.

[0155] In some cases, the capture element includes three or more capture electrodes, such as four or more, five or more, ten or more, or twenty or more, In such cases, the capture electrodes may be configured to form two or more bipolar electrode pairs, e.g., three or more pairs, four or more pairs, five or more pairs, or ten or more pairs.

[0156] In some cases, the individual entity fusion region is configured to reduce shear forces experienced by one or more individual entities trapped within the individual entity fusion region, where the shear forces are caused by carrier fluid flowing through the individual entities, and where if the shear forces are strong enough, the one or more individual entities are unintentionally forced from the individual entity fusion region. Stated another way, the individual entity fusion region contains one or more features configured to reduce shear forces experienced by one or more individual entities trapped within the individual entity fusion region, where the feature can be a geometric change in the dimensions of the first outlet flow path, a recess, a change in cross-sectional area, a change in dimension, a flow obstruction, a flow divider, a stacked fluid inlet, a valve, or a combination thereof.

[0157] The individual entity fusion region and capture elements, e.g., one or two capture electrodes capable of exerting a dielectric force, are configured such that the force applied by the capture electrodes is sufficient to capture multiple individual entities in the individual entity fusion region for a time sufficient to combine the multiple individual entities in the individual entity fusion region to form a combined individual entity.

[0158] The time that a first individual entity is captured within the individual entity fusion region before contacting a second individual entity depends on factors including, but not limited to, the rate at which the individual entities are sorted and the fraction of the individual entities passing through the sorting flow path that contains the desired content for the second individual entity.

[0159] As an example, the captured first individual entity may contain a lysis reagent, and the desired second individual entity may contain a single cell. In such an example, the desired second individual entity may contain a single cell, for example, the cell may be lysed by the lysis reagent in the first individual entity upon formation of the combined individual entity. In such an example, the sorting element may sort the predicted second individual entities at a rate of approximately 1,000 Hz, with approximately 3% of the predicted second individual entities containing the desired single cell. Thus, on average, 1 / 30 seconds - 0.033 seconds - 33 ms elapses before the sorting element sorts the second individual entity containing the desired cell. Thus, in such a case, the first individual entity is captured within the individual entity fusion region for approximately 0.033 seconds before combining with the second individual entity.

[0160] In some cases, the sorting element sorts the individual entities at a rate of at least 10 Hz, e.g., at least 100 Hz, at least 500 Hz, at least 1,000 Hz, at least 2,000 Hz, or at least 10,000 Hz. In some cases, only 50% or less of the individual entities contain the desired content in a second individual entity, such as 25% or less, 10% or less, 5% or less, 1% or less, or 0.1% or less. In some cases, the individual entity fusion region and capture element are configured to capture a first individual entity in 0.1 ms or more, such as 1 ms or more, 5 ms or more, 10 ms or more, 25 ms or more, 50 ms or more, 100 ms or more, 500 ms or more, 1,000 ms or more, or 5,000 ms or more. In some cases, the first individual entity is captured within the individual entity fusion region for 0.1 ms or more, e.g., 1 ms or more, 10 ms or more, 100 ms or more, or 1,000 ms or more, before the second individual entity enters the region.

[0161] In some cases, the expected second individual entity includes two or more types of contents. As an example, the expected second individual entity may contain a lysis reagent (e.g., the first individual entity), a single cell (e.g., the desired second individual entity), and a sequencing reagent (e.g., the desired third individual entity). Thus, the time at which the first individual entity is captured within the individual entity fusion region also depends on whether different types of individual entities are being sorted.

[0162] In some cases, the capture electrodes are configured to provide an electric field that affects the surface of the individual entities so that the individual entities may more easily fuse, e.g., so that the individual entities spontaneously fuse. In some cases, the application of the electric field is sufficient to provide a destabilizing effect on the individual entities to facilitate fusion.

[0163] To facilitate the above operations, the present disclosure provides, in some embodiments, a substrate including individually controllable electrodes. The substrate can be configured so that individual electrodes can be selectively activated and deactivated, for example, by applying or removing a voltage or current to selected electrodes. In this manner, specific individual entities captured via a force applied by the electrodes can be selectively released. The electrodes of the array can be embedded in the substrate material (e.g., a suitable polymeric material), e.g., beneath the surface of the substrate to which the individual entities are attached via the application of force. A variety of suitable conductive materials, including various metals, are known in the art and can be utilized in connection with the disclosed electrode arrays. Liquid electrodes, such as those described previously herein, can also be used for such applications.

[0164] The methods and devices can include various numbers and configurations of electrodes. In some cases, the sorting element includes a zero electrode, e.g., the sorting element includes an acoustic sorter or a valve, and the capture element includes a single electrode. In some cases, the sorting element includes a zero electrode and the capture element includes two electrodes as a bipolar electrode pair, e.g., one of the capture electrodes is also a shielding electrode that can at least partially shield undesired electromagnetic fields. In some cases, the sorting element includes one electrode and the capture element includes a bipolar electrode pair. In some cases, the sorting element and the capture element each include a bipolar electrode pair, e.g., one electrode of each pair is also a shielding electrode. In some cases, the device includes any one of the numbers and types of sorting and capture electrodes, and the device further includes one or more additional shielding electrodes, e.g., one shielding electrode or two shielding electrodes. In some cases, the additional shielding electrode is located proximate the inlet flow channel, the individual entity creating device, or both. In some cases, the one or more shielding electrodes are connected to each other. In some cases, the one or more shielding electrodes are different portions of a single piece of metal.

[0165] In some cases, the electrodes are connected to an AC power source at a frequency of about 1 kHz or greater. In some cases, the electrodes are connected to a power source having a voltage of about 10 V to 10,000 V.

[0166] In some cases, the capture element includes two electrodes, e.g., two electrodes that exert a dielectrophoretic force. In some cases, the distance between the first and second capture electrodes is between about 20 μm and about 500 μm, e.g., between about 50 μm and about 200 μm, or between about 10 μm and about 50 μm.

[0167] In certain embodiments, the liquid electrode is energized using a power supply or a high-voltage amplifier. In some embodiments, the liquid electrode flow channel includes an inlet port so that a conductive liquid can be added to the liquid electrode flow channel. The conductive liquid can be added to the liquid electrode flow channel, for example, by connecting a liquid-filled tube to the inlet port and applying pressure. In certain embodiments, the liquid electrode flow channel also includes an outlet port for expelling the conductive liquid from the flow channel.

[0168] In some cases, the capture element includes a single electrode, i.e., a monopolar capture configuration. In some cases, the single electrode can have a high voltage, e.g., 1 kV at 10 kHz. Thus, energizing the electrode creates electric field lines that create an electric field gradient within the individual entity fusion region. This, in turn, provides non-uniform polarization of the individual entities within the microfluidic channel, facilitating capture and combination.

[0169] In some cases, the capture element includes two electrodes, i.e., a bipolar electrode pair. One electrode can have a positive voltage, while the other electrode acts as a ground, thereby creating electric field lines between the two electrodes. Thus, the second electrode, i.e., the ground electrode, can be considered to be configured to shape the electric field within the individual entity fusion region to facilitate capture and combination of the individual entities, relative to a monopolar configuration. In some cases, the second electrode is positioned on the same side of the flow path as the first electrode, while in other cases, the electrodes are positioned on opposite sides of the flow path.

[0170] Furthermore, the number of electrodes or bipolar electrode pairs is not limited to only one or two electrodes. In contrast, to facilitate capture and combination, many electrodes or electrode pairs may be present. In some cases, both bipolar electrode pairs and unpaired electrodes are present. In some cases, there are three or more capture electrodes, including four or more, five or more, ten or more, or twenty or more. In some cases, there are two or more pairs of bipolar electrode pairs, including three or more, four or more, or five or more.

[0171] In some cases, the shortest distance between the two capture electrodes is between about 20 μm and about 500 μm, e.g., between about 50 μm and about 200 μm, between about 75 μm and about 150 μm, between about 100 μm and about 150 μm, or between about 120 μm and about 140 μm. In some cases, the shortest distance between the capture electrode and the interior of the first outlet channel is between about 5 μm and about 100 μm, e.g., between about 10 μm and about 50 μm, between about 20 μm and about 40 μm, between about 25 μm and about 35 μm, or between about 28 μm and about 32 μm.

[0172] Analysis of combined individual entities The combined individual entities can be imaged before they are released from the individual entity capture region. In some cases, imaging includes capturing an image that shows fluorescence. In some cases, imaging involves capturing an image that does not include fluorescence. In some cases, imaging allows, for example, to ascertain the number of cells in the combined individual entities or otherwise measure the combined individual entities.

[0173] The combined individual entities can be released from the individual entity capture region, for example, by reducing or eliminating the force exerted by the capture element on the combined individual entities. In some cases, releasing the individual entities involves reducing or eliminating the dielectrophoretic force from one or more capture electrodes by decreasing power to the one or more capture electrodes. In some cases, the combined individual entities leave the individual entity fusion region within 0.1 ms to 100 ms, e.g., 0.2 ms to 10 ms, 0.5 ms to 5 ms, or 0.75 ms to 2.5 ms, of the change in capture force. In some cases, the individual entities leave the individual entity fusion region in 10 ms or less, such as 5 ms or less, 2 ms or less, or 1 ms or less.

[0174] After the combined individual entities are released from the individual entity fusion region, the method may include analyzing the combined individual entities or components therein.

[0175] In some cases, the analysis includes one or more of the following: single-cell functional assays, measurement of cell-cell communication, selective RNA-seq, 3D cell culture, small-scale 3D cell culture, potency assays, drug screening, screening of engineered cell libraries, neo-antigen screening, CRISPR screening, multi-step manipulation, and sorting-off function assays. In some cases, the analysis includes imaging the combined individual entities, for example, to detect fluorescent tags therein.

[0176] In some cases, the multi-step operation involves creating and releasing a combined individual entity containing multiple cells, incubating the cells for, e.g., 4 to 24 hours, and then returning the incubated cells to the device, where they can be combined with a lysis buffer and other reagents for RNA sequencing.

[0177] Selective execution of reactions by selectively combining individual entities The present disclosure also provides methods for selectively performing a reaction by combining two or more individual entities, as described above, where the reaction occurs between one or more components from each individual entity, which may be one or more cells, one or more products derived from cells, one or more reagents, or a combination thereof.

[0178] In some cases, a suitable method includes combining a cell and one or more reagents. As an example, FIG. 4 illustrates the combination of four individual entities, three of which each contain a different reagent, and the fourth individual entity contains a single cell. Thus, FIG. 4 illustrates that the microfluidic devices described herein can be used to selectively combine different individual entities, resulting in the formation of a combined individual entity containing, for example, three reagents and a cell. In some cases, the reagents can include cell lysis reagents, PCR reagents, reagents for analyzing DNA or RNA in cells, antibodies, or combinations thereof. In such cases, the method can further include collecting genomic data from the contents of the individual entities or the combined individual entities.

[0179] In some cases, the one or more products derived from the cell include cell lysate, DNA, RNA, or a combination thereof. Thus, the method can involve analysis of a product from the cell (e.g., cell lysate), even though the cell itself is included in any of the separate entities.

[0180] Thus, the method for selectively carrying out a reaction can include a combination of two or more individual entities, for example, three or more and four or more entities. In some cases, the number of individual entities that include at least one cell is zero, one, two, or three or more individual entities. In some cases, the number of cells in an individual entity is one. In some cases, none of the combined individual entities includes a cell.

[0181] Selectively combining individual entities each containing at least one cell The present disclosure provides methods for selectively combining two or more individual entities, each individual entity comprising one or more cells. In some cases, one or more cells in a first individual entity can have one or more differences, e.g., be of a different type than one or more cells in a second individual entity. Different types of cells can be distinguished from one another based on one or more characteristics, e.g., cancer cells versus non-cancerous cells, engineered cells versus unengineered cells, cells with different genomes, cells with different functions (e.g., blood cells versus fat cells), cells labeled with a fluorescent label versus unlabeled cells, live cells versus dead cells, etc.

[0182] As an example, a first individual entity may include one type of cell, and a second individual entity may include a second type of cell. Thus, using the methods and microfluidic devices of the present disclosure, the first and second types of cells can be combined into a single combined individual entity. In addition, the method may further include analyzing the interaction between the two cells. In some cases, the combined cells may include three or more (e.g., four or more) different cells.

[0183] In addition, the method allows for the selective combination of certain types of cells, while other types of cells are not included in the combined individual entities. As an example, first, second, and third types of cells can be included in first, second, and third individual entities. For example, based on the detection of each individual entity by a detector, two of the three cells can be selectively combined (e.g., second and third types of cells), while the remaining cells can be selectively excluded from the combined individual entity (e.g., first type of cells). Thus, the method allows for the selective combination of specific cells. In some cases, the method includes the selective combination of two or more cells and one or more reagents, along with the selective exclusion of one or more reagents or cells.

[0184] The method has applications in a variety of fields, including oncology, immunology, neurology, and other fields where it is desirable to selectively combine specific cells (e.g., while selectively excluding other cells). Thus, the method allows for, and optionally includes, the study of cell-cell interactions between selected cells. The present disclosure provides a method for selectively studying cell-cell interactions. In some cases, two or more combined cells are cells present in the nervous system, such as neurons.

[0185] The method can be used to study the interaction between cancer cells and immune cells. For example, the method can be used to screen a library of engineered T cells, such as chimeric antigen receptor T cells (CAR-T cells), for their effectiveness in fighting or killing cancer cells. In some cases, the method also involves evaluating the side effects or toxicity of the engineered cells on normal cells, such as non-cancerous cells. In some cases, determining efficacy, side effects, toxicity, or a combination thereof involves imaging the cells, obtaining genomic data on the cells, or a combination thereof. In some cases, the method includes studying the interaction between the engineered T cells and cancer cells in the presence of a chemotherapeutic composition, for example, as a combination therapy. In some cases, the chemotherapeutic composition includes a checkpoint inhibitor.

[0186] In some cases, the method involves forming a plurality of combined individual entities, each of which includes two or more cells, e.g., one engineered T cell and one cancer cell. In some cases, the number of each type of cell is equal, e.g., one immune cell and one cancer cell, or two immune cells and two cancer cells. In some cases, the multiple types of cells are combined in unequal numbers or ratios. As an example, one immune cell can be combined with 10 cancer cells, e.g., to test the ability of the immune cell to persistently kill multiple cancer cells. In some cases, the ratio of the first type of cell to the second type of cell is 1.1:1.0 or greater, e.g., 2:1 or greater, 5:1 or greater, 10:1 or greater, or 25:1 or greater. The number of cells can be 2:1, 2:1 or greater, 5:1 or greater, 10:1 or greater, or 25:1 or greater. In other cases, three or more types of cells are combined in unequal ratios or numbers. The ratio or number of each pair of cells may be those numbers and ratios listed above.

[0187] In some cases, the methods involve determining not only the type of cell within the individual entities, but also the number of cells of that type within the individual entities. Thus, even if some individual entities contain one cell and others contain two cells, the methods allow for the creation of combined individual entities having a particular number or ratio of cells (e.g., five cancer cells and one immune cell).

[0188] The present disclosure also provides a method for producing a three-dimensional cell culture (3D cell culture) using selectively selected cells. In some cases, the three-dimensional cell culture is an organoid. In some cases, the three-dimensional cell culture is a spheroid. Creating a three-dimensional cell culture allows cells to be studied in conditions more similar to physiological / in vivo conditions than two-dimensional cell cultures. In some cases, the method involves sorting and combining cells so that each cell in the culture is of the same type, or so that a substantial majority of the cells are of the same type (e.g., 90% or more, 95% or more, 98% or more, or 99% or more). Alternatively, the method may include sorting and combining cells so that the cell culture contains a substantial proportion of two or more cell types (e.g., the cell culture contains at least 10% or more of a first cell type and at least 10% or more of a second cell type). When the cell culture contains a substantial proportion of two or more cell types, the method may include combining individual entities, each of which contains each of the desired cell types. In some cases, methods for making three-dimensional cell cultures include printing combined individual entities onto a substrate, as described, for example, in U.S. Patent No. 2018 / 0056288, which is incorporated herein by reference for its disclosure of printing one or more individual entities onto a substrate.

[0189] Accordingly, the present disclosure provides a method for selectively sorting and combining individual entities, each of which includes at least one cell, analyzing one or more properties of the one or more cells, such as cell-cell interactions, and determining part or all of a genomic analysis of the one or more cells, which method may further include correlating the analyzed properties with genomic data.

[0190] In some cases, the number of individual entities combined is 10 or more, including 50 or more, 250 or more, 1,000 or more, 5,000 or more, or 10,000 or more. The number of cells combined may be the same or different from the number of individual entities combined. In some cases, the number of cells combined is 10 or more, including 50 or more, 250 or more, 1,000 or more, 5,000 or more, or 10,000 or more.

[0191] In some cases, the method involves selectively combining the individual entities such that the resulting combined individual entities include two or more cells, including three or more cells, four or more cells, five or more cells, six or more cells, seven or more cells, eight or more cells, nine or more cells, ten or more cells, or fifteen or more cells.

[0192] Microfluidic Devices and Systems As noted above, embodiments of the disclosed subject matter employ systems and / or devices, including microfluidic devices and systems. Devices of the subject disclosure include all of those described above in connection with the subject methods. Microfluidic devices and systems of the present disclosure can be characterized in various ways.

[0193] As mentioned above, a microfluidic device may include one or more flow paths, eg, flow paths through which individual entities may enter, exit, and / or pass.

[0194] In some cases, each channel in the described devices is a microchannel, i.e., the channel can have at least one cross-sectional dimension on the order of a millimeter or less (e.g., about 1 millimeter or less). In some cases, each channel in the described devices, e.g., the inlet channel, the sorting channel, the first outlet channel, the first spacer oil inlet, has at least one cross-sectional dimension on the order of about 500 μm or less, e.g., about 100 μm or less, about 50 μm or less, or about 10 μm or less. As mentioned above, the present disclosure provides systems including a microfluidic device and other elements separate from the microfluidic device, e.g., a temperature control module, an incubator, and a sequencer. Because the separate elements are part of the described systems but not part of the microfluidic device, the channels in the separate elements do not necessarily have at least one cross-sectional dimension on the order of a millimeter.

[0195] In some embodiments, systems and / or devices are provided that include one or more individual entity creating devices (e.g., droplet creating devices) configured to generate individual entities, e.g., droplets, and / or one or more flow paths, e.g., as described herein. In some embodiments, the one or more flow paths are operatively, e.g., fluidly connected to the one or more droplet creating devices and / or configured to receive one or more droplets therefrom. In some cases, the individual entity creating devices contain a T-junction.

[0196] As noted above, in certain embodiments, the flow channels are one or more "micro" flow channels. In view of the above, it should be understood that some of the principles and design features described herein can be scaled to larger devices and systems, including devices and systems employing flow channels that reach millimeter or even centimeter-scale flow cross sections. Thus, when certain devices and systems are described as "microfluidic," the description is intended to apply equally to certain larger-scale devices, in certain embodiments.

[0197] When referring to a microfluidic "device," it is generally intended to refer to a single entity in which one or more channels, reservoirs, stations, etc. share a continuous substrate (which may or may not be unitary). A microfluidic device embodiment includes the presence of one or more fluid flow paths (e.g., channels) having dimensions discussed herein. A microfluidic "system" may include one or more microfluidic devices and associated fluid connections, electrical connections, control / logic functions, etc.

[0198] The present disclosure also provides systems that include, for example, a microfluidic device as described above, and one or more additional components, such as (a) a temperature control module operably connected to the microfluidic device; (b) a detector configured to detect individual entities in an input flow path, wherein the microfluidic device is configured to sort individual entities in a sorting flow path based on detection by the detector; (c) an incubator operably connected to the microfluidic device or an individual entity fabrication apparatus; (d) a sequencer operably connected to the microfluidic device; (e) a device configured to fabricate a plurality of individual entities, i.e., an individual entity fabrication apparatus, disposed within the microfluidic device or disposed separately from the microfluidic device; and (f) one or more conveyors configured to transport particles (e.g., cells) or individual entities, wherein the individual entities can, in some cases, include particles between any combination of the incubator, the device configured to fabricate a plurality of individual entities, the microfluidic device, and the sequencer.

[0199] In various embodiments, the microfluidic devices of the present disclosure provide a continuous flow of a fluid medium. Fluids flowing through channels within a microfluidic device exhibit many unique properties. Typically, the dimensionless Reynolds number is extremely low, resulting in a flow that always remains laminar. Furthermore, in this regime, two fluids that combine do not readily mix, and diffusion alone may facilitate the mixing of the two compounds.

[0200] Additionally, the subject devices, in some embodiments, include one or more temperature and / or pressure control modules. Such modules may be capable of regulating the temperature and / or pressure of the carrier fluid in one or more flow paths of the device. More specifically, the temperature control modules may be one or more thermal cyclers. In some cases, the microfluidic device includes a salt solution to generate the electric field gradient used for dielectrophoretic deflection and to limit stray electric fields that may cause unintended droplet fusion.

[0201] In some cases, the device is configured to produce 5 or more combined individual entities per minute, including 10 or more, 25 or more, 50 or more, 75 or more, 100 or more, 150 or more, 200 or more, or 300 or more. In some cases, the device is configured to produce 300 or more combined individual entities per hour, including 1,500 or more, 3,000 or more, 4,500 or more, 6,000 or more, 9,000 or more, 12,000 or more, or 21,000 or more.

[0202] In some cases, the device is configured to selectively combine the individual entities such that the resulting combined individual entities include two or more cells, including three or more cells, four or more cells, five or more cells, six or more cells, seven or more cells, eight or more cells, nine or more cells, ten or more cells, or fifteen or more cells.

[0203] The present disclosure also provides an electrode system, for example, an electrode system including: individually controllable electrodes, each electrode may be positioned proximate a sorting channel or an individual entity fusion region of a microfluidic device; a power source; and a controller configured to selectively enable or disable an electrical connection between the power source and each individually controllable electrode in the array, thereby providing a respective inactive electrode, wherein each active electrode proximate the sorting channel can sort an individual entity to a first exit channel or a second exit channel, and wherein each active electrode proximate the individual entity fusion region can capture a plurality of individual entities in the individual entity fusion region for a time sufficient for the plurality of individual entities to combine to form a combined individual entity.

[0204] Incubation and sequencing In some cases, the method includes selectively combining two or more individual entities into a combined individual entity and releasing the combined individual entity, wherein the combined individual entity includes at least one cell. The method further includes incubating at least one combined individual entity including at least one cell. The method further includes, for example, sequencing one or more cells from the incubated cells after incubation, using a composition to create one or more individual entities, which are then passed through a microfluidic device and sequenced. This is performed by injecting the incubated individual entities into a microfluidic device and selectively binding them with individual entities containing a lysis buffer and sequencing reagents such as RNA capture beads.

[0205] Various features and examples of microfluidic device components suitable for use with the present disclosure will now be described.

[0206] manufacturing In some embodiments of the present disclosure, microfluidic devices are fabricated using microfabrication techniques, which may be used to fabricate integrated circuits (ICs), microelectromechanical systems (MEMS), display devices, etc. Among the types of microfabrication processes that may be used to generate small dimension patterns in microfluidic device fabrication are photolithography (including X-ray lithography, electron beam lithography, etc.), self-aligned deposition and etching techniques, anisotropic deposition and etching processes, self-assembled mask formation (e.g., forming a layer of hydrophobic-hydrophilic copolymers), etc.

[0207] According to disclosed embodiments, microfabrication processes vary depending on the type of material used for the substrate and / or the desired production volume. For low-volume production or prototypes, fabrication techniques include LIGA, powder blasting, laser ablation, mechanical machining, electrical discharge machining, photoforming, etc. For mass production of microfluidic devices, either lithography or master-based replication processes may be used. Lithography processes for fabricating substrates from silicon / glass include both wet and dry etching techniques commonly used in semiconductor device manufacturing. For mass production of plastic substrates, injection molding and hot embossing are typically used.

[0208] Surface Treatments and Coatings Surface modifications can be useful for controlling the functional mechanisms (e.g., flow control) of microfluidic devices and can be applied in accordance with the subject disclosure, for example, to prevent adsorption of fluidic species to channel walls or to attach antibodies to surfaces for detecting biological components.

[0209] In particular, polymer devices tend to be hydrophobic, making filling the channels difficult. The hydrophobic nature of polymer surfaces can also make it difficult to control electroosmotic flow rates (EOF). One technique for coating polymer surfaces according to the present disclosure is the application of polyelectrolyte multilayers (PEMs) to the channel surface. PEMs involve sequentially filling the channel with alternating solutions of positive and negative polyelectrolytes, which allows the multilayers to form electrostatic bonds. The layers typically do not bond to the channel surface but can completely cover the channel, even after long-term storage. Another technique for applying a hydrophilic layer on a polymer surface according to the present disclosure involves UV grafting of a polymer onto the channel surface. Initial grafting site radicals are created on the surface by exposing the surface to UV radiation while simultaneously exposing the device to a monomer solution. The monomers react to form covalently bonded polymers at the reaction sites. In some cases, for example, when fluorinated fluids are passed through the channel, the channel of a device or system is coated with fluorosilane.

[0210] In some embodiments, glass channels according to the subject disclosure generally have a high level of surface charge, which can cause proteins to adsorb and potentially interfere with the separation process. In some situations, the present disclosure includes applying a polydimethylsiloxane (PDMS) and / or surfactant coating to the glass channels. Other polymers that can be used to retard surface adsorption include polyacrylamide, glycol groups, polysiloxane, glyceroglycidoxypropyl, poly(ethylene glycol), and hydroxyethylated poly(ethyleneimine). Furthermore, the subject electroosmotic devices can include a coating with a charge of adjustable magnitude by manipulating the conditions (e.g., pH) inside the device. The coating can be either positively or negatively charged, so that the direction of flow can be selected based on the coating.

[0211] Special coatings according to the present disclosure can also be applied to immobilize specific species on the channel surface. This process is called "surface functionalization." For example, polymethyl methacrylate (PMMA) surfaces can be coated with amines to facilitate the attachment of various functional groups or targets. Alternatively, PMMA surfaces can be made hydrophilic through an oxygen plasma treatment process.

[0212] Microfluidic Elements Microfluidic systems and devices according to the subject disclosure can contain one or more fluid channels, such as microchannels, valves, pumps, reactors, mixers, and other / or components, some of which, and their general structures and dimensions, are described below.

[0213] Various types of valves can be applied for flow control in the microfluidic devices of the present disclosure. These include, but are not limited to, passive valves and check valves (membrane, flap, bivalve, leak, etc.). The flow rate through these valves depends on various physical characteristics of the valve, such as surface area, flow channel size, and valve material. Valves also have associated operational and manufacturing advantages / disadvantages that can be considered when designing a microfluidic device.

[0214] Embodiments of the subject devices include one or more micropumps. Micropumps, like other microfluidic components, are subject to manufacturing constraints. Typical considerations in pump design include handling air bubbles, clogging, and durability. Micropumps that can be included in the subject devices include, but are not limited to, electric charge pumps, fixed-stroke microdisplacement pumps, peristaltic micromembrane pumps, and / or pumps with integrated check valves.

[0215] Macrodevices rely on turbulent forces, such as rocking and stirring, to mix reagents. In comparison, such turbulent forces are not practically achievable in microdevices such as those disclosed herein; instead, mixing in microfluidic devices is generally achieved through diffusion. Because mixing by diffusion can be slow and inefficient, microstructures, such as those used with the disclosed subject matter, are often designed to enhance the mixing process. These structures manipulate fluids in a manner that increases the interfacial surface area between fluid domains, thereby speeding up diffusion. In certain embodiments, microfluidic mixers are used. Such mixers may be provided upstream from, or in some cases integrated with, the microfluidic separation devices and / or sorters of the present disclosure.

[0216] In some embodiments, the devices and systems of the present disclosure include micromixers. Micromixers can be divided into two general categories: active mixers and passive mixers. Active mixers operate by actively controlling flow regimes (e.g., changes in pressure gradients, electrical charges, etc.). Passive mixers do not require input energy; instead, they use only "hydrodynamics" (e.g., pressure) to drive fluid flow at a constant rate. One example of a passive mixer involves stacking two fluid streams separated by a plate on top of each other. When the separating plate is removed, the streams come into contact with each other. Stacking the two fluids increases the contact area and reduces the diffusion length, thereby enhancing the diffusion process. Mixing and reaction devices can be connected to a heat transfer system if thermal management is required. Similar to macroheat exchangers, microheat exchangers can have either cocurrent, countercurrent, or once-through flow schemes. Microfluidic devices can have channel widths and depths ranging from about 10 μm to about 10 cm. One channel structure includes a long main separation channel and three shorter "off-shoot" side channels that terminate in either buffer, sample, or waste reservoirs. The separation channel is several centimeters long, while the three side channels are typically several millimeters long. Of course, the actual length, cross-sectional area, shape, and branching design of a microfluidic device depend on the application and other design considerations, such as throughput (which depends on flow resistance), velocity profile, and residence time.

[0217] The microfluidic devices described herein may include one or more electric field generators for performing certain steps of the methods described herein, including, but not limited to, pico-injection, droplet coalescence, selective droplet fusion, and droplet sorting. In certain embodiments, the electric field is generated using metal electrodes. In certain embodiments, the electric field is generated using liquid electrodes. In certain embodiments, the liquid electrodes include liquid electrode channels filled with a conductive liquid (e.g., saline or buffer solution) and located at the location within the microfluidic device where the electric field is desired. In certain embodiments, the liquid electrodes are energized using a power supply or high-voltage amplifier. In some embodiments, the liquid electrode channels include an inlet port so that a conductive liquid can be added to the liquid electrode channel. The conductive liquid can be added to the liquid electrode channel by, for example, connecting a liquid-filled tube to the inlet port and applying pressure. In certain embodiments, the liquid electrode channels also include an outlet port for expelling the conductive liquid from the channel. In certain embodiments, liquid electrodes are used in the pico-injection, droplet coalescence, selective droplet fusion, and / or droplet sorting aspects of the microfluidic devices described herein. Liquid electrodes can be used, for example, when the material to be injected is not charged via the application of an electric field.

[0218] In certain embodiments, the width of one or more microchannels of the microfluidic device (e.g., input microchannel, mating microchannel, pyoinjection microchannel, and / or one or more upstream or downstream channels of these channels) is 100 microns or less, e.g., 90 microns or less, 80 microns or less, 70 microns or less, 60 microns or less, 50 microns or less, e.g., 45 microns or less, 40 microns or less, 39 microns or less, 38 microns or less, 37 microns or less, 36 microns or less, 35 microns or less, 34 microns or less, 33 microns or less, 32 microns or less, 31 microns or less, 30 microns or less, 29 microns or less, 28 microns or less, 27 microns or less, 26 microns or less, 25 microns or less, 20 microns or less, 15 microns or less, 10 microns or less. In some embodiments, the width of one or more of the microchannels is about 10 microns to about 15 microns, about 15 microns to about 20 microns, about 20 microns to about 25 microns, about 25 microns to about 30 microns, about 30 microns to about 35 microns, about 35 microns to about 40 microns, about 40 microns to about 45 microns, or about 45 microns to about 50 microns, about 50 microns to about 60 microns, about 60 microns to about 70 microns, about 70 microns to about 80 microns, about 80 microns to about 90 microns, or about 90 microns to about 100 microns.

[0219] Additional description of various microchannel structures and features that may be utilized in connection with the disclosed methods and devices is provided in PCT Publication No. WO 2014 / 028378, the disclosure of which is incorporated herein by reference in its entirety and for all purposes.

[0220] Glass, silicon and other "hard" materials (lithography, etching, deposition) According to embodiments of the disclosed subject matter, microcanals and microcavities can be fabricated from glass, silicon, and other "hard" materials using a combination of lithography, etching, and / or deposition techniques. Techniques based on the above can be applied to the fabrication of devices on the scale of 0.1 to 500 micrometers.

[0221] Microfabrication techniques based on semiconductor fabrication processes are generally performed in cleanrooms. Cleanroom quality is classified by the number of particles per cubic inch less than 4 μm in size. A typical cleanroom class for MEMS microfabrication can be 1000-10,000.

[0222] In certain embodiments, photolithography can be used for microfabrication. In photolithography, photoresist deposited on a substrate is exposed to a light source through an optical mask. Traditional photoresist methods allow for structure heights of up to 10-40 μm. If taller structures are required, thicker photoresists such as SU-8 or polyimide can be used, resulting in heights of up to 1 mm.

[0223] After transferring the pattern on the mask to the photoresist-coated substrate, the substrate is then etched using either a wet or dry process. In wet etching, the substrate, areas not protected by the mask, are subjected to chemical bombardment in the liquid phase. The liquid reagents used in the etching process depend on whether the etching is isotropic or anisotropic. In isotropic etching, acids are generally used to create three-dimensional structures such as spherical cavities in glass or silicon. Anisotropic etching uses highly basic solvents to create flat surfaces such as wells and canals. Wet anisotropic etching on silicon creates diagonal channel profiles.

[0224] Dry etching involves bombarding a substrate with ions in either the gas or plasma phase. Dry etching techniques can be used to create rectangular channel cross sections and arbitrary channel paths. Various types of dry etching can be used, including physical etching, chemical etching, physicochemical etching (e.g., RIE), and physicochemical etching with inhibitors. Physical etching uses ions accelerated through an electric field to bombard the surface of the substrate and "etch" the structure. Chemical etching can use an electric field to drive chemical species to the surface of the substrate. The chemical species then react with the substrate surface to create voids and volatile species.

[0225] In certain embodiments, deposition is used in microfabrication. Deposition techniques can be used to create layers of metals, insulators, semiconductors, polymers, proteins, and other organic materials. Many deposition techniques fall into one of two major categories: physical vapor deposition (PVD) and chemical vapor deposition (CVD). In one approach to PVD, a substrate target is contacted with a holding gas (which can be generated, for example, by evaporation). Certain species in the gas adsorb to the surface of the target, forming a layer that constitutes the deposit. In another approach commonly used in the microelectronics processing industry, a target containing the material to be deposited is sputtered using an argon ion beam or other appropriately energy source. The sputtered material is then deposited on the surface of a microfluidic device. In CVD, species in contact with the target react with the surface, forming chemically bonded components of the object. Other deposition techniques include spin coating, plasma spraying, plasma polymerization, dip coating, casting, and Langmuir-Blodgett film deposition. In plasma spraying, a fine powder containing particles up to 100 μm in diameter is suspended in a carrier gas. The particle-containing mixture is accelerated and heated through a plasma jet. The molten particles are propelled onto the substrate and freeze to form a dense coating. Plasma polymerization produces polymer films (e.g., PMMA) from plasma containing organic vapors.

[0226] Once microchannels, microcavities, and other features are etched into the glass or silicon substrate, the etched features are typically sealed to ensure the "watertight" nature of the microfluidic device. When sealing, adhesives can be applied to all surfaces that come into contact with each other. The sealing process can use fusion techniques such as those developed for glass-to-silicon, glass-to-glass, or silicon-to-silicon bonding.

[0227] Anodic bonding can be used to bond glass to silicon. A voltage is applied between the glass and silicon, and the temperature of the system is increased to induce sealing of the surfaces. The electric field and high temperature induce migration of sodium ions in the glass to the glass-silicon interface. The sodium ions in the glass-silicon interface are highly reactive with the silicon surface, forming a solid chemical bond between the surfaces. The type of glass used can have a thermal expansion coefficient close to that of silicon (e.g., Pyrex Corning 7740).

[0228] Fusion bonding can be used for glass or silicon sealing. The substrates are first forced together and aligned by applying a high contact force. Once in contact, atomic forces (primarily van der Waals forces) hold the substrates together, and they can then be placed in a furnace and annealed at high temperatures. Depending on the material, the temperatures used range from about 600 to 1100°C.

[0229] Polymers / Plastics A variety of techniques can be used to micromachine plastic substrates according to subject embodiments, including laser ablation, stereolithography, oxygen plasma etching, particle jet ablation, and microelectroerosion. Some of these techniques can also be used to shape other materials (glass, silicon, ceramics, etc.).

[0230] Replication techniques are used to generate multiple copies of microfluidic devices. The techniques involve first fabricating a master or mold insert containing the pattern to be replicated. The master is then used to mass-produce polymer substrates through a polymer replication process.

[0231] In the replication process, a master pattern contained in a mold is replicated onto a polymer structure. In one particular embodiment, a polymer and hardener mixture is poured into the mold at an elevated temperature. After the mixture cools, the polymer contains the pattern of the mold and is then removed from the mold. Alternatively, plastic can be injected into a structure containing a mold insert. In microinjection, plastic heated to a liquid state is injected into the mold. After cooling, the plastic retains the shape of the mold.

[0232] PDMS (polydimethylsiloxane), a silicon-based organic polymer, can be used in molding processes to form microfluidic structures. Due to its elastic properties, PDMS is suitable for microchannels of approximately 5 μm to 500 μm. Specific properties of PDMS that make it suitable for microfluidic purposes include: 1) It is optically transparent, allowing for flow visualization. 2) PDMS, when mixed with the appropriate amount of reticulating agent, has elastomeric properties that facilitate keeping microfluidic connections "watertight." 3) Membrane-based valves and pumps can be fabricated with PDMS due to its elasticity. 4) Untreated PDMS is hydrophobic and becomes temporarily hydrophilic after surface oxidation with oxygen plasma or immersion in strong base. The oxidized PDMS will adhere to glass, silicon, or polyethylene as long as those surfaces are exposed to oxygen plasma. 5) PDMS is permeable to gases. Because air bubbles are forcibly removed from the material, it is easy to fill the channels with liquid even when there are air bubbles in the canal. Furthermore, PDMS is permeable to non-polar organic solvents.

[0233] Microinjection can be used to form plastic substrates for a wide range of microfluidic designs. In this process, liquid plastic material is first injected into a mold under vacuum and pressure at a temperature above the glass transition temperature of the plastic. The plastic is then cooled below its glass transition temperature. After the mold is removed, the resulting plastic structure is a negative of the mold pattern.

[0234] Yet another replication technique is hot embossing, in which a polymer substrate and master are heated above the polymer's glass transition temperature, Tg (around 100-180 °C for PMMA or PC). The embossed master is then pressed against the substrate with a pre-set compression force. The system is then cooled below Tg, and the mold and substrate are then separated.

[0235] Typically, polymers experience the highest physical forces when separated from the mold tool, especially when the microstructure contains high aspect ratios and vertical walls. To avoid damaging the polymer microstructure, the material properties of the substrate and mold tool can be considered. These properties include sidewall roughness, sidewall angle, the chemical interface between the embossing master and the substrate, and the temperature coefficient. High sidewall roughness on the embossing tool can damage the polymer microstructure because the roughness contributes to frictional forces between the tool and the structure during the separation process. If the frictional forces are greater than the local tensile strength of the polymer, the microstructure may be destroyed. Friction between the tool and the substrate can be significant for microstructures with vertical walls. The chemical interface between the master and the substrate can also be problematic. Because the embossing process exposes the system to high temperatures, chemical bonds can form within the master-substrate interface. These interfacial bonds can impede the separation process. Differences in the thermal expansion coefficients of the tool and the substrate can result in additional frictional forces.

[0236] Various techniques can be used to form molds, embossed masters, and other masters containing the patterns used to replicate plastic structures through the replication process described above. Examples of such techniques include LIGA (described below), ablation techniques, and various other machining techniques. Similar techniques can also be used to create low-volume masks, prototypes, and microfluidic structures. Materials used for mold tools include metals, metal alloys, silicon, and other hard materials.

[0237] Laser ablation can be used to create microstructures on a substrate directly or through the use of a mask. This technique typically uses a precision-guided laser with a wavelength between the infrared and ultraviolet. Laser ablation can be performed on glass and metal substrates, as well as polymer substrates. Laser ablation can be performed either by moving the substrate surface relative to a fixed laser beam, or by moving the beam relative to a fixed substrate. A variety of microwells, canals, and high-aspect structures can be created by laser ablation.

[0238] Certain materials, such as stainless steel, can be micromachined to create durable mold inserts and create structures down to the 10 μm range. Various other micromachining techniques exist for microfabrication, including μ-electrical discharge machining (μ-EDM), μ-milling, and focused ion beam milling. μ-EDM allows for the fabrication of three-dimensional structures in conductive materials. In μ-EDM, material is removed by a high-frequency discharge generated between an electrode (positive tool) and a workpiece (negative tool). Both the workpiece and tool are immersed in a dielectric fluid. This technique produces a relatively rough surface, but allows for flexibility in terms of materials and geometry.

[0239] For example, electroplating can be used to create a replica mold tool / master from a nickel alloy. The process begins with a photolithography step, in which a photoresist is used to define the structure to be electroplated. The areas to be electroplated are free of resist. For structures with high aspect ratios and low roughness requirements, LIGA can be used to create the electroplated form. LIGA is an acronym for the German words Lithographic, Galvanoformung, and Abformung. In one approach to LIGA, a thick PMMA layer is exposed to X-rays from a synchrotron source. The surface created by LIGA has low roughness (approximately 10 nm RMS), and the resulting nickel tool has good surface chemistry for many polymers.

[0240] Similar to glass and silicon devices, polymer microfluidic devices must be sealed before they can function. Common problems in the bonding process for microfluidic devices include blocking of the flow channels and alteration of the physical parameters of the flow channels. Lamination is one method used to seal plastic microfluidic devices. In one lamination process, a PET foil (approximately 30 μm) coated with a molten adhesive layer (typically 5 μm–10 μm) is rolled onto the microstructure with a heated roller. Through this process, the lid foil is sealed onto the flow channel plate. Some research groups have reported bonding via interfacial polymerization, whereby the structure is heated and force is applied to the opposite side to seal the flow channels. However, excessive force can damage the microstructure. Both reversible and irreversible bonding techniques exist for plastic and plastic-glass interfaces. One method of reversible sealing involves first thoroughly rinsing the PDMS substrate and glass plate (or the second portion of PDMS) with methanol and then bringing the surfaces into contact with each other before drying. The microstructure is then dried in an oven at 65 °C for 10 minutes. This process does not require a clean room. The irreversible seal is achieved by first thoroughly rinsing the pieces with methanol and then drying them separately in a stream of nitrogen. The two pieces are then placed in an air plasma cleaner and oxidized at high power for approximately 45 seconds. The substrates are then brought into contact with each other, spontaneously forming an irreversible seal.

[0241] Other available techniques include laser welding and ultrasonic welding. In laser welding, polymers are bonded together through laser-generated heat. This method has been used in the manufacture of micropumps. Another joining technique that may be used in some applications is ultrasonic welding.

[0242] One of the nucleic acid amplification techniques described herein is polymerase chain reaction (PCR). However, in certain embodiments, non-PCR amplification techniques can be used, such as various isothermal nucleic acid amplification techniques (e.g., real-time strand displacement amplification (SDA), rolling circle amplification (RCA), and multiple displacement amplification (MDA)).

[0243] For a PCR amplification module, the module will need to be provided with at least the building blocks for amplifying nucleic acids (e.g., sufficient concentrations of four nucleotides), primers, polymerase (e.g., Taq), and an appropriate temperature control program. The polymerase and nucleotide building blocks can be provided to the amplification module via an external port or in a buffer provided from an upstream source. In certain embodiments, the buffer stream provided to the sorting module contains some of all the ingredients for nucleic acid amplification. Especially for PCR, precise temperature control of the reaction mixture is crucial to achieving high reaction efficiency. One method of on-chip thermal control is Joule heating, in which electrodes are used to heat fluids within the module at defined locations. Fluid conductivity can be used as temperature feedback for power control.

[0244] In certain embodiments, individual entities (e.g., microdroplets) containing a PCR mixture may be flowed through a flow path that incubates the individual entities under conditions effective for PCR. Flowing the individual entities through a flow path may include a meandering flow path through various temperature zones maintained at temperatures effective for PCR. The flow path may, for example, cycle through two or more temperature zones, with at least one zone maintained at about 65°C and at least one zone maintained at about 95°C. As the individual entities move through the zones, their temperatures are cycled as needed for PCR. The exact number of zones, and the respective temperatures of each zone, can be readily determined by one of skill in the art to achieve the desired PCR amplification.

[0245] Exemplary Non-Limiting Aspects of the Disclosure The above-described aspects (including embodiments) of the present subject matter may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain numbered, non-limiting aspects of the present disclosure are provided below. As will be apparent to one of ordinary skill in the art upon reading this disclosure, each individually numbered aspect can be used or combined with any of the preceding aspects or any of the aspects following the individually numbered aspect. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0246] Aspect 1. a) inlet channel; b) a sorting channel in fluid communication with the inlet channel; c) a first outlet channel and a second outlet channel in fluid communication with the sorting channel, the first outlet channel containing an individual entity merging region; d) a sorting element disposed proximate the sorting channel, the sorting element configured to sort individual entities in the sorting channel to a first outlet channel; and e) Capture elements placed in close proximity to the individual entity fusion area. Contains A microfluidic device, wherein the capture element and the individual entity fusion region are configured to capture a plurality of individual entities within the individual entity fusion region for a time sufficient to combine the plurality of individual entities to form a combined individual entity.

[0247] Aspect 2. 2. The microfluidic device of embodiment 1, wherein the sorting element comprises a sorting electrode that exerts an electromagnetic force sufficient to sort individual entities in the sorting channel to the first outlet channel.

[0248] Aspect 3. Aspect 3. The microfluidic device according to any one of aspects 1 to 2, wherein the electromagnetic force is a dielectrophoretic force.

[0249] Aspect 4. Aspect 3. The microfluidic device according to any one of aspects 1 to 2, wherein the electromagnetic force is an electrophoretic force.

[0250] Aspect 5. The microfluidic device of any one of embodiments 1 to 4, further comprising a second sorting electrode.

[0251] Aspect 6. 6. The microfluidic device of embodiment 5, further comprising a third sorting electrode.

[0252] Aspect 7. 7. The microfluidic device of any one of aspects 5-6, wherein the first and second sorting electrodes are configured such that the first and second sorting electrodes form a bipolar electrode pair and the first capture electrode is positively charged.

[0253] Aspect 8. 8. The microfluidic device of any one of embodiments 5 to 7, wherein the first and second sorting electrodes are positioned on opposite sides of the sorting channel.

[0254] Aspect 9. 9. The microfluidic device of any one of aspects 5 to 8, wherein the first sorting electrode is positioned closer to the sorting flow path than the second sorting electrode, or the second sorting electrode is positioned closer to the sorting flow path than the first sorting electrode.

[0255] Aspect 10. 10. The microfluidic device according to any one of aspects 5 to 9, wherein the distance between the end of the first sorting electrode, the second sorting electrode, or both, and the inner wall of the sorting channel is between about 1 μm and about 100 μm.

[0256] Aspect 11. 11. The microfluidic device according to any one of aspects 5 to 10, wherein the distance between the first sorting electrode and the second sorting electrode is about 25 μm to about 500 μm.

[0257] Aspect 12. 12. The microfluidic device according to any one of aspects 5 to 11, wherein the first sorting electrode and the second sorting electrode are connected to an AC power supply having a frequency of about 0.1 kHz to about 100 kHz and a voltage of about 10 V to about 10,000 V.

[0258] Aspect 13. 13. The microfluidic device of any one of embodiments 2 to 12, wherein each sorting electrode comprises a liquid electrode.

[0259] Aspect 14. 14. A microfluidic device according to embodiment 13, wherein each sorting liquid electrode is embedded in the microfluidic device and contains one or more liquid flow paths filled with a conductive medium.

[0260] Aspect 15. 2. The microfluidic device of embodiment 1, wherein the sorting element comprises a valve, a surface wave sorting element, an acoustic streaming element, or a combination thereof.

[0261] Aspect 16.

[0019] A microfluidic device according to any one of embodiments 1-14, wherein the capture element exerts an electromagnetic force, a mechanical force, or a combination thereof sufficient to capture the plurality of individual entities within the individual entity fusion region for a time sufficient to combine the plurality of individual entities to form a combined individual entity.

[0262] Aspect 17. 17. The microfluidic device of any one of aspects 1-16, wherein the capture element comprises a first capture electrode that exerts an electromagnetic force sufficient to capture the plurality of individual entities within the individual entity fusion region for a time sufficient to combine the plurality of individual entities to form a combined individual entity.

[0263] Aspect 18. 18. The microfluidic device of embodiment 17, wherein the electromagnetic force is a dielectrophoretic force.

[0264] Aspect 19. 18. The microfluidic device according to embodiment 17, wherein the electromagnetic force is an electrophoretic force.

[0265] Aspect 20. 20. The microfluidic device of any one of embodiments 17 to 19, further comprising a second capture electrode.

[0266] Aspect 21. 21. The microfluidic device according to embodiment 20, further comprising a third capture electrode.

[0267] Aspect 22. 22. The microfluidic device of any one of aspects 20-21, wherein the first and second sorting electrodes are configured such that the first and second sorting electrodes form a bipolar electrode pair and the first capture electrode is positively charged.

[0268] Aspect 23. 23. The microfluidic device of any one of embodiments 20 to 22, wherein the first and second sorting electrodes are disposed on the same side of the sorting channel.

[0269] Aspect 24. 24. The microfluidic device of any one of aspects 20 to 23, wherein the first capture electrode is positioned closer to the first outlet flow path than the second capture electrode, or the second capture electrode is positioned closer to the first outlet flow path than the first capture electrode.

[0270] Aspect 25. 25. The microfluidic device of any one of aspects 20 to 24, wherein the distance between the end of the first capture electrode, the second capture electrode, or both, and the inner wall of the first outlet channel is between about 10 μm and about 50 μm.

[0271] Aspect 26. 26. The microfluidic device according to any one of aspects 20 to 25, wherein the distance between the first capture electrode and the second capture electrode is about 25 μm to about 500 μm.

[0272] Aspect 27. 27. The microfluidic device of embodiment 26, wherein the distance is between about 50 μm and about 200 μm.

[0273] Aspect 28. 28. The microfluidic device of any one of aspects 20 to 27, wherein the first capture electrode and the second capture electrode are connected to an AC power supply having a frequency of about 0.1 kHz to about 100 kHz and a voltage of about 10 V to about 10,000 V.

[0274] Aspect 29. 29. The microfluidic device according to embodiment 28, wherein the frequency is from about 1 kHz to about 50 kHz.

[0275] Aspect 30. 30. The microfluidic device of any one of embodiments 17 to 29, wherein each capture electrode contains a liquid electrode.

[0276] Aspect 31. 31. A microfluidic device according to embodiment 30, wherein each trapped liquid electrode is embedded in the microfluidic device and contains one or more liquid flow channels filled with a conductive medium.

[0277] Aspect 32. A microfluidic device according to any one of aspects 20 to 31, wherein the first capture electrode extends along the first outlet flow path downstream of the individual entity merging region, or the second capture electrode extends along the first outlet flow path downstream of the individual entity merging region.

[0278] Aspect 33. Aspect 33. The microfluidic device of any one of aspects 1-32, wherein the sorting flow path defines a concentric or near-concentric flow path, and wherein a portion of the first sorting electrode is located at the center of the concentric or near-concentric flow path.

[0279] Aspect 34. 34. The microfluidic device of embodiment 33, wherein the first sorting electrode is positioned closer to the first outlet channel than to the second outlet channel.

[0280] Aspect 35.

[0039] Aspect 34. The microfluidic device of any one of aspects 1-33, wherein the microfluidic device further comprises a partial-height flow distributor disposed within the sorting channel, the partial-height flow distributor configured to direct individual entities toward the first outlet channel or the second outlet channel.

[0281] Aspect 36. 36. The microfluidic device of embodiment 35, wherein the height of the partial-height flow distributor is about 50% to 75% of the height of the sorting channel.

[0282] Aspect 37. 37. The microfluidic device of any one of aspects 1-36, wherein the separate entity fusion region contains a feature selected from a geometric change in a dimension of the first outlet channel, a flow obstacle, a flow divider, a stacked fluid inlet, a valve, or a combination thereof.

[0283] Aspect 38. Aspect 38. The microfluidic device of any one of aspects 1-37, wherein the separate entity fusion region comprises a geometric change in a dimension of the first outlet channel, wherein the geometric change comprises an increase in a cross-sectional area of ​​the first outlet channel.

[0284] Aspect 39. 39. The microfluidic device of any one of embodiments 1-38, wherein the separate entity merging region comprises a geometric variation, the geometric variation comprising a recess in a wall of the first outlet channel.

[0285] Aspect 40. A microfluidic device according to any one of aspects 1 to 39, wherein the individual entity fusion region contains a stacking fluid inlet channel, and wherein stacking fluid flowing through the stacking fluid inlet channel is configured to direct individual entities in the individual entity fusion region towards a capture electrode.

[0286] Aspect 41. A microfluidic device as described in embodiment 40, wherein the individual entity fusion region further contains a flow distributor, and the stacked fluid inlet channel and flow distributor are configured so that stacked fluid flowing through the stacked fluid inlet channel directs individual entities in the individual entity fusion region toward the capture electrode.

[0287] Aspect 42. the first inlet channel includes an upstream region located between the sorting channel and the individual entity merging region; and the change in cross-sectional area is such that the individual entity fusion region has a larger cross-sectional area than the upstream region; 42. The microfluidic device according to any one of embodiments 38 to 41.

[0288] Aspect 43.

[00111] A microfluidic device according to any one of embodiments 1-42, wherein the discrete entity fusion region has a triangular, approximately triangular, trapezoidal, or approximately trapezoidal shape defined by a channel wall of the microfluidic device.

[0289] Aspect 44. A microfluidic device according to any one of aspects 1 to 43, wherein the individual entity fusion region contains a valve, the valve being a membrane valve configured to hinder the flow of individual entities through the individual entity fusion region while allowing the flow of carrier fluid through the individual entity fusion region in a first state, and the membrane valve configured to release the individual entities or combined individual entities in a second state.

[0290] Aspect 45. 45. The microfluidic device of any one of embodiments 1-44, wherein the first outlet channel contains an angled turn in the channel wall downstream of the separate entity merging region.

[0291] Aspect 46. 46. ​​The microfluidic device of any one of aspects 1-45, further comprising a partial-height flow diverter disposed in the first outlet channel, the partial-height flow diverter configured to direct individual entities towards a capture electrode in the individual entity fusion region.

[0292] Aspect 47. 47. The microfluidic device of embodiment 46, wherein the height of the partial-height flow distributor is about 50%-75% of the height of the first outlet channel.

[0293] Aspect 48.

[00182] A microfluidic device according to any one of embodiments 1-47, comprising a spacer fluid channel in fluid communication with the inlet channel, the spacer fluid channel being configured such that flow of the spacer fluid through the spacer fluid channel positions the spacer fluid between two individual entities flowing through the inlet channel, thereby maintaining or increasing the distance between the two individual entities, thereby allowing each of the two individual entities to be sorted or not sorted independently.

[0294] Aspect 49. 49. The microfluidic device according to embodiment 48, wherein the spacer fluid is an oil.

[0295] Aspect 50. 50. The microfluidic device of any one of aspects 1-49, comprising a bias fluid channel in fluid communication with the sort channel, the bias fluid channel configured such that bias fluid flowing through the bias fluid channel moves individual entities closer to a second sidewall of the sort channel and away from a first sidewall of the sort channel.

[0296] Aspect 51. 51. The microfluidic device of embodiment 50, wherein the biasing fluid is oil.

[0297] Aspect 52. 52. The microfluidic device of any one of aspects 1-51, wherein the first outlet channel is configured to receive an individual entity positioned closer to a first sidewall of the sorting channel than to a second sidewall of the sorting channel, and the second outlet channel is configured to receive an individual entity positioned closer to a second sidewall of the sorting channel than to the first sidewall of the sorting channel.

[0298] Aspect 53.

[0082] Aspect 53. The microfluidic device of any one of aspects 1-52, wherein the device is configured such that when the sorting element does not exert a force on the individual entities flowing through the sorting channel, the individual entities flow into the second outlet channel.

[0299] Aspect 54. Aspect 54. The microfluidic device of any one of aspects 1-53, wherein the first outlet channel is configured such that individual entities flowing through the first outlet channel are directed to a first sidewall of the first outlet channel.

[0300] Aspect 55. 55. The microfluidic device of any one of embodiments 1-54, wherein the discrete entities are droplets.

[0301] Aspect 56.

[0049] A microfluidic device according to any one of embodiments 1-55, wherein the discrete entities comprise one or more cells, one or more beads, one or more particles, one or more reagents, one or more media, one or more drugs, one or more extracellular matrices, one or more hydrogels, or a combination thereof.

[0302] Aspect 57. 57. The microfluidic device of any one of embodiments 1-56, wherein the discrete entities contain RNA capture beads.

[0303] Aspect 58. 58. The microfluidic device of any one of embodiments 1-57, wherein the discrete entities comprise immunoassay beads.

[0304] Aspect 59. 59. The microfluidic device of any one of embodiments 1-58, wherein the discrete entities contain reagents, drugs, extracellular matrices, or combinations thereof.

[0305] Aspect 60. 60. The microfluidic device of any one of embodiments 1-59, wherein the discrete entities contain one or more cells.

[0306] Aspect 61. 61. The microfluidic device of any one of embodiments 1-60, wherein the discrete entity contains a single cell.

[0307] Aspect 62. 61. The microfluidic device of any one of embodiments 1-60, wherein the discrete entities contain two or more cells.

[0308] Aspect 63. 61. The microfluidic device of embodiment 60, wherein one or more cells are labeled with a fluorescent tag.

[0309] Aspect 64. 64. The microfluidic device of any one of embodiments 60, 62, or 63, wherein the one or more cells are pretreated with a surface functionalization configured to promote cell clumping within the droplet.

[0310] Aspect 65. 65. The microfluidic device of any one of embodiments 1-64, wherein one or more of the discrete entities is devoid of cells or devoid of one or more cells.

[0311] Aspect 66. 65. The microfluidic device of any one of aspects 1-64, wherein one or more of the discrete entities contain reagents but lack cells, and one or more of the discrete entities contain cells but lack reagents.

[0312] Aspect 67.

[0066] A microfluidic device according to any one of the preceding aspects, wherein one or more of the individual entities contain cells and one or more of the individual entities contain a hydrogel, an extracellular matrix, or a combination thereof.

[0313] Aspect 68. 68. The microfluidic device of any one of aspects 1-67, wherein one or more of the separate entities contain an RNA capture bead and one or more of the separate entities contain a reagent, the reagent being an oligonucleotide configured to hybridize to the RNA capture bead.

[0314] Aspect 69. 69. The microfluidic device of any one of embodiments 1-68, wherein the separate entities comprise a drug and an oligonucleotide.

[0315] Aspect 70. 70. The microfluidic device of any one of embodiments 1-69, wherein the individual entities have dimensions of between about 1 μm and about 1,000 μm.

[0316] Aspect 71. 71. The microfluidic device of embodiment 70, wherein the individual entities have a diameter of between about 1 μm and 1,000 μm.

[0317] Aspect 72. the sorting element includes a first sorting electrode and a second sorting electrode; the capture element includes a first capture electrode and a second capture electrode; the sorting flow path defines a concentric or nearly concentric flow path, and a portion of the first sorting electrode is located at a center of the concentric or nearly concentric flow path; the microfluidic device contains a bias fluid sorting channel in fluid communication with the sorting channel, the bias fluid sorting channel configured such that fluid flowing through the bias fluid sorting channel moves the individual entities closer to the second sidewall of the inlet channel and away from the first sidewall of the inlet channel; the separate entity blending region includes a recess in a wall of the first outlet channel; the individual entity fusion region has a triangular or approximately triangular shape; and the first outlet channel contains an angled turn in the channel wall downstream of the separate entity blending region; 2. The microfluidic device according to embodiment 1.

[0318] Aspect 73. the sorting element includes a first sorting electrode and a second sorting electrode disposed on opposite sides of the inlet channel; the capture element includes a first capture electrode and a second capture electrode disposed on the same side of the first outlet channel; the sorting flow path defines a concentric or nearly concentric flow path, and a portion of the first sorting electrode is located at a center of the concentric or nearly concentric flow path; the first capture electrode and the second capture electrode have different shapes; a first capture electrode extending along the first outlet flow path downstream of the individual entity merging region, or a second capture electrode extending along the first outlet flow path downstream of the individual entity merging region; the microfluidic device contains a bias fluid sorting channel in fluid communication with the sorting channel, the bias fluid sorting channel configured such that fluid flowing through the bias fluid sorting channel moves the individual entities closer to the second sidewall of the inlet channel and away from the first sidewall of the inlet channel; The individual entity fusion area is (a) a recess in the wall of the first outlet channel; (b) a stacked fluid inlet channel, the stacked fluid inlet channel configured such that fluid flowing through the stacked fluid inlet channel directs individual entities within the individual entity fusion region toward a capture electrode; and (c) Flow divider. 2. The microfluidic device of embodiment 1, comprising:

[0319] Aspect 74. 74. The microfluidic device of any one of embodiments 1 to 73, further comprising a second inlet channel in fluid communication with the sorting channel.

[0320] Aspect 75. 75. The microfluidic device of any one of embodiments 1 to 74, wherein the second outlet channel is a waste outlet channel.

[0321] Aspect 76.

[0072] A microfluidic device according to any one of embodiments 1-75, comprising a combined individual entity outlet channel in fluid communication with the first outlet, the combined individual entity outlet channel being configured to receive the combined individual entities.

[0322] Aspect 77. a) the microfluidic device of any one of embodiments 1 to 76, and b) one or more or all of the following: i) an individual entity fabrication device configured to fabricate a plurality of individual entities, the individual entity fabrication device being located within the microfluidic device or separate from the microfluidic device; ii) a distinct entity library containing two or more types of distinct entities; iii) a detector configured to detect individual entities in the input flow path, wherein the microfluidic device is configured to sort the individual entities in the sort flow path based on detection by the detector; iv) a temperature control module operably connected to the microfluidic device; v) an incubator operably connected to the microfluidic device; vi) an imaging device configured to image the combined individual entities; and vii) a sequencer operably connected to the microfluidic device or the incubator A system including:

[0323] Aspect 78. 78. The system of embodiment 77, comprising an individual entity creation device.

[0324] Aspect 79. 79. The system of any one of aspects 77-78, wherein the individual entity creating device is a droplet creating device.

[0325] Aspect 80. Aspect 80. The system of any one of aspects 77-79, wherein the individual entity creation device is configured to create the individual entities by moving the individual entity fluid while cycling the valve.

[0326] Aspect 81. The system of embodiment 80, wherein the valve is a piezoelectric actuator.

[0327] Aspect 82. 82. The system of any one of aspects 77-81, wherein the individual entity creation device is configured to create the individual entities by exposing the individual entity fluid to light, magnetic force, or electric force.

[0328] Aspect 83. 83. The system of any one of embodiments 77-82, comprising an individual entity library containing two or more types of individual entities.

[0329] Aspect 84. 84. The system of any one of embodiments 77 to 83, wherein the individual entity library contains a first type of individual entity that includes cells, and a second type of individual entity that includes reagents.

[0330] Aspect 85. 85. The system of any one of embodiments 77 to 84, wherein the individual entity library contains a first type of individual entity that contains a first type of cell, and a second type of individual entity that contains a second type of cell.

[0331] Aspect 86. 86. The system of any one of embodiments 77 to 85, wherein the individual entity library contains a first type of individual entity that contains cells, and a second type of individual entity that contains hydrogels, and extracellular matrices, or a combination thereof.

[0332] Aspect 87. 87. The system of any one of aspects 77 to 86, comprising a detector configured to detect individual entities in the input channel, and wherein the microfluidic device is configured to sort individual entities in the sorting channel based on detection by the detector.

[0333] Aspect 88. 88. The system of any one of embodiments 77-87, comprising an incubator operably connected to the microfluidic device.

[0334] Aspect 89. The system of embodiment 88, further comprising an imaging device configured to image the combined individual entities within the incubator.

[0335] Aspect 90. 90. The system of any one of embodiments 77 to 89, comprising a sequencer operably connected to the microfluidic device or the incubator.

[0336] Aspect 91. a) flowing two distinct entities in a carrier fluid through an inlet channel into a sorting channel of the microfluidic device or system of any one of aspects 1-90, wherein the two distinct entities are insoluble, immiscible, or a combination thereof in the carrier fluid; b) selectively sorting two distinct entities in the sorting channel to a first outlet channel; and c) capturing the two distinct entities within the distinct entity fusion region for a time sufficient for the two distinct entities to combine to form a combined distinct entity; A method for selectively combining at least two separate entities, including:

[0337] Aspect 92. d) flowing a third distinct entity in a carrier fluid through an inlet channel into a sorting channel of the microfluidic device, wherein the third distinct entity is insoluble, immiscible, or a combination thereof in the carrier fluid; e) selectively sorting a third individual entity in the sorting flow path to the first outlet flow path; and f) capturing a third individual entity within the individual entity fusion region to be combined with the combined individual entity created from the first individual entity and the second individual entity; further comprising step d) occurs before, simultaneously with, or after each of steps b) and c); Step e) occurs before, at the same time as, or after step c). The method of embodiment 91.

[0338] Aspect 93. A method according to any one of aspects 91 to 92, further comprising releasing the combined individual entities from the individual entity fusion region by disabling, reducing, or reversing the capture element so that the combined individual entities flow out of the first outlet channel.

[0339] Aspect 94. A method according to any one of aspects 91 to 93, comprising imaging the individual entities or combined individual entities within the individual entity fusion region.

[0340] Aspect 95. 95. The method of any one of embodiments 91-94, wherein the releasing comprises disabling the capture electrode.

[0341] Aspect 96. 96. The method of any one of aspects 91 to 95, further comprising repeating said steps at least once.

[0342] Aspect 97. Aspects 97. The method of any one of aspects 91-96, wherein at least one distinct entity flows through the first inlet channel and at least one distinct entity flows through the second inlet channel.

[0343] Aspect 98. Creating multiple distinct entities; and Storing multiple distinct entities for a period of time before the flow step 98. The method of any one of embodiments 91 to 97, further comprising:

[0344] Aspect 99. Create multiple individual entities, Multiple individual entities may be directed to the inlet channel without being stored for a period of time. 99. The method of any one of embodiments 91 to 98, comprising:

[0345] Aspect 100. 99. The method of any one of embodiments 98-99, wherein the time period is 1 minute.

[0346] Aspect 101. Aspects 99. The method of any one of aspects 91-99, wherein the creating step comprises selecting and combining two or more types of individual entities from an individual entity library.

[0347] Aspect 102. 102. The method of any one of embodiments 91-101, wherein at least two of the separate entities each contain a different reagent and selectively react.

[0348] Aspect 103. 103. The method of any one of embodiments 91-102, wherein at least one distinct entity contains a cell and lacks a reagent, and at least one distinct entity comprises a reagent but lacks a cell and selectively performs a reaction on a cell.

[0349] Aspect 104. A method according to any one of aspects 91 to 103, wherein the reagent is a cell lysis reagent.

[0350] Aspect 105. Aspect 94. The method of any one of aspects 91 to 103, wherein the reagents are polymerase chain reaction (PCR) reagents.

[0351] Aspect 106. A method according to any one of aspects 91 to 103, wherein the reagent is a drug.

[0352] Aspect 107. 107. The method of any one of embodiments 91-106, wherein at least one distinct entity contains a cell and at least one distinct entity contains a hydrogel or an extracellular matrix.

[0353] Aspect 108. 108. The method of any one of embodiments 91-107, wherein at least one distinct entity contains cells of the first type and lacks cells of the second type, and at least one distinct entity contains cells of the second type and lacks cells of the first type.

[0354] Aspect 109. 109. The method according to any one of embodiments 91 to 108, wherein the surface of at least one cell is functionalized with an oligonucleotide.

[0355] Aspect 110. 110. The method of any one of embodiments 91-109, wherein the first distinct entity contains an oligonucleotide and the second distinct entity contains an RNA capture bead configured to hybridize with the oligonucleotide.

[0356] Aspect 111. 111. The method of any one of embodiments 91-110, further comprising detecting individual entities in the inlet channel and sorting the individual entities based on the detection.

[0357] Aspect 112. 112. The method of any one of aspects 91-111, wherein the individual entities are not detected before reaching the inlet channel.

[0358] Aspect 113. 113. The method of any one of embodiments 91-112, wherein detecting comprises optical detection of the individual entities.

[0359] Aspect 114. 114. The method of embodiment 113, wherein the optical detection comprises fluorescence measurement.

[0360] Aspect 115. 115. The method of any one of embodiments 91-114, wherein at least one distinct entity comprises a cell, and further comprising culturing the cells, and selectively culturing one or more of the cells.

[0361] Aspect 116. 116. The method of embodiment 115, wherein the culturing is carried out in an incubator operably connected to the microfluidic device.

[0362] Aspect 117. 117. The method according to any one of embodiments 115 to 116, wherein the culturing is carried out for at least 12 hours.

[0363] Aspect 118. 117. The method of any one of embodiments 91 to 116, further comprising sequencing the genome, proteome, transcriptome, or a combination thereof, of cells from the individual entities or combined individual entities, thereby selectively sequencing the cells.

[0364] Aspect 119. 120. The method of embodiment 118, further comprising selectively combining the incubated cells with one or more sequencing reagents using a device, wherein the selective combining occurs prior to the sequencing step.

[0365] Aspect 120. 120. The method of embodiment 119, wherein the one or more sequencing reagents contain a lysis buffer.

[0366] Aspect 121. 121. The method of any one of embodiments 119-120, wherein the one or more sequencing reagents comprise RNA sequencing reagents.

[0367] Aspect 122. 121. The method of any one of embodiments 118-120, wherein the one or more sequencing reagents comprise barcoded RNA capture beads.

[0368] Aspect 123. 119. The method of any one of embodiments 91 to 118, further comprising measuring the effect of the agent on the cell, wherein the effect of the agent on the cell is selectively measured.

[0369] Aspect 124. 124. The method of any one of aspects 91 to 123, further comprising collecting cell-cell interaction data regarding an interaction between a first cell and a second cell, wherein the cell-cell interaction data is selectively collected.

[0370] Aspect 125. 125. The method of embodiment 124, wherein the first cell is an immune cell and the second cell is a cancer cell.

[0371] Aspect 126. 126. The method of embodiment 125, wherein the immune cell is an engineered T cell.

[0372] Aspect 127. 127. The method of embodiment 126, wherein the engineered T cell is a chimeric antigen receptor T cell (CAR-T cell).

[0373] Aspect 128. 128. The method of any one of embodiments 124 to 127, wherein the cell-cell interaction data comprises the efficacy of the engineered T cells in killing cancer cells.

[0374] Aspect 129. 129. The method of any one of embodiments 124 to 128, wherein the cell-cell interaction data comprises genomic data of one or more of the cells.

[0375] Aspect 130. 130. The method of any one of embodiments 91-129, further comprising producing a three-dimensional cell culture from at least one combined individual entity.

[0376] Aspect 131. The method of embodiment 130, wherein the three-dimensional cell culture is an organoid.

[0377] Aspect 132. 131. The method of embodiment 130, wherein the three-dimensional cell culture is a spheroid.

[0378] Aspect 133. The method of any one of embodiments 130 to 132, wherein at least one cell is a nervous system cell.

[0379] Aspect 134. 134. The method of embodiment 133, wherein the nervous system cell is a neuron. [Example]

[0380] The following examples are presented to provide those of skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures). However, some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp (base pairs), kb (kilobase), pl (picoliter), s or sec (seconds), min (minute), h or hr (hour), aa (amino acid), nt (nucleotide), etc.

[0381] Example 1: Fabrication of a microfluidic device Microfluidic devices were fabricated using standard soft lithography procedures. The device geometry was drawn using AutoCAD and printed onto a photographic negative. 3D micromolds were fabricated by spin-coating and photoexposing layers of SU8 photoresist. After developing the micromold to remove unpolymerized SU8, the microfluidic geometry was fabricated by casting PDMS over the micromold. After curing in an oven at 60 °C for 2 h, the PDMS device was bonded to a clean glass slide using oxygen plasma. After bonding, the microfluidic channels were hydrophobized through treatment with fluorosilane. Alternative approaches for fabricating microfluidic devices include hot embossing, micromachining, and injection molding. Electrodes were formed by filling the microfluidic channels with conductive materials such as salt water, liquid metal, molten solder, or conductive ink that was subsequently annealed.

[0382] For example, one fabricated microfluidic device, such as that shown in Figure 2, includes an inlet channel, a first spacer oil inlet, a second spacer oil inlet, a sorting channel, a fluorescence detector, first and second sorting electrodes, first and second outlet channels, an upstream region, a separate entity merging region, a downstream region, a recess, a flow divider, a stacked inlet, and first and second capture electrodes. As noted above, some of the elements are part of other listed elements, e.g., the separate entity merging region is part of the first outlet channel.

[0383] Example 2: Selective Combination of Individual Entities A mixed emulsion of individual entities in a carrier fluid was introduced into the inlet channel of a microfluidic device. Just upstream of the sorting channel, the individual entities were detected by an optical detector. Additionally, elements of the microfluidic device (e.g., a spacer oil inlet) caused the individual entities to preferentially migrate to one side of the inlet and sorting channels. Based on detection, undesired components of the mixed emulsion flowed unimpeded through the concentric sorting channel to a second outlet channel. Individual entities within the concentric sorting channel containing desired contents (e.g., selected cells, beads, and reagents) were actively sorted into the first outlet channel by dielectrophoretic forces from two sorting electrodes.

[0384] As each individual entity flows along the first outlet channel, it is directed along the wall of the first outlet channel closest to the capture electrode due to the channel geometry (e.g., recess, flow divider, and stacked oil inlet). Upon reaching the individual entity fusion region, the selectively actuatable bipolar droplet capture electrode exerts an attractive dielectrophoretic force on each individual entity, combining the individual entities and capturing them within the individual entity fusion region for a time and under conditions sufficient to form a combined individual entity.

[0385] Example 3: Selective Reactions via Selective Combinations of Individual Entities A microfluidic device with an individual entity capture region, as shown in Figure 4, was fabricated according to the method described above. The first, second, and third reagent aqueous streams contained 100 μM dextran-conjugated Cascade Blue dye, along with 1 μM, 10 μM, and 100 μM dextran-conjugated Alexa Fluor 647 dye, respectively. Jurkat cell suspensions were stained with calcein and resuspended in PBS containing 100 μM Cascade Blue at a concentration of 100,000 cells / mL. Four distinct groups of fluorescently labeled 300-picoliter droplets were pre-fabricated using a flow-focusing droplet generator with an aqueous phase flow rate of 500 μL / hr and an oil flow rate of 1,000 μL / hr, and collected in a common reservoir where the individual entities within each group contained exactly one aliquot of the first reagent, second reagent, third reagent, and cell suspension. Due to the cell density, one in approximately 33 individual entities formed from the cell suspension contains a single cell, while most of the remaining entities contain only PBS.

[0386] Each type of individual entity was then flowed through the microfluidic device and fluorescently detected immediately upstream of the sorting channel. The individual entities were injected at a flow rate of 100 μL / hr, and fluorinated oil (HFE7500) containing 0.2% fluorosurfactant was introduced into the spacer fluid and bias fluid inlets at 2,000 μL / hr. The unique combination and concentration of fluorescent dyes used allowed for easy recognition of the entity types in the fluorescent detection data. Combined individual entities were formed as follows: The capture electrode was activated, and a series of single individual entities containing cells, reagent 1, reagent 2, and reagent 3 were sorted and flowed into the individual entity fusion region. As shown in Figure 4, the four individual entities combined to form a combined individual entity, which was then released from the individual entity fusion region by turning off the power to the capture electrode.

[0387] Example 4: Selective combination of three cells A microfluidic device was fabricated as described above. Individual entities, each containing cells, were created by uniquely labeling three Jurkat cell populations with Calcein Blue, Calcein, and CellTracker Red dyes. They were then resuspended in PBS at 100,000 cells / mL, formed into 300 pL droplets, and flowed into the microfluidic device. The individual entities were sorted and captured in an attempt to obtain combined individual entities containing three differently colored single cells per combined individual entity. Figure 6 shows an image of the resulting combined individual entities, and Figure 7 provides a graph showing the number of combined individual entities generated by a microfluidic device according to the present disclosure, which was able to generate triple-combined droplets with 60% efficiency. Failure was largely due to incomplete cell dissociation into a single-cell suspension, resulting in the presence of more than one clump of cells within the sorted individual entity. By randomly loading cells from a denser suspension containing an average of one cell of each color per droplet volume, Poisson statistics predict that only 5% of droplets will contain the desired combination, with the remaining 95% of the droplet population containing a wide range of combination conditions (Figure 8). When combining droplets in more standard dilute cell suspension conditions with a 10% single-cell occupancy, the probability of randomly assembling a droplet containing three distinct single cells was approximately 0.1%, while the expected proportion of triple-combined droplets based on random combinations of individual entities was only approximately 5%.

[0388] Example 5: Speed ​​and Time to Combine Individual Entities The speed and duration with which a device according to the present disclosure can selectively combine individual entities containing one or more cells was evaluated. A microfluidic device was fabricated as described above. It was found that the device was capable of producing combined individual entities containing 1 to 50 cells. It was also found that the device could operate continuously for 90 minutes while producing approximately 10,000 combined individual entities.

[0389] The maximum speed at which combined individual assemblies can be constructed depends on factors such as the maximum sorting rate, the frequency of the individual entities of interest, the migration time of the sorted entities to the capture location, and the release rate of the combined individual entities. Sorting has been demonstrated by Sciambi et al. at up to 30 kHz using dielectrophoresis methods similar to those described in this application. Cells and beads are typically encapsulated into individual entities at less than 10% occupancy, which reduces the effective single-cell sorting frequency by an order of magnitude. Low-frequency cell subpopulations, such as natural killer cells (5%) from peripheral blood mononuclear cell (PBMC) suspensions, increase the time one must wait until the appropriate individual entity is detected and sorted, and require any previously sorted individual entities to be maintained in the fusion zone for extended periods of time. In some situations, the migration time of sorted droplets from the sorter to the capture zone is on the order of 10 mS, assuming a 1 mm long, 40 μm square channel and a carrier oil flow rate of 1 mL / hr. The capture electrodes may be activated at frequencies above 10 kHz, but the time required for the combined individual entities to release from the non-activated capture is approximately 1 mS.

[0390] Example 6: Development of an intercellular functional assay Microfluidic devices were fabricated as described above. Multiple individual entities containing a single CAR-T cell, a single RAJI cell, or a single interferon gamma cytokine detection bead encapsulated with a secondary antibody and Sytox viability stain were generated. All individual entities were fluorescently labeled for unique identification. The mixed individual entities were then flowed into the microfluidic device, where they were combined to form a single CAR-T cell, a single RAJI cell, and a single cytokine detection bead. The assembled entities were captured in microcentrifuge tubes and incubated at 37°C for 24 hours. After incubation, the assembled individual entities were dispensed into microwell arrays, where they were fluorescently imaged.

[0391] Example 7: Multi-step workflow After the assembled individual entities described in Example 6 are incubated to allow for a functional response, they can be re-injected into the microfluidic device for further processing. In addition to the incubated individual entities, a second population of individual entities containing lysis buffer and barcoded RNA capture beads is created and re-injected into the device. The incubated cell entities and lysis buffer / RNA capture bead entities are sorted into each combined entity, after which the cells are lysed and hybridized onto the RNA capture beads for downstream processing.

[0392] Example 8: On-chip imaging and real-time barcoding The assembled individual entities are incubated to allow a functional response, creating a population of individual entities containing lysis buffer and barcoded RNA capture beads. Further, a population of individual entities is formed from a solution containing known oligonucleotide barcodes and labeled with a unique combination of fluorescent dyes. All entities are injected into the device as described above. The cell-containing entities are first delivered to the capture location, then imaged on the device, and the image is saved. Next, the lysis buffer / RNA capture beads are sorted for capture, followed by sorting of known entity combinations containing known unique combinations of oligonucleotide barcodes. These barcodes hybridize onto the RNA capture beads along with the cell-derived RNA, providing a method for correlating imaging dates with sequencing data. The combined individual entities are collected and processed downstream.

[0393] Example 9: Selective single-cell RNA sequencing based on cell type Individual entities are created from a PBMC population treated with fluorescently labeled antibodies. Additional lysis buffer / RNA capture bead individual entities are also created. The mixed individual entity population is injected into the device, and equal numbers of B cells, natural killer cells, and dendritic cells are individually sorted into the capture region and fused with the lysis buffer / RNA capture bead individual entities. The combined individual entities are collected and processed downstream. Selecting less abundant cell types for sequencing eliminates the need to oversample more abundant cell types in the mixed sample, reducing sequencing costs.

[0394] Example 10: Two bead assembly processes Using standard flow-focusing droplet microfluidics, two batches of input droplets were prepared and collected in a common 1 mL syringe. The first and second batches contained a dilute suspension of either blue or red fluorescent microparticles (Spherotech) and either 1 μm or 0.5 μm AlexaFluor 488 10 kDa Dextran (AF488Dex) (Thermo Fisher), respectively. The droplets were primarily empty and contained one red or one blue bead (Figure 21A). Sorting gates for droplets with red and blue fluorescent beads were drawn using the AF488Dex droplet dye and the relative fluorescence signature of the bead fluorescence intensity. For each assembled droplet, exactly one blue bead droplet and one red bead droplet were sorted and fused. 17,500 droplets were assembled in this manner in 1 hour and 25 minutes, and these assembled droplets were collected in 200 μL of dummy PBS droplet emulsion. The composition of the input emulsion and assembled droplet emulsions was assessed using fluorescence microscopy. A 5 μL sample of each emulsion was sampled and imaged (Leica, Dmi8 Thunder) in a cell counting slide (Thermo Fisher) (Figure 21B). The contents of each imaged droplet were then quantified using a custom Image J script. Of the 104 combined droplets, 93 / 104 contained exactly one blue bead and one red bead (Figure 21C).

[0395] Example 11: CAR-T Functional Assay Workflow Cells were fluorescently stained the night before emulsification. CAR-T cells and RAJI cells were stained with CellTracker Green CMFDA and CellTracker Orange CMRA for 10 minutes at 37°C, respectively. Cells were then washed in complete culture medium, incubated overnight, and washed again. Input droplets with a diameter of 40 μm were generated using standard flow-focusing droplet microfluidics. Three batches of input droplets were prepared using standard flow-focusing droplet microfluidics and collected in a common 1 mL syringe (BD Biosciences). The first two batches consisted of either stained CAR-T cells or stained RAJI cells, loaded at 5e6 cells / mL, and contained 3 μM or 1 μM Cascade Blue 10 kDa dextran (CB-Dex) (Thermo Fisher), respectively. The third batch of droplets contained assay reagent components consisting of IFNλ detection microparticles, biotinylated IFNλ detection antibody, streptavidin AlexaFluor 647, and 6 μM CB-Dex. Prior to the cell-cell interaction test, cytokine detection microparticles were prepared. Briefly, 4 mg of carboxylated and fluorescent polystyrene particles were functionalized with IFNλ capture antibody using carbodiimide chemistry. The functionalized particles were washed three times and stored in PBS until use. Droplet assembly was performed on a 40 μm microfluidic assembler device. Sorting gates were defined to isolate droplets containing CAR-T, RAJI, or detection microparticles based on the relative CB-Dex and fluorescence signatures of the cells and microparticles (Figure 22A). The droplets were then sorted and fused to assemble droplets containing one CAR-T, one RAJI, and two detection microparticles and associated reagents (Figure 22B). In this way, 15,000 droplets, approximately 65 μm in diameter, were assembled in 1 hour and 30 minutes and collected in 200 μL of blank PBS droplet emulsion in a 3 mL syringe. This collected emulsion contained 500 μL of HFE7500 containing 5% surfactant to stabilize the droplets during incubation. The assembled droplet emulsion was incubated for 12 hours at 37 °C to allow for cell-cell interactions and cytokine secretion.Assembled droplet sorting was performed on an 80 μm droplet assembler microfluidic device. Sorting gates were defined using CB-Dex to detect the presence of assembled droplets and AF647 to threshold and sort droplets, where IFNλ pulldown onto the detection microparticles produces a bead-localized spike in fluorescence (Figure 22C). Assay-positive and assay-negative droplet populations were then collected in 200 μL emulsions of dummy PBS droplets for downstream processing.

Claims

1. a) inlet channel; b) a sorting channel in fluid communication with the inlet channel; c) a first outlet flow path and a second outlet flow path in fluid communication with the sorting flow path, the first outlet flow path including an upstream region adjacent to and in fluid communication with the sorting flow path, a separate entity merging region adjacent to and in fluid communication with the upstream region, and a downstream region adjacent to and in fluid communication with the separate entity merging region, and a first wall partially defining the upstream region, the separate entity merging region, and the downstream region; d) a sorting element disposed proximate the sorting channel, the sorting element configured to sort individual entities within the sorting channel to a first outlet channel; and e) a capture element positioned proximate to the individual entity fusion region; Contains the capture element and the individual entity fusion region are configured to capture the plurality of individual entities within the individual entity fusion region for a time sufficient to combine the plurality of individual entities to form a combined individual entity; the discrete entity blending region contains a feature selected from a geometric change in the dimension of the first outlet flow path, a flow obstacle, a flow divider, a stacked fluid inlet, a valve, or a combination thereof; Microfluidic devices.

2. 10. The microfluidic device of claim 1, wherein the sorting element comprises a sorting electrode that exerts an electromagnetic force sufficient to sort individual entities in the sorting channel to the first outlet channel.

3. The microfluidic device according to any one of claims 1 to 2, wherein the electromagnetic force is a dielectrophoretic force.

4. The microfluidic device according to any one of claims 1 to 2, wherein the electromagnetic force is an electrophoretic force.

5. The microfluidic device of any one of claims 1 to 4, further comprising a second sorting electrode.

6. The microfluidic device of claim 5 further comprising a third sorting electrode.

7. 7. The microfluidic device of claim 5, wherein the first and second sorting electrodes are configured such that the first and second sorting electrodes form a bipolar electrode pair and the first capture electrode is positively charged.

8. The microfluidic device according to any one of claims 5 to 7, wherein the first and second sorting electrodes are positioned on opposite sides of the sorting channel.

9. The microfluidic device according to any one of claims 5 to 8, wherein the first sorting electrode is positioned closer to the sorting flow path than the second sorting electrode, or the second sorting electrode is positioned closer to the sorting flow path than the first sorting electrode.

10. 10. The microfluidic device according to claim 5, wherein the distance between the end of the first sorting electrode, the second sorting electrode, or both ends and the inner wall of the sorting channel is between about 1 μm and about 100 μm.

11. The microfluidic device according to any one of claims 5 to 10, wherein the distance between the first sorting electrode and the second sorting electrode is from about 25 μm to about 500 μm.

12. 12. The microfluidic device according to claim 5, wherein the first sorting electrode and the second sorting electrode are connected to an AC power source having a frequency of about 0.1 kHz to about 100 kHz and a voltage of about 10 V to about 10,000 V.

13. The microfluidic device of any one of claims 2 to 12, wherein each sorting electrode comprises a liquid electrode.

14. 14. The microfluidic device of claim 13, wherein each sorting liquid electrode contains one or more liquid flow channels embedded in the microfluidic device and filled with a conductive medium.

15. The microfluidic device of claim 1 , wherein the sorting element comprises a valve, a surface wave sorting element, an acoustic streaming element, or a combination thereof.

16. 15. The microfluidic device of claim 1, wherein the capture element exerts an electromagnetic force, a mechanical force, or a combination thereof sufficient to capture the plurality of individual entities within the individual entity fusion region for a time sufficient to combine the plurality of individual entities to form a combined individual entity.

17. 17. The microfluidic device of claim 1, wherein the capture element comprises a first capture electrode that exerts an electromagnetic force sufficient to capture the plurality of individual entities within the individual entity merging region for a time sufficient to combine the plurality of individual entities to form a combined individual entity.

18. 18. The microfluidic device of claim 17, wherein the electromagnetic force is a dielectrophoretic force.

19. 18. The microfluidic device of claim 17, wherein the electromagnetic force is an electrophoretic force.

20. The microfluidic device of any one of claims 17 to 19, further comprising a second capture electrode.

21. 21. The microfluidic device of claim 20, further comprising a third capture electrode.

22. 22. The microfluidic device of claim 20, wherein the first and second sorting electrodes are configured such that the first and second sorting electrodes form a bipolar electrode pair and the first capture electrode is positively charged.

23. The microfluidic device of any one of claims 20 to 22, wherein the first and second sorting electrodes are located on the same side of the sorting channel.

24. 24. The microfluidic device of claim 20, wherein the first capture electrode is positioned closer to the first outlet flow path than the second capture electrode, or the second capture electrode is positioned closer to the first outlet flow path than the first capture electrode.

25. 25. The microfluidic device of claim 20, wherein the distance between the end of the first capture electrode, the second capture electrode, or both, and the inner wall of the first outlet channel is between about 10 μm and about 50 μm.

26. The microfluidic device of any one of claims 20 to 25, wherein the distance between the first capture electrode and the second capture electrode is from about 25 μm to about 500 μm.

27. 27. The microfluidic device of claim 26, wherein the distance between the first capture electrode and the second capture electrode is between about 50 μm and about 200 μm.

28. 28. The microfluidic device of claim 20, wherein the first capture electrode and the second capture electrode are connected to an AC power source having a frequency of about 0.1 kHz to about 100 kHz and a voltage of about 10 V to about 10,000 V.

29. 29. The microfluidic device of claim 28, wherein the frequency is from about 1 kHz to about 50 kHz.

30. A microfluidic device according to any one of claims 17 to 29, wherein each capture electrode comprises a liquid electrode.

31. 31. The microfluidic device of claim 30, wherein each trapped liquid electrode contains one or more liquid flow channels embedded in the microfluidic device and filled with a conductive medium.

32. 32. The microfluidic device of claim 20, wherein the first capture electrode extends along the first outlet flow path downstream of the individual entity merging region, or the second capture electrode extends along the first outlet flow path downstream of the individual entity merging region.

33. 33. The microfluidic device of claim 1, wherein the sorting channels define concentric or nearly concentric channels, and a portion of the first sorting electrode is located at the center of the concentric or nearly concentric channels.

34. 34. The microfluidic device of claim 33, wherein the first sorting electrode is positioned closer to the first outlet channel than to the second outlet channel.

35. 34. The microfluidic device of claim 1, further comprising a flow divider disposed within the sorting channel, the flow divider configured to direct individual entities toward the first outlet channel or the second outlet channel.

36. 36. The microfluidic device of claim 35, wherein the height of the flow distributor is about 50% to 75% of the height of the sorting channel.

37. The microfluidic device of any one of claims 1 to 36, wherein the discrete entity merging region comprises a geometric change in the dimensions of the first outlet channel.

38. 38. The microfluidic device of any one of claims 1 to 37, wherein the individual entity merging region comprises a geometric change in the dimensions of the first outlet channel, the geometric change comprising an increase in the cross-sectional area of ​​the first outlet channel.

39. 39. The microfluidic device of any one of claims 1 to 38, wherein the discrete entity merging region comprises a geometric variation, the geometric variation comprising a recess in a wall of the first outlet channel.

40. 40. The microfluidic device of any one of claims 1 to 39, wherein the individual entity merging region contains a stacking fluid inlet channel, and wherein stacking fluid flowing through the stacking fluid inlet channel is configured to direct individual entities in the individual entity merging region towards a capture electrode.

41. 41. The microfluidic device of claim 40, wherein the individual entity merging region further contains a flow divider, and the stack fluid inlet channel and flow divider are configured such that stack fluid flowing through the stack fluid inlet channel directs individual entities in the individual entity merging region toward the capture electrode.

42. the first inlet channel includes an upstream region located between the sorting channel and the individual entity merging region; and the separate entity fusion region has a larger cross-sectional area than the upstream region; The microfluidic device according to any one of claims 38 to 41.

43. 43. The microfluidic device of any one of claims 1 to 42, wherein the individual entity fusion region has a triangular, approximately triangular, trapezoidal, or approximately trapezoidal shape defined by a channel wall of the microfluidic device.

44. 44. A microfluidic device according to any one of claims 1 to 43, wherein the individual entity fusion region contains a valve, the valve being a membrane valve configured to prevent the flow of individual entities through the individual entity fusion region while allowing the flow of carrier fluid through the individual entity fusion region in a first state, and the membrane valve configured to release the individual entities or combined individual entities in a second state.

45. 45. The microfluidic device of any one of claims 1 to 44, wherein the first outlet channel contains an angled turn in the channel wall downstream of the discrete entity merging region.

46. 46. ​​The microfluidic device of any one of claims 1 to 45, further comprising a flow diverter disposed within the first outlet channel, the flow diverter configured to direct individual entities towards a capture electrode within the individual entity merging region.

47. 47. The microfluidic device of claim 46, wherein the height of the flow distributor is about 50% to 75% of the height of the first outlet channel.

48. 48. The microfluidic device of any one of claims 1 to 47, comprising a spacer fluid channel in fluid communication with the inlet channel, the spacer fluid channel configured such that flow of the spacer fluid through the spacer fluid channel positions the spacer fluid between two individual entities flowing through the inlet channel, thereby maintaining or increasing the distance between the two individual entities, thereby allowing each of the two individual entities to be sorted or not sorted independently.

49. 49. The microfluidic device of claim 48, wherein the spacer fluid is an oil.

50. 50. The microfluidic device of any one of claims 1 to 49, comprising a bias fluid channel in fluid communication with the sort channel, the bias fluid channel configured such that bias fluid flowing through the bias fluid channel urges individual entities closer to a second sidewall of the sort channel and away from a first sidewall of the sort channel.

51. 51. The microfluidic device of claim 50, wherein the biasing fluid is an oil.

52. 52. The microfluidic device of claim 1, wherein the first outlet channel is configured to receive an individual entity positioned closer to a first sidewall of the sort channel than to a second sidewall of the sort channel, and the second outlet channel is configured to receive an individual entity positioned closer to a second sidewall of the sort channel than to the first sidewall of the sort channel.

53. 53. A microfluidic device according to any preceding claim, configured such that when the sorting element does not exert a force on the individual entities flowing through the sorting channel, the individual entities flow into the second outlet channel.

54. 54. The microfluidic device of any one of claims 1 to 53, wherein the first outlet channel is configured such that individual entities flowing through the first outlet channel are directed towards a first sidewall of the first outlet channel.

55. A microfluidic device according to any preceding claim, wherein the discrete entities are droplets.

56. 56. The microfluidic device of any one of claims 1 to 55, wherein the discrete entities comprise one or more cells, one or more beads, one or more particles, one or more reagents, one or more media, one or more drugs, one or more extracellular matrices, one or more hydrogels, or a combination thereof.

57. 57. The microfluidic device of any one of claims 1 to 56, wherein the discrete entities comprise RNA capture beads.

58. 58. The microfluidic device of any one of claims 1 to 57, wherein the discrete entities comprise immunoassay beads.

59. 59. The microfluidic device of any one of claims 1 to 58, wherein the discrete entities comprise reagents, drugs, extracellular matrices, or combinations thereof.

60. 60. The microfluidic device of any one of claims 1 to 59, wherein the discrete entities contain one or more cells.

61. 61. The microfluidic device of any one of claims 1 to 60, wherein the discrete entities contain single cells.

62. 61. The microfluidic device of any one of claims 1 to 60, wherein the discrete entities contain two or more cells.

63. 61. The microfluidic device of claim 60, wherein one or more cells are labeled with a fluorescent tag.

64. 64. The microfluidic device of any one of claims 60, 62, or 63, wherein the one or more cells are pretreated with a surface functionalization configured to promote cell clumping within the droplet.

65. 65. The microfluidic device of any one of claims 1 to 64, wherein one or more of the discrete entities is devoid of cells or devoid of one or more cells.

66. 65. The microfluidic device of any one of claims 1 to 64, wherein one or more of the discrete entities contains a reagent but lacks cells, and one or more of the discrete entities contains cells but lacks a reagent.

67. 65. The microfluidic device of any one of claims 1 to 64, wherein one or more of the discrete entities contain cells and one or more of the discrete entities contain a hydrogel, an extracellular matrix, or a combination thereof.

68. 68. The microfluidic device of any one of claims 1 to 67, wherein one or more of the individual entities contain RNA capture beads and one or more of the individual entities contain a reagent, the reagent being an oligonucleotide configured to hybridize to the RNA capture beads.

69. The microfluidic device of any one of claims 1 to 68, wherein the discrete entities comprise a drug and an oligonucleotide.

70. 70. The microfluidic device of any one of claims 1 to 69, wherein the individual entities have dimensions of from about 1 μm to about 1,000 μm.

71. 71. The microfluidic device of claim 70, wherein the individual entities have a diameter of between about 1 μm and 1,000 μm.

72. the sorting element includes a first sorting electrode and a second sorting electrode; the capture element includes a first capture electrode and a second capture electrode; the sorting flow path defines a concentric or nearly concentric flow path, and a portion of the first sorting electrode is located at a center of the concentric or nearly concentric flow path; the microfluidic device contains a bias fluid sorting channel in fluid communication with the sorting channel, the bias fluid sorting channel configured such that fluid flowing through the bias fluid sorting channel moves the individual entities closer to the second sidewall of the inlet channel and away from the first sidewall of the inlet channel; the separate entity blending region includes a recess in a wall of the first outlet channel; the individual entity fusion region has a triangular or approximately triangular shape; and the first outlet channel contains an angled turn in the channel wall downstream of the separate entity blending region; The microfluidic device of claim 1 .

73. the sorting element includes a first sorting electrode and a second sorting electrode disposed on opposite sides of the inlet channel; the capture element includes a first capture electrode and a second capture electrode disposed on the same side of the first outlet channel; the sorting flow path defines a concentric or nearly concentric flow path, and a portion of the first sorting electrode is located at a center of the concentric or nearly concentric flow path; the first capture electrode and the second capture electrode have different shapes; a first capture electrode extending along the first outlet flow path downstream of the individual entity merging region, or a second capture electrode extending along the first outlet flow path downstream of the individual entity merging region; the microfluidic device contains a bias fluid sorting channel in fluid communication with the sorting channel, the bias fluid sorting channel configured such that fluid flowing through the bias fluid sorting channel moves the individual entities closer to the second sidewall of the inlet channel and away from the first sidewall of the inlet channel; The individual entity fusion area is (a) a recess in the wall of the first outlet channel; (b) a stacked fluid inlet channel, the stacked fluid inlet channel configured such that fluid flowing through the stacked fluid inlet channel directs individual entities within the individual entity merging region toward a capture electrode; and (c) Flow divider. The microfluidic device of claim 1 , comprising:

74. 74. The microfluidic device of any one of claims 1 to 73, further comprising a second inlet channel in fluid communication with the sorting channel.

75. 75. The microfluidic device of any one of claims 1 to 74, wherein the second outlet channel is a waste outlet channel.

76. 76. The microfluidic device of any one of claims 1 to 75, comprising a combined individual entity outlet channel in fluid communication with the first outlet, the combined individual entity outlet channel configured to receive the combined individual entities.

77. a) a microfluidic device according to any one of claims 1 to 76, and b) one or more or all of the following: i) an individual entity fabrication device configured to fabricate a plurality of individual entities, the individual entity fabrication device being located within the microfluidic device or separate from the microfluidic device; ii) a discrete entity library containing two or more types of discrete entities; iii) a detector configured to detect individual entities in the input flow path, wherein the microfluidic device is configured to sort the individual entities in the sort flow path based on detection by the detector; iv) a temperature control module operably connected to the microfluidic device; v) an incubator operably connected to the microfluidic device; vi) an imaging device configured to image the combined individual entities; and vii) a sequencer operably connected to the microfluidic device or the incubator. A system including:

78. 78. The system of claim 77, including an individual entity creating device.

79. 79. The system of any one of claims 77 to 78, wherein the individual entity creating device is a droplet creating device.

80. 80. The system of any one of claims 77 to 79, wherein the individual entity creation device is configured to create the individual entities by moving the individual entity fluid while cycling the valves.

81. 81. The system of claim 80, wherein the valve is a piezoelectric actuator.

82. 82. The system of any one of claims 77 to 81, wherein the discrete entity creation device is configured to create discrete entities by exposing the discrete entity fluid to light, magnetic force, or electric force.

83. A system according to any one of claims 77 to 82, comprising a distinct entity library containing two or more types of distinct entities.

84. 84. The system of any one of claims 77 to 83, wherein the individual entity library contains a first type of individual entity that includes cells and a second type of individual entity that includes reagents.

85. 85. The system of any one of claims 77 to 84, wherein the individual entity library contains a first type of individual entity that contains a first type of cell, and a second type of individual entity that contains a second type of cell.

86. 86. The system of any one of claims 77-85, wherein the individual entity library contains a first type of individual entity that contains cells, and a second type of individual entity that contains hydrogels, and extracellular matrices, or combinations thereof.

87. 87. The system of any one of claims 77 to 86, including a detector configured to detect individual entities in the input flow path, and wherein the microfluidic device is configured to sort individual entities in the sort flow path based on detection by the detector.

88. 88. The system of any one of claims 77 to 87, comprising an incubator operably connected to the microfluidic device.

89. 90. The system of claim 88, further comprising an imaging device configured to image the combined individual entities within the incubator.

90. 90. The system of claims 77-89, comprising a sequencer operably connected to the microfluidic device or the incubator.

91. a) flowing two distinct entities in a carrier fluid through an inlet channel into a sorting channel of a microfluidic device or system according to any one of claims 1 to 90, wherein the two distinct entities are insoluble, immiscible, or a combination thereof in the carrier fluid; b) selectively sorting two distinct entities in the sorting channel to a first outlet channel; and c) capturing the two separate entities within the separate entity fusion region for a time sufficient for the two separate entities to combine to form a combined separate entity; 1. A method for selectively combining at least two separate entities, comprising:

92. d) flowing a third distinct entity in a carrier fluid through an inlet channel into a sorting channel of the microfluidic device, wherein the third distinct entity is insoluble, immiscible, or a combination thereof in the carrier fluid; e) selectively sorting a third individual entity in the sorting flow path to the first outlet flow path; and f) capturing a third individual entity within the individual entity fusion region to be combined with the combined individual entity created from the first individual entity and the second individual entity; further comprising step d) occurs before, simultaneously with, or after each of steps b) and c); Step e) occurs before, simultaneously with, or after step c).

92. The method of claim 91.

93. 93. The method of any one of claims 91-92, further comprising releasing the combined individual entities from the individual entity fusion region by disabling, reducing, or reversing the capture element such that the combined individual entities flow out the first outlet channel.

94. 94. A method according to any one of claims 91 to 93, comprising imaging the individual entities or combined individual entities within an individual entity fusion region.

95. 95. A method according to any one of claims 91 to 94, wherein releasing comprises disabling a capture electrode.

96. 96. The method of any one of claims 91 to 95, further comprising repeating steps a), b) and c) at least once.

97. 97. A method according to any one of claims 91 to 96, wherein at least one distinct entity flows through a first inlet flow path and at least one distinct entity flows through a second inlet flow path.

98. Creating multiple distinct entities; and Storing multiple distinct entities for a period of time before the flow step 98. The method of any one of claims 91 to 97, further comprising:

99. Create multiple individual entities, Multiple individual entities may be directed to the inlet channel without being stored for a period of time. The method of any one of claims 91 to 98, comprising:

100. A method according to any one of claims 98 to 99, wherein the period of time for storing the plurality of distinct entities before the flow step is one minute.

101. A method according to any one of claims 91 to 99, wherein the creating step comprises selecting and combining two or more types of individual entities from a library of individual entities.

102. 102. The method of any one of claims 91 to 101, wherein at least two of the separate entities each contain a different reagent to selectively react.

103. 103. The method of any one of claims 91 to 102, wherein at least one distinct entity contains a cell and lacks a reagent, and at least one distinct entity contains a reagent but lacks a cell, and selectively performs a reaction on the cell.

104. The method of any one of claims 91 to 103, wherein the reagent is a cell lysis reagent.

105. 104. The method of any one of claims 91 to 103, wherein the reagents are polymerase chain reaction (PCR) reagents.

106. The method of any one of claims 91 to 103, wherein the reagent is a drug.

107. 107. The method of any one of claims 91 to 106, wherein at least one separate entity contains a cell and at least one separate entity contains a hydrogel or an extracellular matrix.

108. 108. The method of any one of claims 91 to 107, wherein at least one distinct entity contains cells of a first type and lacks cells of a second type, and at least one distinct entity contains cells of a second type and lacks cells of the first type.

109. 109. The method of claim 108, wherein the surface of at least one cell is functionalized with an oligonucleotide.

110. 110. The method of any one of claims 91 to 109, wherein the first distinct entity comprises an oligonucleotide and the second distinct entity comprises an RNA capture bead configured to hybridize with the oligonucleotide.

111. 111. The method of any one of claims 91 to 110, further comprising detecting individual entities in the inlet flow path and sorting the individual entities based on the detection.

112. A method according to any one of claims 91 to 111, wherein the individual entities are not detected before they reach the inlet flow path.

113. A method according to any one of claims 91 to 112, wherein the detection comprises optical detection of the individual entities.

114. 114. The method of claim 113, wherein the optical detection comprises fluorescence measurement.

115. 115. The method of any one of claims 91-114, wherein at least one distinct entity comprises a cell, and further comprising culturing the cells, selectively culturing one or more of the cells.

116. 116. The method of claim 115, wherein the culturing is carried out in an incubator operably connected to the microfluidic device.

117. The method of claims 115 to 116, wherein the culturing is carried out for at least 12 hours.

118. 117. The method of any one of claims 91-116, further comprising sequencing the genome, proteome, transcriptome, or combination thereof, of cells from the individual entities or combined individual entities, to selectively sequence the cells.

119. 119. The method of claim 118, further comprising selectively combining the incubated cells with one or more sequencing reagents using a device, wherein the selective combination occurs prior to the sequencing step.

120. 120. The method of claim 119, wherein the one or more sequencing reagents comprise a lysis buffer.

121. 121. The method of any one of claims 119 to 120, wherein the one or more sequencing reagents comprise RNA sequencing reagents.

122. 121. The method of any one of claims 118-120, wherein one or more sequencing reagents comprise barcoded RNA capture beads.

123. 109. The method of claim 108, further comprising measuring the effect of the agent on the cell, wherein the effect of the agent on the cell is selectively measured.

124. 124. The method of any one of claims 91 to 123, further comprising collecting cell-cell interaction data regarding an interaction between a first cell and a second cell, wherein the cell-cell interaction data is selectively collected.

125. 125. The method of claim 124, wherein the first cell is an immune cell and the second cell is a cancer cell.

126. 126. The method of claim 125, wherein the immune cell is an engineered T cell.

127. 127. The method of claim 126, wherein the engineered T cells are chimeric antigen receptor T cells (CAR-T cells).

128. 128. The method of any one of claims 124 to 127, wherein the cell-cell interaction data comprises the effectiveness of the engineered T cells in killing cancer cells.

129. 129. The method of any one of claims 124 to 128, wherein the cell-cell interaction data comprises genomic data of one or more of the cells.

130. 130. The method of any one of claims 91 to 129, further comprising producing a three-dimensional cell culture from at least one combined individual entity.

131. 131. The method of claim 130, wherein the three-dimensional cell culture is an organoid.

132. 131. The method of claim 130, wherein the three-dimensional cell culture is a spheroid.

133. 133. The method of any one of claims 130 to 132, wherein at least one cell is a neural cell.

134. 134. The method of claim 133, wherein the nervous system cell is a neuron.

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