WO2023183461A1

WO2023183461A1 - Microfluidic cartridges

Info

Publication number: WO2023183461A1
Application number: PCT/US2023/016024
Authority: WO
Inventors: Ian Walton; Russell Cole; Scott West; Kyle BULLOCH; Justin MADRIGAL; Andrew Williams
Original assignee: Scribe Biosciences, Inc.
Priority date: 2022-03-24
Filing date: 2023-03-23
Publication date: 2023-09-28

Abstract

Provided herein are systems, devices, and methods for using microfluidic cartridges (e.g., to characterize cell-cell interactions in droplets). In certain embodiments, the microfluidic cartridges comprise at least one microfluidic chips (e.g., selected from a drop making chip, a drop assembly chip, and a drop sorting chip) attached to a base substrate, and are configured to be removably inserted into a cartridge receiving instrument. In some embodiments, the drop assembly chip comprises a sorting region, a droplet merger region, and a trapping element (e.g., composed of trapping electrodes) and is configured for deterministic sorting and trapping of at least two droplets to form a combined droplet (e.g., containing one cell type from each of the droplets).

Description

MICROFLUIDIC CARTRIDGES

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Patent Application 63/323,374, filed March 24, 2023, the entirety of which is incorporated by reference.

FIELD OF THE INVENTION

Provided herein are systems, devices, and methods for using microfluidic cartridges (e.g., to characterize cell-cell interactions in droplets). In certain embodiments, the microfluidic cartridges comprise at least one microfluidic chip (e.g., selected from a drop making chip, a drop assembly chip, and a drop sorting chip) attached to a base substrate, and are configured to be removably inserted into a cartridge receiving instrument. In some embodiments, the drop assembly chip comprises a sorting region, a droplet merger region, and a trapping element (e.g., composed of trapping electrodes) and is configured for deterministic sorting and trapping of at least two droplets to form a combined droplet (e.g., containing one cell type from each of the droplets).

BACKGROUND

The immune system relies on a massive number of individual cell-cell interaction events. These interactions are key to distinguishing self from non-self and foundational to emerging cancer immunotherapies such as immune checkpoint blockade, adoptive T cell therapy, and cancer vaccines. Nevertheless, despite the centrality of cell interactions in biology , few technologies are available for characterizing them at scale. Instead, interactions are usually deciphered from bulk cultures based on cytokine release, cytotoxicity, surface marker presentation, or single-cell transcriptional profiling. While useful for characterizing synergistic behavior between cell types and overall response, bulk data lacks the detail necessary for precisely mapping cellular interactions at the heart of many biological systems, especially the immune system.

Characterizing cell-cell interactions is thus a large and unmapped frontier that has the potential to impact the treatment of numerous health maladies, especially cancer and autoimmunity. Although this has motivated new technologies, isolating specific interactions at scale remains difficult. For example, microfluidic approaches with droplets, microchambers, and wells exploit picoliter volumes to quantitate secreted cytokines and barcoding strategies to analyze DNA, messenger RNA, and proteins. By leveraging the inherent throughput of these methods, tens of thousands of cells can be analyzed, providing rich data. However, these approaches do not control cell loading, yielding mostly empty compartments. While this inefficiency is generally acceptable for single-cell studies, cell-cell interactions require combinations, which are impractical to generate randomly. For example, with a common loading of —5%, high-throughput approaches with 100,000 microcompartments capture ~5,000 single cells but just ~100 random cell-cell pairs. By implementing controlled cell loading with microfluidic cell pairing, printed droplets, or light- induced dielectrophoresis, every compartment can contain the needed number of specific cells, greatly increasing efficiency. These approaches generally perform well within their intended usages and function to analyze thousands of pairs. However, they were not designed with the intention of profiling the complexity of systems such as the immune repertoire, which comprises millions of T cells. Thus, there remains a need for technologies capable of rapidly generating controlled cell combinations. Such an approach would open the way for characterizing cellular interactions at scale and be valuable for numerous studies across fields including cancer, immunology, and microbiology.

SUMMARY OF THE INVENTION

In some embodiments, provided herein are devices and systems comprising: a) a base substrate, b) at least one microfluidic chip operably attached to the base substrate, wherein the at least one microfluidic chip is selected from: i) a drop making chip, ii) a drop assembly chip, and iii) a drop sorting chip; c) a plurality of micro or macro fluidic channels ; wherein the device or system is in the form of a cartridge that is configured to be inserted and removed from a cartridge receiving instrument.

In additional embodiments, the device or system comprises: a) a base substrate, b) at least one microfluidic chip operably attached to said base substrate, wherein said at least one microfluidic chip is selected from: i) a drop making chip, ii) a first drop assembly chip, iii) a second drop assembly chip; and iv) a drop sorting chip; and c) a plurality of micro or macro fluidic channels; wherein said device or system is in the form of a cartridge that is configured to be inserted and removed from a cartridge receiving instrument. In certain embodiments, the at least one microfluidic chip comprises said first drop assembly chip and said second assembly chip. In further embodiments, the second assembly chip is the last microfluidic chip present on the cartridge in the direction of fluid flow. In additional embodiments, the at least one microfluidic chip comprises said drop making chip, said first drop assembly chip, and said second assembly chip.

In particular embodiments, the at least one microfluidic chip is the drop assembly chip. In further embodiments, the at least two of the microfluidic chips are present and are operably attached to the base substrate, and wherein the plurality of micro or macro fluidic channels operably fluidically interconnect the at least two microfluidic chips. In additional embodiments, the at least two microfluidic chips include the drop making chip and the drop assembly chip. In some embodiments, the at least two microfluidic chips include the drop assembly chip and the drop sorting chip. In further embodiments, all three of the microfluidic chips are present and are operably attached to the base substrate, and wherein the plurality of micro or macro fluidic channels operably fluidically interconnect all three of the microfluidic chips.

In certain embodiments, the drop assembly chip comprises: i) a sorting region, ii) a droplet merger region, and iii) a trapping element (e g., comprising electrodes). In particular embodiments, the drop assembly chip is configured for deterministic sorting and trapping of at least two droplets to form a combined droplet (see, e.g., Figure 5). In other embodiments, the systems and devices further comprise: at least one input oil reservoir fluidically linked to the droplet making chip. In other embodiments, the systems and devices further comprise: a plurality of input sample tubes for receiving reagents or cells to be combined into droplets formed by the droplet making chip. In other embodiments, the sy stems and devices further comprise: droplet making selection valve fluidically linked to the droplet making chip by at least some of the plurality of macro or micro fluidic channels.

In additional embodiments, the systems and devices further comprise: a sample droplet reservoir and/or a dummy droplet reservoir, which are fluidically linked to the droplet making selection valve by at least some of the plurality of macro or micro fluidic channels. In other embodiments, the systems and devices further comprise: a droplet flow control valve which is fluidically linked to the sample droplet reservoir, and/or the dummy droplet reservoir, by at least some of the plurality of macro or micro fluidic channels. In some embodiments, the droplet flow control valve is fluidically linked to the droplet assembly chip by at least some of the plurality of macro or micro fluidic channels.

In other embodiments, the systems and devices further comprise: at least one oil input channel fluidically linked to the droplet assembly chip or to the droplet making chip (or at least one oil input channel fluidically linked to each of the droplet assembly chip or droplet making chip). In further embodiments, the at least one oil input channel is at least partially formed in the base substrate and/or are fluidically linked to the cartridge-receiving instrument.

In other embodiments, the systems and devices further comprise: a plurality of electrodes operably linked to the droplet assembly chip. In additional embodiments, the systems and devices further comprise: an assembly flow control valve fluidically linked to the droplet assembly chip and/or the droplet sorting chip by at least some of the plurality of macro or micro fluidic channels. In additional embodiments, the systems and devices further comprise: a sort negative channel or reservoir, and a sort positive channel or reservoir, which are fluidically linked to the droplet sorting chip by at least some of the plurality of macro or micro fluidic channels.

In particular embodiments, the systems and devices further comprise: a sort oil control valve, which is fluidically linked to the droplet sorting chip and/or the sort negative channel or reservoir and/or the sort positive channel or reservoir. In additional embodiments, the systems and devices further comprise: at least one oil waste port. In some embodiments, the at least one oil waste port is formed in the base substrate.

In certain embodiments, the plurality of micro or macro fluidic channels are formed in the base substrate (e.g., formed in the base substrate and then covered to form the top of such channels). In other embodiments, the plurality of micro or macro fluidic channels are the macro channels and have a diameter of about 0.5 to 1.5 mm. In particular embodiments, the plurality of micro or macro fluidic channels are the micro channels and have a cross section of about 0. 1 to 0.25 mm.

In additional embodiments, the systems further comprise: the cartridge-receiving instrument. In other embodiments, the cartridge-receiving instrument comprises at least one of the following components: i) a detector for detecting a sort region present on the droplet assembly chip and/or the droplet sorting chip; ii) an oil reservoir; iii) a pressurized gas source; iv) a thermal incubation source; and v) a waste oil collection bin. In some embodiments, the droplet assembly chip further comprises a sorting channel and a first outlet channel, and wherein the trapping electrodes comprise a first sorting electrode that exert an electromagnetic force sufficient to sort a droplet in the sorting channel to the first outlet channel. In certain embodiments, the electromagnetic force is a dielectrophoretic force. In additional embodiments, the electromagnetic force is an electrophoretic force.

In further embodiments, the sorting region comprises a sorting channel and wherein the trapping element comprise first and second sorting electrodes configured such that the first and second sorting electrodes form a bipolar electrode pair and the first trapping electrode is positively charged. In some embodiments, the first and second sorting electrodes are positioned on opposite sides of the sorting channel. In particular embodiments, at least one of the following applies: i) the first sorting electrode is positioned closer to the sorting channel than the second sorting electrode, ii) the second sorting electrode is positioned closer to the sorting channel than the first sorting electrode, iii) the distance between an end of the first sorting electrode, the second sorting electrode, or both and an interior wall of the sorter channel is between approximately 1 pm and approximately 100 pm, iv) the distance between the first sorting electrode and the second sorting electrode is approximately 25 pm to approximately 500 pm, v) the first sorting electrode and the second sorting electrode are connected to an alternating current electrical source with a frequency of approximately 0. 1 kHz to approximately 100 kHz and a voltage of approximately 10 V to approximately 10,000 V, vi) each sorting electrode comprises a liquid electrode, and vii) each sorting liquid electrode comprise one or more liquid channels imbedded in the base substrate and filled with conductive media.

In additional embodiments, the discrete entities (e.g., droplets) employed with the system and devices have a diameter of from about 1 pm to 1000 pm (e.g., 1 ... 100 ... 300 ... 700 ... 1000 pm). In other embodiments, the combined discrete entity (e.g., combined droplets) has a volume of from about 1 femtoliter to about 1000 nanoliters, or from 10 to 800 picoliters (e.g., 1 femtoliter .... 10 nanoliters ... 1000 nanoliters).

DESCRIPTION OF THE FIGURES

Figure 1 shows an exemplary droplet assembly workflow (e.g., to be performed partially or totally with the microfluidic cartridges herein) which allows for precisely defined assay droplets to be built, incubated, and sorted based on phenotypic readout (e.g., cytokine secretion). (A) Droplet assembly deterministically combines single-cell-containing droplets and reagent-containing droplets to create thousands of identical, defined assay droplets. (B) Assay droplets are incubated for a defined period, and secreted cytokine (or other molecule of interest) is captured onto the surface of a detection bead. (C) Assay droplets are sorted for downstream processing based on the presence of cytokine (or other molecule of interest).

Figure 2 shows a top-down view of an exemplary microfluidics cartridge.

Figure 3 shows an isometric view of an exemplary microfluidics cartridge.

Figure 4 provides an image of part of an exemplary droplet assembly chip having a spacer fluid channel, a bias fluid channel, a laminating oil inlet channel, a concentric sorter channel, a flow divider, and a recess according to embodiments of the present disclosure.

Figure 5 provides images of a part of an exemplary droplet assembly chip having a concentric sorter channel, a recess, and an approximately triangular downstream region according to embodiments of the present disclosure.

Figure 6, panels i-iv, show a zoomed-out view of part of an exemplary droplet assembly chip that may form part of a microfluidic cartridge. A droplet (e.g., containing a first cell) is detected as it enters the droplet sorting region (i), the sorting electrode is actuated to redirect the drop towards the assembly lane (n), and the sorted droplet merges with the droplet-in-assembly at the DEP (dielectrophoretic) trap to form a combined droplet (iii) (e.g., containing a second cell). Following assembly, the DEP trap is turned off to release the combined droplet (iv). Panels v-viii show a close-up of the merging process. Four droplets (e.g., containing a total of two cells) are sorted by their fluorescent signature and directed to the DEP trap for merging (v). As the droplets encounter the actuated trap, they are sequentially merged into the assembled droplet (vi-vii). The electrode is then temporarily turned off so the assembled droplet may be released and recovered downstream (viii) (e.g., after being sorted for the presence of live cells).

Figure 7 provides a schematic flow diagram of a method of selectively combining discrete entities using part of a droplet assembly chip.

Figures 8 provides a schematic showing example configurations for trapping a discrete entity (e.g., droplet) on part of an droplet assembly chip. Panel i) shows a bipolar electrode pair embedded in the same side wall of a channel. Panel ii) shows a bipolar electrode pair embedded on opposite sides of channel Panel iii) shows bipolar electrode pair embedded in the floor or ceiling of a channel.

Figure 9 provides a schematic showing exemplary configurations for directing discrete entities to a discrete entity merger region on an exemplary droplet assembly chip. Panel i) shows application of a lamination flow to confine the laminar flow containing the droplet to the side wall of the channel. Panel ii) shows a partial height flow divider that allows fluid, but not droplets to enter the center portion of the channel. Panel hi) shows a configuration where a groove of similar height to the droplet dimensions is patterned near the side wall of a channel, while the rest of the channel is constructed with a reduced height to exclude droplets. Panel iv) shows a porous flow divider that allows fluid, but not droplets to enter the center portion of the channel. Panel v) shows a partial height flow dividers that direct droplets to a trap at the center of the microfluidic channel.

Figure 10 provides a schematic showing an exemplary' embodiment wherein trapping is facilitated by a mechanical valve which may be present on an exemplary droplet assembly chip. Panel i) shows an initial stage where the discrete entities are trapped by the valve. Panel ii) shows a second stage wherein the discrete entities have been combined, e.g. due to electrical, chemical, or other means. Panel iii) shows a third stage where the combined discrete entity is released by opening the valve and carried downstream.

Figure 11 provides a schematic showing exemplary embodiments with different channel geometries (which may be used in an exemplary microfluidic cartridge) in proximity to an electromagnetic trapping element that may be part of a droplet assembly chip. Panel i) shows a discrete entity merger region upstream of a bend in the channel wall. Panel ii) shows a discrete entity merger region in a lateral facet in the channel wall. Panel iii) shows a discrete entity being trapped in a region that is vertically taller than the main channel.

Figure 12A shows first and second exemplary antibody-oligonucleotides and how they bind in proximity to each other on different epitopes of a target protein such that a template structure is formed. An oligonucleotide probe hybridizes to the template structure, which then allows the nickase enzyme to cleave the probe releasing the fluorophore and quencher. In this regard, the signal from the fluorophore is no longer quenched and is detectable, indicating that the target protein has been detected. This cycle repeats as more and more oligonucleotide probes are cleaved. Figure 12B shows certain parts of the oligo template structure labeled, along with a nickase enzyme and an oligonucleotide probe.

DETAILED DESCRIPTION

Provided herein are systems, devices, and methods for using microfluidic cartridges (e.g., to characterize cell-cell interactions in droplets). In certain embodiments, the microfluidic cartridges comprise at least one microfluidic three chip (e.g., selected from a drop making chip, a drop assembly chip, and a drop sorting chip) attached to a base substrate, and are configured to be removably inserted into a cartridge receiving instrument. In some embodiments, the drop assembly chip comprises a sorting region, a droplet merger region, and a trapping element (e.g., composed of trapping electrodes) and is configured for deterministic sorting and trapping of at least two droplets to form a combined droplet (e.g., containing one cell type from each of the droplets).

Provided herein are microfluidic cartridges that facilitate the types of droplet assembly, assays, and sorting shown in the exemplary workflow described in Figure 1. Exemplary cartridges are shown in Figures 2 and 3, which may have individual microfluidic chips (e g., 3 chips) that are in sequence In particular embodiments, the microfluidic cartridges comprise a drop making chip, a drop assembly chip, and a drop sorting chip, all operably attached to a base substrate, where the microfluidic cartridge is configured to removably inserted into a cartridge receiving instrument. In certain embodiments, the chips are bonded (e.g., laser welding, pressure sensitive adhesives, adhesives, other chemical bonding methods) to a base substrate (e.g., solid planar platform on which the chips are assembled). The entire cartridge can also be molded, for example, as a monolithic piece. Chips may be made, for example, from COC (Cyclic olefin copolymer) or COP (Cyclic olefin copolymer), or from any other suitable plastic (e.g., PMMA, polycarbonate, polypropylene etc.).

The cartridges herein may have reservoirs for input sample reagents and oils (See, Figures 2 and 3). There are, in some embodiments, reservoirs for each stage of the process (e.g., process like shown in Figure 1). For example, a reservoir for sample drops and dummy (spacer) drops. There may also be a reservoir for the assembled drops, and two reservoirs for the sorted drops (e.g., which may be either assay positive of assay negative, depending on the assay run in the drops).

In particular embodiments, the cartridge has molded macro-channels between micro channel chips and macro reservoirs and valves. The macro channels may be, for example, about 0.5 mm to about 1 mm, or 0.25 to 1.5 mm, in cross section. In certain embodiments, the macro-channels are molded into the base substrate and then sealed with a polymer film. The film can be, for example, laser welded, bonded with pressure sensitive adhesives, thermally bonded etc.

In certain embodiments (e g., as shown in Figures 2 and 3), in between chips and reservoirs there are macro-scale valves the control flow for each stage to the next. These valves may be, for example, elastomer bonded or molded to the base substrate. The valves are generally actuated by the cartridge-receiving instrument. The valves allow the user to control drop making, drop assembly and drop sorting. The assembly and sorting process geneally uses more oil by volume (e.g., 20X) than the input sample drops. This excess oil may be drained from collected assembly and sort reservoirs. Valves at the output also allow the user to control any excess oil used at any step.

In some embodiments, the cartridge allows for viewing of the sort region of each chip by the detection optics (e.g., fluorescent detection optics) in the cartridge-receiving instrument. In particular embodiments, a multiple line laser illuminates the sort region of the chip, and the assay signal (e.g., fluorescent signal) from each dop event is detected by collection PMTs in the cartridge-receiving instrument. The chips can also be viewed, for example, with a high speed camera on the cartridge-receiving instrument. The base substrate of the cartridge may also provide electrode connections to the various chips. The connections can go, for example, to the sort region of the chips and the trap region of the chips.

Input system oil can be introduced in wells on the cartridge or can be introduced from the cartridge-receiving instrument at input points on the cartridge. The oil for making drops is a small volume and can be on the cartridge or on the cartridge-receiving instrument. The oil used for flow during assembly and sort is generally as large volume and can be stored on the cartridge-receiving instrument.

In certain embodiments, waste ports on the cartridge allow excess oil to flow out of the collection reservoirs. Each reservoir can be connected to a pressurized gas source (e.g., on the cartridge-receiving instrument), that can be controlled for temperature, relative humidity and CO2 composition. The cartridge can also be temperature controlled, heated or cooled (e g., by equipment in the cartridge-receiving instrument).

I. Exemplary Cartridges and Droplet Flow in Figures 2 and 3

The following provides an exemplary description of the components of the exemplary cartridges shown in Figures 2 and 3, which allow, for example the steps in Figure 1 to be carried out.

Input oil for creating drops is added to the Input Oil Reservoirs. Samples, cells, reagents etc. are added to the Input Sample Reservoirs. In certain embodiments, 1 to 5 (or 3- 8) reservoirs are present. More lanes can also be added. Pressure is then provided to the input samples and oil to drive the reagents into the Drop Making Chip. The output drops from the Drop Making Chip are collected in the Sample Drop Reservoirs. All lanes can be run simultaneously or individual lanes may be run. Lanes are controlled with the Drop Making Selection Valve which can run each lane at one time. Instead of pressure to the input reservoirs, vacuum can be applied to the Sample Drop Reservoirs and this will provide the needed flow. During the drop making process the Drop Flow Control Valve is closed. Dummy drops can also be created and they flow into the Dummy Drop Reservoir.

Once drop making is completed, the Drop Making Selection Valve closes all lanes. The Drop Flow Control Valve is opened. A pressurized gas mixture is applied to the Sample Drop Reservoir and Dummy Drop Reservoir and the Assembly Reservoir. The pressure at the Assembly Reservoir is lower than at the Sample and Dummy drop reservoirs so that the drops can flow into the Drop Assembly Chip. In addition Spacer and Bias oils from the instrument flow in via Oil Input ports. These oils are used to control drop timing during assembly. The fluorescence signals of drops are measured and the appropriate drops are sorted by the dielectrophoretic sort region of the chip and then trapped (e.g., in the dielectrophoretic trap) in the chip to create the desired assembly. Electrodes bring the appropriate high voltages to the chips.

Once an assembly drop is created it is released from its dielectrophoretic trap. The process is repeated for each assembly until all input drops are depleted. As assembled drops are collected (in Assembly Reservoir), excess oil will flow either out a waste channel or through the Assembly Reservoir. The Assembly Reservoir has an Assembly Flow Control Valve. This valve has at least 3 positions: closed, open to waste and closed to Sort Chip, and finally open to Sort Chip (closed to waste). The Assembly Flow Control Valve is opened to waste at specific times to allow excess oil to drain via the Oil Waste Ports. The excess oil drains from the bottom of the reservoir as drops float on top. When assembly is completed the Assembly Flow Control Valve is closed.

The assembled drops, cells reagents etc. are then incubated while appropriate biochemical reactions occur. During incubation the cartridge can be removed and placed in an incubation environment. The cartridge can also be left in the cartridge-receiving instrument and the incubation environment can be applied to the reservoirs by the appropriate controlled gas mixture or other means. Temperature of the cartridge can also be controlled.

Once incubation is complete the assemblies are then sorted. The Assembly Flow Control Valve is opened so assembly drops can flow into the Sort Chip. A pressurized gas mixture is applied to the Assembly Reservoir and the Positive Sort Reservoir and Negative Sort Reservoir. The Assembly Reservoir pressure is higher so drops can flow. Spacer and Bias oil are also pressurized and flow via Oil input. Drops and oil flow through the Sort Chip. Fluorescent signal is detected from each drop. Assay Positive or Assay Negative assemblies are sorted by the dielectrophoretic sorter region into the appropriate channel. Electrodes provide the high voltage connection to the chip. As excess oil fills each sort reservoir the Sort Oil Control Valve is opened and closed to drain excess oil via the Oil Waste Ports. Once all drops are sorted the Sort Oil Control Valve is closed and sorted drops can be collected.

II. Cartridge Components and Methods

In some embodiments, the discrete entities (e.g., droplets) herein are flowed in a microfluidic cartridge, which may be used to combine multiple drops such that all the reagents are combined into a single discrete entity. In certain embodiments, the components of the microfluidic cartridges are described in PCT application W02020232072A1 and Cole et al., Proc. Natl. Acad. Sci., 114(33): 8728-8733, 2017, which are both incorporated by reference herein in their entireties. In certain embodiments, the microfluidic cartridge comprises a combination of deterministic single-cell droplet sorter chip (droplet sorting chip) and droplet-assembler chip (drop making chip) that can selectively assemble cells and reagents. In certain embodiments, the microfluidic cartridges perform a cyclic buildup and release of designer droplets through the merging of select droplets on a defined dielectrophoretic trapping position inside the microfluidic device (e.g., Fig. 6). This approach is advantageous because it is less prone to contamination, higher throughput, and requires fewer moving parts than other devices. The flexible of this platform makes it a well-suited technology to perform integrated and functional cell-cell, cell-ECM interaction analysis and link any perturbations to select expressed gene sequences or transcriptome profiles at a single cell level. Essentially, cartridges herein allow for precise, flexible, scalable liquid handling that can build a large number of predetermined reaction conditions.

In certain embodiments, droplet manipulation and sorting is achieved by electro wetting, the modification of the wetting properties of a surface with an applied electric field. Electrowetting manipulation of droplets in a microfluidic device may be achieved through the application of differential voltages to different regions in an electrode grid (see, US Pat. 6,911,132, herein incorporated by reference). Alternatively, droplet actuation and sorting can be achieved using opto-electrowetting, where localized electric fields are triggered through the selective application of light to a photoconductive layer (see, US Pat. 6,958,132, which is herein incorporated by reference in its entirety).

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

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

Exemplary double and multiple emulsions are described in U.S. Pat. 9,238,206, which is herein incorporated by reference in its entirety, particularly for such double and multiple emulsions. In general, a multiple emulsion describes larger droplets that contain one or more smaller droplets therein. In a double emulsion, the larger droplets may, in turn, be contained within another fluid, which may be the same or different than the fluid within the smaller droplet. In certain embodiments, larger degrees of nesting within the multiple emulsion are possible. For example, an emulsion may contain droplets containing smaller droplets therein, where at least some of the smaller droplets contain even smaller droplets therein, etc. Multiple emulsions can be useful for encapsulating species such as pharmaceutical agents, cells, antibodies, proteins, chemicals, or the like. In certain embodiments, a double emulsion is produced, i.e., a carrying fluid, containing a second fluidic droplet, which in turn contains a first fluidic droplet therein. In some cases, the carrying fluid and the first fluid may be the same. The fluids may be of varying miscibilities, e.g., due to differences in hydrophobicity. For example, the first fluid may be water soluble, the second fluid oil soluble, and the carrying fluid water soluble. This arrangement is often referred to as a w/o/w multiple emulsion (“water/oil/water”). Another double emulsion may include a first fluid that is oil soluble, a second fluid that is water soluble, and a carrying fluid that is oil soluble. This type of double emulsion is often referred to as an o/w/o double emulsion (“oil/water/oil”). It should be noted that the term “oil” in the above terminology merely refers to a fluid that is generally more hydrophobic and not miscible in water, as is known in the art. Thus, the oil may be a hydrocarbon in some embodiments, but in other embodiments, the oil may comprise other hydrophobic fluids.

In certain embodiments, the discrete entities herein (e.g., droplets) comprise a hydrogel shell or microcapsule, such as exemplified in U.S. Pat. 10,710,045 and U.S. Pat. Pub. 20140127290, both of which are herein incorporated by reference in their entireties, particularly for such hydrogel shells or microcapsules. In certain embodiments, the hydrogel shells for the discrete entities, or microcapsules, comprise a liquid core, and at least one external envelope totally encapsulating the liquid core at its periphery, said external envelope being able to retain the liquid core when the capsule is immersed into a gas and comprising at least one gelled polyelectrolyte and/or a stiffened biopolymer. In certain embodiments, such microcapsules contain a cell and/or other reagents discussed herein. In certain embodiments, a microcapsule refers to a particle or capsule having a mean diameter of about 50 pm to about 1000 pm, formed of a cross-linked hydrogel shell surrounding a biocompatible matrix. The microcapsule may have any shape suitable for cell encapsulation. The microcapsule may contain one or more cells dispersed in the biocompatible matrix, cross-linked hydrogel, or combination thereof, thereby “encapsulating” the cells.

In some embodiments, the subject discrete entities (e.g., droplets) have a dimension, e.g., a diameter, of or about 1.0 pm to 1000 pm, inclusive, such as 1.0 pm to 750 pm, 1.0 pm to 500 pm, 1.0 pm to 100 pm, 1.0 pm to 10 pm, or 1.0 pm to 5 pm, inclusive. In some embodiments, discrete entities as described herein have a dimension, e.g., diameter, of or about 1.0 pm to 5 pm, 5 pm to 10 pm, 10 pm to 100 pm, 100 pm to 500 pm, 500 pm to 750 pm, or 750 pm to 1000 pm, inclusive. Furthermore, in some embodiments, discrete entities as described herein have a volume ranging from about 1 fL to 1 nL, inclusive, such as from 1 fL to 100 pL, 1 fL to 10 pL, 1 fL to 1 pL, 1 fL to 100 fL, or 1 fL to 10 fL, inclusive. In some embodiments, discrete entities as described herein have a volume of 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. In addition, discrete entities as described herein may have a size and/or shape such that they may be produced in, on, or by a microfluidic device and/or flowed from or applied by a microfluidic device.

In some embodiments, the discrete entities as described herein are droplets. The terms “drop,” “droplet,” and “microdroplet” are used interchangeably herein, to refer to small, generally spherically structures, containing at least a first fluid phase, such as an aqueous phase (e.g., water), bounded by a second fluid phase (e.g., oil) which is immiscible with the first fluid phase. In some embodiments, 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, such as water). In some embodiments, the second fluid phase is an immiscible phase carrier fluid. Thus, droplets according to the present disclosure may be provided as aqueous-in-oil emulsions or oil in aqueous emulsions. Droplets may be sized and/or shaped as described herein for discrete entities. For example, droplets according to the present disclosure generally range from 1 pm to 1000 pm, inclusive, in diameter. Droplets according to the present disclosure may be used to encapsulate cells, e.g., cells, reagents for making oligo template structures, nickases, quenched oligonucleotide probes, nucleic acids (e.g., DNA), enzymes, reporter dyes, reagents, and a variety of other components. The term droplet may be used to refer to a droplet produced in, on, or by a microfluidic device and/or flowed from or applied by a microfluidic device.

As used herein, the term “di electrophoretic force” refers to the force exerted on an uncharged particle caused by the polarization of the particle by and interaction with a nonuniform electric field. A dielectrophoretic force can be directed towards (i.e. “attractive dielectrophoretic force”), away from (i.e. “repulsive dielectrophoretic 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.

As used herein, the term “electrophoretic force” refers to the force exerted on a charged particle caused by interaction with an electric field. An electrophoretic force can be directed towards (i.e. “attractive electrophoretic force”) away from (i.e. “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.

As used herein, the term “carrier fluid” refers to a fluid configured or selected to contain one or more discrete entities (e.g., droplets) as described herein. A carrier fluid may include one or more substances and may have one or more properties (e.g., viscosity), which allow it to be flowed through a microfluidic device or a portion thereof. In some embodiments, carrier fluids include, for example: oil or water, and may be in a liquid or gas phase.

The present disclosure provides methods of selectively moving and/or combining discrete entities using microfluidic cartridges (e.g., to combine target and effector cells), as shown in Figures 2 and 3.

FIG. 4 presents a schematic representation of part of a droplet assembly chip. In some cases, a discrete entity merger region includes a recess, such as shown as recess 107 in FIG. 4. In some cases, the discrete entity merger region includes a flow divider, such as shown as flow divider 113 in FIG. 4. In some cases, the droplet assembly chip further includes a laminating oil inlet, such as shown as laminating oil inlet 112 in FIG. 4. In some cases, a trapping element is present that includes two electrodes that have a significantly different shape from one another, such as shown as electrodes 109 in FIG. 4. In some cases, the trapping element includes two electrodes that produce a region of high electric field gradients that extends into the microfluidic channel. In some cases, the discrete entity merger region includes a change in the angle of flow between an adjacent upstream region and the discrete entity merger region, e.g. as shown in FIG. 5. In some cases, the droplet assemblychip includes a spacer fluid inlet. As an example, the chip in FIG. 4 includes spacer fluid channel 110 in fluid communication with the inlet channel 101. The spacer fluid channel can be configured such that flowing spacer fluid through the spacer fluid channel causes spacer fluid to be located between two discrete entities flowing through the inlet channel, thereby maintaining or increasing the distance between the two discrete entities, thereby allowing each of the two discrete entities to be independently sorted or not sorted.

In some cases, the droplet assembly chip further includes a bias fluid inlet. As an example, the droplet assembly chip in FIG. 4 includes bias fluid channel 111 in fluid communication with sorter channel 102. The bias fluid channel can be configured such that flowing bias fluid through the bias fluid channel will cause a discrete entity to move closer to a second side wall of the sorter channel and farther away from a first side wall of the sorter channel. Thus, as an example, the spacer fluid inlet 111 would cause the discrete entity to move closer to the wall of the inlet channel that is closer to the bottom of the figure, and further away from the wall closer to the top of the figure. As such, one or more bias fluid channels can be configured such that a discrete entity will preferentially flow to a first outlet location or a second outlet location in the absence of a force from a sorting element. In some cases, the bias fluid inlet channel can be configured such that a discrete entity will preferentially flow to a second outlet channel in the absence of a dielectrophoretic force from a sorting electrode. As an example, the bias fluid inlet 111 in FIG. 4 causes a discrete entity to preferentially flow to second outlet channel 105 in the absence of a force exerted on the discrete entity by the sorting electrodes 103.

In some cases, the droplet assembly chip includes a detector configured to detect a discrete entity in the input channel (e.g., to detect if it contain a target protein), wherein the droplet assembly chip is configured to sort a discrete entity in the sorting channel based on the detection by the detector. As an example, FIG. 4 shows an embodiment in which a discrete entity in detection region 114 of inlet channel 101 can be detected by a detector, after which sorting electrodes 103 can sort the discrete entity into the first outlet channel 104 or the second outlet channel 105. The FIG. 4 droplet assembly chip also includes shielding electrodes 115a, 115b, 115c, and 115d. As used herein, the term “shielding electrode” is used interchangeably with “moat electrode.” Each shielding electrode can be configured to perform one or more functions including: at least partially shielding discrete entities from undesired electromagnetic fields, assisting with the sorting of discrete entities, and assisting with the trapping of discrete entities.

As such, as used herein, shielding electrodes can also be referred to as sorting electrodes or trapping electrodes if such electrodes are configured to participate in the sorting or trapping of discrete entities. Hence, shielding electrode 115a can also be referred to as a sorting electrode if it is configured to form a bipolar electrode pair with sorting electrode 103 to facilitate the sorting of discrete entities. Similarly, shielding electrode 115d can also be referred to as a trapping electrode if it is configured to form a bipolar electrode pair with trapping electrode 109 to facilitate the trapping of discrete entities.

In some cases, a shielding electrode can generate an electromagnetic field such that discrete entities in the droplet assembly chip is at least partially shielded from undesired electromagnetic fields. Such undesired electromagnetic fields can originate from outside the microfluidic device or from within the microfluidic cartridge. In some cases, the undesired electromagnetic fields are those fields that are not generated by a sorting electrode or by a trapping electrode. By at least partially shielding discrete entities in the droplet assembly chip, the shielding electrodes can inhibit the unintended merging of discrete entities (i.e. merging of discrete entities outside the discrete entity merger region). In some cases, shielding electrodes 115a, 115b, and 115c can be used to at least partially shield discrete entities from electromagnetic fields that are not generated by the sorting electrode or the trapping electrode.

In some cases, shielding electrodes can assist with the sorting of discrete entities. As an example, shielding electrode 115a can interact with sorting electrode 103 in order to facilitate sorting, such as by forming a bipolar electrode pair with sorting electrode 103. In some cases, sorting electrode 103 can be the charged electrode (e g. positively charged), and shielding electrode 115a can be a ground. Stated in another manner, shielding electrode 115a can be configured to influence the shape of the electromagnetic field generated by sorting electrode 103 in order to facilitate sorting.

In some cases, shielding electrodes can assist with the trapping of discrete entities. As an example, shielding electrode 115d can interact with trapping electrode 109 in order to facilitate trapping, such as by forming a bipolar electrode pair with trapping electrode 109. In some cases, sorting electrode 109 can be the charged electrode (e g. positively charged), and shielding electrode 115d can be a ground. Stated in another manner, shielding electrode 115d can be configured to influence the shape of the electromagnetic field generated by trapping electrode 109 in order to facilitate sorting.

In some cases, one or more of the shielding electrodes are separate elements, such as when all 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, such as part of a single piece of metal. In some cases, one or more of the shielding elements are attached to ground.

As shown in FIG. 4, in some cases, the droplet assembly chip includes one or more shielding electrodes. In some cases, the droplet assembly chip includes zero shielding electrodes, such as when the discrete entities are sorted using a single sorting electrode and the discrete entities are trapped using a single trapping electrode.

As such, discrete entities are sorted and selectively combined within a droplet assembly chip. Stated in another manner, the discrete entities may be sorted and combined without leaving microfluidic sized channels and regions. As reviewed above, the trapping element and the sorting element can be electrodes that exert a dielectrophoretic force on the discrete entity. In some cases, the electrodes are microfluidic channels containing a conductive material (e.g. salt water, liquid metal, molten solder, or a conductive ink to be annealed later). In some cases, the electrodes are patterned on the substrate of the microfluidic device (e.g. a patterned indium tin oxide (ITO) glass slide). In some cases, the trapping element includes two electrodes. In some cases, the trapping element is a selectively actuatable bipolar droplet trapping electrode. In some cases, the sorting element includes two electrodes. In some cases, the sorting element includes a selectively actuatable bipolar droplet sorting electrode.

In some cases, the sorting channel includes a partial height flow divider. In some cases, the sorting channel has a concentric or essentially concentric flow path and a portion of the sorting electrode is positioned at the center of the arc of the concentric or essentially concentric flow path.

In some embodiments, the discrete entity (e.g., droplet) includes particles (e.g., cells, reagents, etc.). In some embodiments, the discrete entity includes a chemical reagent (e.g. a lysing agent or a PCR reagent). In some embodiments, the discrete entity includes both a cell and a chemical reagent.

In some cases, the sorting is passive sorting. In some cases, the sorting is active sorting (i.e., the sorting element sorts a discrete entity into one of at least two locations based on a detected property of the discrete entity or a component within the discrete entity, such as a signal from cleaved oligonucleotide probe). In some cases, the detected property is an optical property (e.g., from a reporter dye no longer quenched) and the device further includes an optical detector (e.g. an optical detector configured to detect an optical property of a discrete entity or a component within in the inlet channel). In some cases, the optical property is fluorescence and the device further includes a source of excitation light. In some cases, the sorting is based on the detected fluorescence of a reporter dye that forms a FRET pair in a dead or dying cell with the dye already present in the dead or dying cell.

In some cases, the discrete entity merger region can include structural elements that are configured to aid in the trapping and combination of discrete entities therein. In some cases, such structural elements are configured to aid in such trapping and combining by changing the speed or direction of the flow of fluid through an area of the discrete entity merger region.

The present disclosure also provides methods of using a microfluidic cartridge that comprises a droplet making chip, a droplet assembly chip, and a droplet sorting chip, and one or more additional components that are part of the microfluidic cartridge and/or cartridgereceiving instrument (a) a temperature control module operably connected to the microfluidic cartridge; (b) a detector configured to detect a discrete entity in the input channel, wherein the microfluidic cartridge is configured to sort a discrete entity in the sorting channel based on the detection by the detector in the cartridge-receiving instrument; (c) an incubator; and (d) a sequencer. In some cases, the methods of using the microfluidic cartridges herein include controlling the temperature of the cartridge using a temperature control module that is part of the cartridge-receiving device. In some cases, the methods include detecting a discrete entity in the input channel of a droplet assembly chip (e.g. detecting an optical property of the discrete entity or a component therein), and sorting the discrete entity based on the detecting. In some cases, the method includes incubating cells in an incubator that is operably connected to microfluidic cartridge. In some cases, the method includes making discrete entities with a drop making chip that is part of the microfluidic cartridge.

The present disclosure also provides steps that can be performed after the release of a combined microfluidic droplet from a discrete entity merger region. In some cases, the method includes recovering a component (e.g. a cell, a chemical compound or a combination thereof), from the combined discrete entity. In cases where a combined discrete entity includes one or more cells, the one or more cells can be analyzed, for example, as shown in Figure 1 to detect target protein(s) that are secreted.

The present methods allow for the selective combination of two or more discrete entities without the need to accurately time the release or to accurately time the sorting of the two or more discrete entities. As such, in some cases, a first discrete entity (e.g., containing a first cell) is trapped in the discrete entity merger region before a second discrete entity (e.g., containing a second cell) to be combined therewith has entered the outlet channel after being sorted. In some cases, the second discrete entity has not entered the sorter channel, has not entered the inlet channel, or has not even been made when the first discrete entity is trapped in the discrete entity merger region.

The present methods allow for the sorting of discrete entities (with a droplet sorting chip that is part of the cartridge) based on whether they contain the selective combination of only those discrete entities that contain the desired components. In some cases, the method involves creating 5 or more combined discrete 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 method involves making 300 or more combined discrete 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. In some cases, the sorting step is performed such that discrete entities are sorted at a rate of 0.01 Hz or more (e.g. 0.1 Hz or more, 1 Hz or more, 10 Hz or more, 100 Hz or more, 1 kHz or more, 10 kHz or more, or 30 kHz or more). In some cases, an electromagnetic sorter is used instead of a mechanical sorter (e.g. a valve, to allow for faster sorting rates). In some cases, the trapping and combining steps are performed such that a combined discrete entity is formed or released at a rate of 1 Hz or more, e.g. 10 Hz or more, 100 Hz or more, or 1,000 Hz or more.

In some cases, a discrete entity is flowed such that it reaches the discrete entity merger region between 0. 1 ms to 1,000 ms after being sorted, such as between 1 ms and 100 ms, between 2 ms and 50 ms, and between 5 ms and 25 ms. In some cases, the first outlet channel is between 0.2 mm long and 5 mm long. In some cases, the first outlet channel has a dimension (e.g., width or height or diameter) of between 5 gm and 500 pm, such as between 10 m and 100 pm. In some cases, the carrier fluid containing the discrete entities is flowed into the inlet channel at a rate of between 1 pl per hour and 10,000 pl per hour, such as between 10 pl per hour and 1,000 pl per hour, 25 pl per hour and 500 pl per hour, and between 50 pl per hour and 250 pl per hour. In some cases, the spacer fluid is injected at a rate of between 100 pl per hour and 20,000 pl per hour, such as 500 pl per hour and 5,000 pl per hour. In some cases, the bias fluid is injected at a rate of between 100 pl per hour and 20,000 pl per hour, such as 500 pl per hour and 5,000 pl per hour. In some cases, the fluid used to create cell-containing discrete entities has a concentration of between 1,000 cells per ml and 10,000,000 cells per ml, such as between 10,000 cells per ml and 1,000,000 cells per ml, and between 50,000 cells per ml and 200,000 cells per ml. In some cases, the discrete 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.

In some cases, the one or more cells from a discrete entity or a combined discrete entity are cultured for at least 30 minutes or more, such as 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. In some cases, the droplet assembly chip can continuously operate by selectively combining discrete entities for 10 minutes or more, such as 30 minutes or more, 45 minutes or more, 90 minutes or more, or 180 minutes or more. In some cases, the device can make at least 100 combined discrete entities while continuously operating, such as 1 ,000 combined discrete entities or more, 10,000 combined discrete entities or more, or 100,000 combined discrete entities or more.

In some cases, the methods include making one or more discrete entities, such as with a droplet making chip that is part of the cartridge. In such cases, the droplet making chip can be part of the cartridge or separate from the cartridge as otherwise described herein. If the droplet making chip is separate from the cartridge, the droplet making chip can be operably connected to the cartridge (e.g., such that discrete entities can flow from the maker to the cartridge), or the discrete entities can be moved to the cartridge. The cartridges can include one or more droplet making chips (comprising a droplet maker) configured to form discrete entities from a fluid stream. Suitable droplet making chip include selectively activatable droplet makers and the methods may include forming one or more discrete entities via selective activation of the droplet maker. The methods may also include forming discrete entities using a droplet maker, wherein the discrete entities include one or more entities which differ in composition. In some cases, the discrete entity maker comprises a T-junction and the method includes T-junction drop-making. In some cases, making the discrete entities includes a step of emulsification. In some cases, the discrete entity maker is made, in part or in whole, of a polymer. In some cases, one or more surfaces of the discrete entity maker are coated with a fluorosilane (e.g. such a discrete entity maker can be used when fluorinated fluids are passed through the discrete entity maker).

In some cases when multiple types of discrete entities are made (e.g., discrete entities that contain different contents, such as one dyed first cell and one with a second dyed cell), the contents can affect the ability of the discrete entity maker to successfully make the discrete entities. As such, in some cases, different conditions for the discrete entity maker are used to make a first group of discrete entities with first contents than for making a second group of discrete entities with second contents.

Aspects of the disclosed methods may include making discrete entities using one or more cells from a biological sample. In such cases, each discrete entity may contain zero, one, or more than one cell. In some cases, such discrete entities can be made by incorporating the biological sample, cells from the biological sample, lysate from cells of the biological sample, or any other sample derived from the biological sample into a mixed emulsion. In some cases, the method further includes separating one or more components of the biological sample or otherwise processing the biological sample (e.g. via centrifugation, filtration, and the like), before making the discrete entities.

In some cases, after the making of the discrete entities but before introducing the discrete entities to an inlet channel of a microfluidic device as described herein, the discrete entities can be further modified (e.g. by adding reagents for making oligo template structures, nickases, quenched oligonucleotide probes, a dyed cell, a reagent, a drug, a hydrogel, an extracellular matrix, a bead, a particle, a biological material, media, or a combination thereof). In some cases, the bead is an RNA capture bead. In some cases, the bead is an immunoassay bead. In some cases, the barcode is an oligonucleotide. In some cases, different types of discrete entities are labeled with different ty pes of barcodes, fluorescent tags (e.g., on the oligonucleotide probe), or a combination thereof. Fluorescent tags (on the oligo probe, and/or dying a cell generally) can be used to image a discrete entity or combined discrete entity in the discrete entity merger region. Fluorescent tags can also be used to identify the particular type of discrete entities that were combined to create a given combined discrete entity. As such, the properties of the combined discrete entity or component thereof can be correlated with the contents that were used to make the original discrete entities. As an example, different types of cells can be labeled with different fluorescent tags and incorporated into discrete entities. After such cell-containing discrete entities are combined with other discrete entities (e g containing other cells), the outcome of the combined discrete entities can be observed. As some of all of the original discrete entities can be labeled with fluorescent tags, the resulting combined discrete entity can have multiple fluorescent tags. In other cases, the combined discrete entity only has one fluorescent tag. Oligonucleotide barcodes can be used in a similar manner to that of fluorescent tags. Instead of detecting optical fluorescence, however, the oligonucleotide barcodes can be sequenced in order to identify the original discrete entities that formed the combined discrete entity.

Methods and devices which may be utilized in the encapsulating of a component from a biological sample are described in PCT Publication No. WO 2014/028378, the disclosure of which is incorporated by reference herein in its entirety and for all purposes. Encapsulation approaches of interest also include, but are not limited to, hydrodynamically -triggered drop formation and those described by Link, et al., Phys. Rev. Lett. 92, 054503 (2004), the disclosure of which is incorporated herein by reference. Other methods of encapsulating cells into droplets may also be applied. Where desired, the cells may be stained with one or more antibodies and/or probes prior to encapsulating them into drops.

One or more lysing agents may also be added to the discrete entities (e.g., droplets), containing a cell, under conditions in which the cell(s) may be caused to burst, thereby releasing their genomes and target proteins. The lysing agents may be added after the cells are encapsulated into discrete entities. Any convenient lysing agent may be employed, such as proteinase K or cytotoxins. In particular embodiments, cells may be co-encapsulated in drops with lysis buffer containing detergents such as Triton X100 and/or proteinase K. The specific conditions in which the cell(s) may be caused to burst will vary' depending on the specific lysing agent used. For example, if proteinase K is incorporated as a lysing agent, the discrete entities (e.g., droplets), may be heated to about 37-60°C for about 20 min to lyse the cells and to allow the proteinase K to digest cellular proteins, after which they may be heated to about 95°C for about 5-10 min to deactivate the proteinase K. In certain aspects, cell lysis may also, or instead, rely on techniques that do not involve addition of lysing agent. For example, lysis may be achieved by mechanical techniques that may employ various geometric features to effect piercing, shearing, abrading, etc. of cells. Other types of mechanical breakage such as acoustic techniques may also be used. Further, thermal energy can also be used to lyse cells. Any convenient methods of effecting cell lysis may be employed in the methods described herein as appropriate.

One or more primers may be introduced into the discrete entities for each of the genes to be detected. Hence, in certain aspects, primers for all target genes (e g., antibody genes) may be present in the discrete entity at the same time, thereby providing a multiplexed assay. The discrete entities may be temperature-cycled so that discrete entities will undergo PCR. In certain embodiments, rolling circle amplification (RCA)-based proximity ligation is employed.

In some embodiments, a surfactant may be used to stabilize the discrete entities. In some cases, the discrete entities or the associated emulsion lack a surfactant. Accordingly, a discrete entity may involve a surfactant stabilized emulsion. Any convenient surfactant that allows for the desired reactions to be performed in the discrete entities, may be used. In other aspects, a discrete entity is not stabilized by surfactants or particles. The surfactant used depends on a number of factors such as the oil and aqueous phases (or other suitable immiscible phases (e.g., any suitable hydrophobic and hydrophilic phases)) used for the emulsions. For example, when using aqueous droplets in a fluorocarbon oil, the surfactant may have a hydrophilic block (PEG-PPO) and a hydrophobic fluorinated block (Krytox® FSH). If, however, the oil was switched to be a hydrocarbon oil, for example, the surfactant would instead be chosen so that it had a hydrophobic hydrocarbon block, like the surfactant ABIL EM90. In selecting a surfactant, desirable properties that may be considered in choosing the surfactant may include one or more of the following: (1) the surfactant has low viscosity; (2) the surfactant is immiscible with the polymer used to construct the device, and thus it doesn’t swell the device; (3) biocompatibility; (4) the assay reagents are not soluble in the surfactant; (5) the surfactant exhibits favorable gas solubility, in that it allows gases to come in and out; (6) the surfactant has a boiling point higher than the temperature used for PCR (e g., 95°C); (7) the emulsion stability; (8) that the surfactant stabilizes drops of the desired size; (9) that the surfactant is soluble in the carrier phase and not in the droplet phase; (10) that the surfactant has limited fluorescence properties; and (11) that the surfactant remains soluble in the carrier phase over a range of temperatures. Other surfactants can also be envisioned, including ionic surfactants. Other additives can also be included in the oil to stabilize the discrete entities including polymers that increase discrete entity stability at temperatures above 35°C.

The discrete entities (e.g., microdroplets) described herein may be prepared as emulsions, such as an aqueous phase fluid dispersed in an immiscible phase carrier fluid (e.g., a fluorocarbon oil or a hydrocarbon oil) or vice versa. In some cases, the carrier fluid comprises a fluorinated compound. In some cases, the carrier fluid is an aqueous fluid. The nature of the microfluidic channel (or a coating thereon) (e.g., hydrophilic or hydrophobic), may be selected so as to be compatible with the type of emulsion being utilized at a particular point in a microfluidic workflow.

Emulsions may be generated using the droplet making chips, which can form emulsions composed of droplets that are uniform in size. The microdroplet generation process may be accomplished by pumping two immiscible fluids, such as oil and water, into a junction in the chip. The junction shape, fluid properties (viscosity, interfacial tension, etc.), and flow rates influence the properties of the microdroplets generated but, for a relatively w ide range of properties, microdroplets of controlled, uniform size can be generated using methods like T-junctions and flow focusing. To vary microdroplet size, the flow rates of the immiscible liquids may be varied since, for T-junction and flow focus methodologies over a certain range of properties, microdroplet size depends on total flow rate and the ratio of the two fluid flow rates. To generate an emulsion with microfluidic methods, the two fluids are normally loaded into two inlet reservoirs (syringes, pressure tubes) (see Figures 2 and 3) and then pressurized as needed to generate the desired flow rates (using syringe pumps, pressure regulators, gravity, etc ). This pumps the fluids through the device at the desired flow rates, thus generating microdroplet of the desired size and rate.

In some cases, a cell in a discrete entity may be labeled (e.g., by a fluorescent label, a barcode, or a combination thereof). In practicing the subject methods, a number of reagents may be incorporated into and/or encapsulated by, the discrete entities 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 methods of adding reagents to the discrete entities may vary' in a number of 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. For instance, a reagent may be added to a discrete entity by a method involving merging a discrete entity with a second discrete entity which contains the reagent(s) in a discrete entity merger region of a droplet assembly chip of a microfluidic cartridge.

One or more reagents may also, or instead, be added using techniques such as droplet coalescence, or picoinjection. In droplet coalescence, a target drop may be flowed alongside a microdroplet containing the reagent(s) to be added to the droplet. The two droplets may be flowed such that they are in contact with each other, but not touching other microdroplets. These drops may then be passed through electrodes or other aspects for applying an electrical field, wherein the electric field may destabilize the microdroplets such that they are merged together. Reagents may also, or instead, be added using picoinjection. In this approach, a target drop may be flowed past a channel containing the reagent(s) to be added, wherein the reagent(s) are at an elevated pressure. Due to the presence of the surfactants, however, in the absence of an electric field, the microdroplet will flow past without being injected, because surfactants coating the microdroplet may prevent the fluid(s) from entering. However, if an electric field is applied to the microdroplet as it passes the injector, fluid containing the reagent(s) will be injected into the microdroplet. The amount of reagent added to the microdroplet may be controlled by several different parameters, such as by adjusting the injection pressure and the velocity of the flowing drops, by switching the electric field on and off, and the like.

In some cases, a discrete entity includes a bead. In some cases, at least one dimension of the bead (e.g., diameter, is between about 0.5 pm and about 500 pm). In some cases, the bead is made of a polymeric material, such as polystyrene. In some cases, the bead is magnetic or contains a magnetic component. In some cases, the bead has a biomolecule attached to its surface, such as an antibody, a protein, an antigen, DNA, RNA, streptavidin, or a combination thereof. In some cases, the bead is an immunoassay bead. In some cases, the bead is an RNA capture bead. As such, the present disclosure provides methods of selectively combining a biomolecule with another compound or cell, wherein the method includes selectively isolating the biomolecule from a composition using the bead, making a discrete entity that includes the bead and biomolecule, and selectively combining the discrete entity containing the bead and biomolecule with one or more other discrete entities that contain one or more other compounds or cells using the microfluidic cartridges described herein. Methods of selectively isolating biomolecules using beads are known in the art, e.g. U.S. 2010/0009383, which is incorporated herein by reference for its disclosure of a method of separating a biomolecule or cell using beads. In some embodiments, the methods, devices, and/or systems described herein can be used to sequence nucleic acid derived from single cells (e.g., once they have been determined to secrete the target protein, such as a monoclonal antibody). For example, individual cells can be encapsulated in the droplets which include the assay reagents as described herein. The cells can then be lysed and subjected to molecular biological processing to amplify and/or tag their nucleic acids with barcodes. The material from all the droplets can then be pooled for all cells and sequenced and the barcodes used to sort the sequences according to single droplets or cells. These methods can be used, for example, to sequence the genomes or transcriptomes of single cells in a massively parallel format.

In certain embodiments, nucleic acid sequence assay components that employ barcoding for labelling individual mRNA molecules, and/or for labeling for cell/well source (e.g., if wells pooled before sequencing analysis), and/or for labeling particular affixed entities (e.g., if droplet from two or more affixed entities are pooled prior to sequencing) are employed. Examples of such barcoding methodologies and reagents are found in Pat. Pub. US2007/0020640, Pat. Pub. 2012/0010091, U.S. Pat. 8,835,358, U.S. Pat. 8,481,292, Qiu et al. (Plant. Physiol., 133, 475-481, 2003), Parameswaran et al. (Nucleic Acids Res. 2007 Oct; 35(19): el30), Craig et al. reference (Nat. Methods, 2008, October, 5(10): 887-893), Bontoux et al. (Lab Chip, 2008, 8:443-450), Esumi et al. (Neuro. Res., 2008, 60:439-451), Hug et al., J. Theor., Biol., 2003, 221 :615-624), Sutcliffe et al. (PNAS, 97(5): 1976-1981; 2000), Hollas and Schuler (Lecture Notes in Computer Science Volume 2812, 2003, pp 55-62), and WO201420127; all of which are herein incorporated by reference in their entireties, including for reaction conditions and reagents related to barcoding and sequencing of nucleic acids.

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

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

In certain embodiments, the barcode tagging and sequencing methods of WO2014201273 (“SCRB-seq” method, herein incorporated by reference) are employed. The necessary reagents for the SCRB-seq method (e g., modified as necessary' for small volumes) are added to the fixed entities, each containing a lysed cell. Briefly, the SCRB-seq method amplifies an initial mRNA sample from cells from a single fixed entity. Initial cDNA synthesis uses a first primer with: i) N6 for cell/well identification, n) N10 for particular molecule identification, iii) a poly T stretch to bind mRNA, and iv) a region that creates a region where a second template-switching primer will hybridize. The second primer is a template switching primer with a poly G 3’ end, and 5’ end that has iso-bases. After cDNA amplification, the tagged cDNA single fixed entity samples are pooled. Then full-length cDNA synthesis occurs with two different primers, and full-length cDNA is purified. Next, a NEXTERA sequencing library is prepared using an i7 primer (adds one of 12 i7 tags to identify particular multi-well plates) and P5NEXTPT5 to add P5 tag for NEXTERA sequencing (P7 tag added to the other end for NEXTERA). The library is purified on a gel, and then NEXTERA sequencing occurs. As a non-liming example, with twelve 17 plate tags, and 384 cell/well-specific barcodes, this allows total of 4,608 single cell transciptomes to be done at once. This method allows for quantification of mRNA transcripts in single fixed entity.

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

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

Sorting approaches which may be utilized in connection with the disclosed methods, systems and devices also include those described herein, and those described by Agresti, et al., PNAS vol. 107, no 9, 4004-4009. For active sorting, the device includes one or more sorting elements and one or more detectors, wherein each detector is configured to detect one or more properties of a discrete entity, or a component within the discrete entity, and each sorting element is configured to sort the discrete entity into one of two or more locations based on the detecting by the detection element. In some cases, a sorting element is positioned in proximity to the sorting channel, such as an electrode in proximity to the sorting channel. In some cases, a sorting element is positioned within the sorting channel, such as a partial height flow divider in a sorting channel. In some cases, the device includes a sorting element positioned within the sorting channel and one or more sorting elements positioned in proximity to the sorting channel. Exemplary structures and methods for active sorting discrete entities are described in Cole et al., PNAS, 2017, 114, 33, 8728-8733; Clark et al., Lab Chip, 2018, 5, 18, 710-713; and Sciambi et al., Lab on a Chip, 2015, 15, 47-51, the disclosures of which are incorporated herein by reference for sorting elements.

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

In some embodiments, the droplet assembly chips herein include directing the discrete entity to a discrete entity merger region. Accordingly, a device as described herein can include a discrete entity merger region and a trapping element positioned in proximity to the discrete entity merger region. The trapping element can trap a plurality of discrete entities in the discrete entity merger region for a time sufficient for the plurality of discrete entities to combine to form a combined discrete 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 sufficient. In some cases, the electromagnetic force is a dielectrophoretic force. In some cases, the electromagnetic force is an electrophoretic force.

In some cases, the discrete entity merger region includes a feature selected from: a geometric change in a dimension of the first outlet channel, a flow obstacle, a flow divider, a laminating 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 channel (e.g., the discrete entity merger 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 channel (e.g., the discrete entity merger region is narrower than the downstream region). In some cases, the geometric change includes a recess in a channel wall. In some cases, the recess includes an area that is not colinear with the flow of fluid from the upstream region, such as shown as item 107 in FIG. 4. In some cases, where a valve is utilized, the valve is configured to switch between at least two states. In some cases, in the first state, the valve impedes the flow of a discrete entity past the discrete entity merger region while allowing flow of the carrier fluid past the discrete entity merger region. In some cases, in the second state, the valve is configured such that the combined discrete entity is not impeded from flowing past the discrete entity region. In some cases, the method includes putting the valve in a first state such that discrete entities can be trapped and combined into a combined discrete entity, and then putting the valve into a second state to release the discrete entity from the discrete entity merger region. In some cases, the valve is a membrane valve.

A laminating fluid inlet functions in a similar manner to certain embodiments of the spacer fluid inlet described above, such as a laminating fluid inlet is configured such that flowing fluid through the laminating fluid mlet will cause a discrete entity to move further away from a first side a channel and closer to a second side of a channel. Stated in another manner, the fluid flowing through the laminating fluid inlet contacts the fluid moving into the discrete entity merger region from an upstream region of the first outlet channel, thereby affecting the flow of fluid coming from the upstream region. In some cases, the fluid is oil, or a fluid which is otherwise immiscible with the fluid of the discrete entity.

FIG. 4 shows an embodiment wherein the discrete entity merger region includes recess 107, flow divider 113, and laminating fluid inlet 112. In FIG. 4, the laminating fluid provides a force pushing a discrete entity into recess 107 and towards trapping electrodes 109. In addition, flow divider 113 in FIG. 4 further affects the interaction of the laminating fluid and the fluid coming from the upstream region, thereby increasing the force pushing the discrete entity into recess 107. As such, a discrete entity merger region according to the present disclosure can include a laminating oil inlet and/or a flow divider, wherein such an element or elements are configured such that flowing oil through the laminating oil inlet channel will produce a force pushing a discrete entity in the discrete entity merger region towards a trapping electrode, a recess, or a combination thereof. In some embodiments, the device can include a flow divider without the laminating fluid inlet.

In some cases, the downstream region of the first outlet channel is configured to aid in the trapping of a discrete entity in the discrete entity merger region. In some cases, the downstream region has a larger cross-sectional area than the discrete entity merger region, which is an example of a geometric change in the first outlet channel. 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 and the discrete entity merger region is located at or near a vertex of the triangle. As an example, in the system of FIG. 5 has downstream region 208 and discrete entity merger region 207. In some cases, the longitudinal axis of the downstream region is parallel to the longitudinal axis of the discrete entity merger region, whereas in other cases such longitudinal axes are not parallel. In some cases, such axes are parallel but not colinear. In some cases, the axes are parallel and colinear. In some cases, the angle between such axes is greater than 0°, such as 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, such an angle is between approximately 15° and approximately 135°. In some cases, such an angle is between approximately 60° and approximately 120°, such as shown in FIG. 5.

In some cases, the sorting element sorts discrete entities at a rate of at least 10 Hz, such as 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 discrete entities contain the contents desired for the second discrete entity, such as 25% or less, 10% or less, 5% or less, 1% or less, or 0. 1% or less. In some cases, the discrete entity merger region and trapping element are configured to trap a first discrete entity for 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, a first discrete entity is trapped in the discrete entity merger region for 0. 1 ms or more before a second discrete entity enters the region, such as 1 ms or more, 10 ms or more, 100 ms or more, or 1,000 ms or more.

The present disclosure provides a method of performing reactions by selectively combining two or more discrete entities, as described above, wherein the reaction occurs between one or more components from each discrete entity (e.g., between a target and effector cell). Such components can be one or more cells, one or more products derived from a cell, one or more reagents (e.g., reagents for making oligo template structures, nickases, quenched oligonucleotide probes, etc ), or a combination thereof. In some cases, a suitable method includes combination of one cell and one or more reagents described herein. As an example, FIG. 6 shows the combination of four discrete entities, wherein three of the discrete entities each contain a different reagent or cell, and the fourth discrete entity contains a single cell. As such, FIG. 6 shows that a microfluidic device as described herein can be used to selectively combine different discrete entities, resulting in the formation of a combined discrete entity, e.g., that contains at least two cells and two reagents as described herein. In some cases, the reagents can include reagents including oligonucleotide probes, cell lysing reagents, PCR reagents, reagents for analyzing the DNA or RNA within a cell, antibodies, or a combination thereof. In such cases, the method can further include collecting genomic data from contents of the discrete entities or combined discrete entities. In some cases, the one or more products derived from a cell include cell lysate, DNA, RNA, or a combination thereof. As such, the method can involve analyzing products from a cell, e g. cell lysate, even though the cell per se is included in any of the discrete entities.

III. Exemplary Droplet Assays and Components

The following provides an exemplary description of exemplary assays that can be conducted in the droplets and combined droplets on the cartridges described herein.

In certain embodiments, the cartridges herein allow for analyzing cell-cell interactions, such as transmembrane proteins binding to surface displayed variable regions, via discrete entity (e.g., droplet) rmcrofluidics (see, e.g., U.S. Pat. application serial number 17/032,922 published as US Pat. Pub. 20210096125, which is herein incorporated by reference in its entirety . In certain embodiments, a plurality' of first discrete entities and a plurality of second discrete entities are merged in an assembly chip to generate a plurality of merged fixed entities (e.g., fixed via an electrical force), each of which contains one cell expressing a transmembrane (TM) protein and labeled clonal cells displaying a heterologous antibody variable region. In certain embodiments, binding of the clonal cells to the TM expressing cell is detected in each merged fixed entity by the sorting chip (e.g., in conjunction with the cartridge-receiving instrument), and the clonal cells found to bind are treated in order to sequence the nucleic acid encoding the variable region.

In some embodiments, the cartridges herein allow for analyzing the interaction of T- cells and neoantigen presenting cells (and other cells) via discrete entity (e.g., droplet) microfluids (see WO2021/081485, which is herein incorporated by reference in its entirety). In certain embodiments, an assembly chip on a cartridge is used to merge a discrete entity (e g., droplet) containing a T-cell, and a discrete entity (e.g., droplet) containing a neoantigen presenting cell, at a merger region via a trapping element in order to generate a combined discrete entity. In particular embodiments, at least one thousand of such combined discrete entities are formed in about one second. In some embodiments, whether the receptor on the T-cell sufficiently binds the neoantigen to activate the T-Cell is detected (e.g., via detection of cytokine release or granzyme B) in a sorting chip (e.g., in combination with a cartridge receiving instrument) that is on the cartridge. In certain embodiments, method are provides for using the cartridges to identify polyfunctional T-cells or NK-cells, as well as methods of screening for such cells that would be cytotoxic if injected into a subject.

In certain embodiments, the cartridges herein allow for barcoding cells, beads, and secreted proteins in discrete entities (e.g. droplets) to allow sequencing data from such components that are separated during processing to be associated via the common barcodes (see, e.g., US Serial number 17/493,312, which is herein incorporated by reference in its entirety). In some embodiments, the barcodes (e.g., in the droplets) are tethered to the cell surface via a lipid, cholesterol, or antibody, or are attached to a surface molecule that moves from one cell to another via trogocytosis. In certain embodiments, such methods allow cellcell interactions or secreted proteins in the discrete entity to be monitored in the cartridges herein.

In some embodiments, the cartridges herein allow for combining a single myeloma cell and a single B-cell (e.g., from an animal exposed to a desired antigen) via discrete entity (e.g., droplet) microfluidics in an assembly chip on the cartridge (see, e.g., U.S. application serial number 17/690,427, which is herein incorporated by reference in its entirety). In certain embodiments, an assembly chip on the cartridge is used to merge a discrete entitycontaining a B-cell, and a discrete entity containing a myeloma cell, and a discrete entity containing gellable material, at a merger region via a trapping element in order to generate a combined discrete entity. In further embodiments, the combined discrete entity is treated such that a gelled discrete entity is formed. In other embodiments, the gelled discrete entity is treated such that the myeloma cell and B-cell fuse, generating a hybridoma, which is assayed (e.g., in a sorting chip in combination with a cartridge-receiving instrument) to determine if a desired monoclonal antibody is secreted therefrom.

In additional embodiments, the cartridges herein allow for detecting cell viability in a discrete entity (e.g., microdroplet). In some embodiments, the methods employ a droplet assembly chip to combine into a combined discrete entity: a first cell stained with a first detectable dye, a second cell stained with a second detectable dye, and a first reporter dye that: A) is membrane-impermeable to live cells, and B) membrane-permeable to dead and dying cells, and C) generates a first detectable signal when in close proximity' to the first detectable dye, but not the second detectable dye. In particular embodiments, the methods further comprise detecting the presence or absence of the first detectable signal from the combined discrete entity in a sorting chip (e.g., in conjunction with a cartridge-receiving instrument). In certain embodiments, the absence of the first detectable signal indicates the first cell is viable (e.g., allowing the combined discrete entity to be sorted into a keep channel). In other embodiments, the presence of the first detectable signal indicates the first cell is dead or dying (e.g., allowing the combined discrete entity to be sorted into a discard channel).

In some embodiments, the cartridges herein allow for detecting the presence or absence of a target protein in a discrete entity comprising: a) generating a discrete entity (e.g., microdroplet, using a droplet making chip) comprising: i) a first cell that may secrete, or surface express, a target protein, ii) a quenched oligonucleotide probe, iii) first and second antibody-oligonucleotide conjugates that bind the target protein in proximity to each to form an oligonucleotide template structure (OTS) (see Figures 12a and 12b), and a nickase enzyme that cleaves the quenched oligonucleotide probe when it is hybridized to the OTS such that a detectable dye (e.g., fluorescent dye) is released and generates a signal; and b) detecting the presence or absence of the signal (e.g., in a sorting chip in conjunction with a cartridgereceiving instrument) from the detectable dye.

All publications and patents mentioned in the present application are herein incorporated by reference. Various modification and variation of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMS We claim:

1. A device or system comprising: a) a base substrate, b) at least one microfluidic chip operably attached to said base substrate, wherein said at least one microfluidic chip is selected from: i) a drop making chip, ii) a drop assembly chip, and iii) a drop sorting chip; and c) a plurality of micro or macro fluidic channels; wherein said device or system is in the form of a cartridge that is configured to be inserted and removed from a cartridge receiving instrument.

2. The device or system of Claim 1, wherein said at least one microfluidic chip is said drop assembly chip.

3. The device or system of Claim 1, wherein at least two of said microfluidic chips are present and are operably attached to said base substrate, and wherein said plurality of micro or macro fluidic channels operably fluidically interconnect said at least two microfluidic chips.

4 The device or system of Claim 3, wherein said at least two microfluidic chips include said drop making chip and said drop assembly chip.

5. The device or system of Claim 3, wherein said at least two microfluidic chips include said drop assembly chip and said drop sorting chip.

6. The device or system of Claim 1, wherein all three of said microfluidic chips are present and are operably attached to said base substrate, and wherein said plurality of micro or macro fluidic channels operably fluidically interconnect all three of said microfluidic chips.

7. The device or system of any one of Claims 2-4, and-6, wherein said drop assembly chip comprises: i) a sorting region, ii) a droplet merger region, and iii) a trapping element.

8. The device or system of Claim 7, wherein said trapping element comprises trapping electrodes.

9. The device of system of any one of Claims 1-8, wherein said drop assembly chip is configured for deterministic sorting and trapping of at least two droplets to form a combined droplet.

10. The system or device of Claim 1-9, further comprising: at least one input oil reservoir fluidically linked to said droplet making chip.

11. The system or device of any one of Claims 1-10, further comprising a plurality of input sample tubes for receiving reagents or cells to be combined into droplets formed by said droplet making chip.

12. The system or device of any one of Claims 1-11, further comprising: droplet making selection valve fluidically linked to said droplet making chip by at least some of said plurality of macro or micro fluidic channels.

13. The system or device of Claim 12, further comprising: a sample droplet reservoir and/or a dummy droplet reservoir, which are fluidically linked to said droplet making selection valve by at least some of the plurality of macro or micro fluidic channels.

14. The system or device of Claim 13, further comprising: a droplet flow control valve which is fluidically linked to said sample droplet reservoir, and/or said dummy droplet reservoir, by at least some of the plurality of macro or micro fluidic channels.

15. The system or device of Claim 15, wherein said droplet flow control valve is fluidically linked to said droplet assembly chip by at least some of said plurality of macro or micro fluidic channels.

16. The system of device of any one of Claims 1-15, further comprising: at least one oil input channel fluidically linked to said droplet assembly chip or to said droplet making chip.

17. The system or device of Claim 16, wherein said at least one oil input channel is at least partially formed in said base substrate and/or are fluidically linked to said cartridgereceiving instrument.

18. The system or device of any of Claims 1-17, further comprising: a plurality of electrodes operably linked to said droplet assembly chip.

19. The system or device of any one of Claims 1-18, further comprising: an assembly flow control valve fluidically linked to said droplet assembly chip and/or said droplet sorting chip by at least some of said plurality of macro or micro fluidic channels.

20. The system or device of any one of Claims 1-19, further comprising: a sort negative channel or reservoir, and a sort positive channel or reservoir, which are fluidically linked to said droplet sorting chip by at least some of said plurality of macro or micro fluidic channels.

21. The system or device of any one of Claims 1-20, further comprising: a sort oil control valve, which is fluidically linked to said droplet sorting chip and/or said sort negative channel or reservoir and/or said sort positive channel or reservoir.

22. The system or device of any one of Claims 1-21, further comprising at least one oil waste port.

23. The system or device of Claim 22, wherein said at least one oil waste port is formed in said base substrate.

24. The system or device of any one of Claims 1-23, wherein said plurality of micro or macro fluidic channels are formed in said base substrate.

25. The system or device of any one of Claims 1-24, wherein said plurality of micro or macro fluidic channels are said macro channels and have a diameter of about 0.5 to 1.5 mm.

26. The system or device of any one of Claims 1-25, wherein said plurality of micro or macro fluidic channels are said micro channels and have a cross section of about 0.1 to 0.25 mm.

27. The system or device of any one of Claims 1-26, further comprising said cartridgereceiving instrument.

28. The system or device of Claim 27, wherein said cartridge-receiving instrument comprises at least one of the following components: i) a detector for detecting a sort region present on said droplet assembly chip and/or said droplet sorting chip; ii) an oil reservoir; iii) a pressurized gas source; iv) athermal incubation source; and v) a waste oil collection bin.

29. The system or device of Claim 8, wherein said droplet assembly chip further comprises a sorting channel and a first outlet channel, and wherein said trapping electrodes comprise a first sorting electrode that exert an electromagnetic force sufficient to sort a droplet in the sorting channel to the first outlet channel.

30. The system or device of Claim 29, wherein the electromagnetic force is a dielectrophoretic force.

31. The system or device of Claim 30, wherein the electromagnetic force is an electrophoretic force.

32. The system or device of Claim 8, wherein said sorting region comprises a sorting channel and wherein said trapping element comprise first and second sorting electrodes configured such that the first and second sorting electrodes form a bipolar electrode pair and the first trapping electrode is positively charged.

33. The system or device of Claim 32, wherein the first and second sorting electrodes are positioned on opposite sides of the sorting channel.

34. The system or device of Claim 33, wherein at least one of the following applies: i) the first sorting electrode is positioned closer to the sorting channel than the second sorting electrode, ii) the second sorting electrode is positioned closer to the sorting channel than the first sorting electrode, iii) the distance between an end of the first sorting electrode, the second sorting electrode, or both and an interior wall of the sorter channel is between approximately 1 pm and approximately 100 pm, iv) the distance between the first sorting electrode and the second sorting electrode is approximately 25 pm to approximately 500 pm, v) the first sorting electrode and the second sorting electrode are connected to an alternating current electrical source with a frequency of approximately 0. 1 kHz to approximately 100 kHz and a voltage of approximately 10 V to approximately 10,000 V, vi) each sorting electrode comprises a liquid electrode, and vii) each sorting liquid electrode comprise one or more liquid channels imbedded in the base substrate and filled with conductive media.

35. A device or system comprising: a) a base substrate, b) at least one microfluidic chip operably attached to said base substrate, wherein said at least one microfluidic chip is selected from: i) a drop making chip, n) a first drop assembly chip, iii) a second drop assembly chip; and iv) a drop sorting chip; and c) a plurality of micro or macro fluidic channels; wherein said device or system is in the form of a cartridge that is configured to be inserted and removed from a cartridge receiving instrument.

36. The device or system of Claim 35, wherein said at least one microfluidic chip comprises said first drop assembly chip and said second assembly chip.

37. The device or system of Claims 35 or 36, wherein said first or second assembly chip is the last microfluidic chip present on the cartridge in the direction of fluid flow.

38. The device or system of Claim 36, wherein said at least one microfluidic chip comprises said drop making chip, said first drop assembly chip, and said second assembly chip.