Systems and methods

Abstract

To provide novel instruments and cartridges for processing droplets.SOLUTION: The invention provides a microfluidic cartridge for automated sorting and dispensing of sorted microfluidic droplets, the cartridge comprising: a sorting input channel coupled to a sorting region, the sorting region comprising two or more sorting output channels coupled to the sorting input channel and a droplet director to selectively direct a droplet from the sorting input channel to a selected one of the sorting output channels; a droplet dispenser coupled to one of the sorting output channels to dispense the selected droplets from the microfluidic cartridge for collection in a reservoir, the droplet dispenser comprising a droplet ejection mechanism to eject droplets from an emulsion flow into a dispenser output channel for collection in the reservoir; and an emulsion flow buffer region between the sorting region and a dispensing region to control an emulsion flow rate to the droplet dispenser.SELECTED DRAWING: Figure 4a

Description

[Technical field]

[0001] The present invention relates generally to systems and methods for delivering droplets containing one or more entities, particularly biological entities. [Background technology]

[0002] Herein, we are interested in emulsions, typically emulsions that contain water-in-oil microdroplets, generally surfactant-stabilized. One or more biological entities, such as one or more living cells, are incorporated into each droplet, and experiments can then be performed within the droplets, for example, to perform biological assays. Microdroplets can be generated and processed at rates potentially exceeding several thousand per second.

[0003] Typically, the oil composition comprises fluorine and / or mineral oil, and preferably a surfactant, for example, in a volume ratio of about 0.5-5%. The use of fluorous oil is particularly advantageous when the microdroplet contains a living entity, since fluorous oils are good at transporting oxygen to the microdroplet. The surfactants can be either polymeric or small molecules, for example surfactants derived from block copolymers of perfluoroethers such as Krytox™ or polyethylene glycol (PEG) can be used. The substances or analytes in the microdroplet can include, for example, cells, DNA, proteins, peptides, beads, particles, crystals, micelles, macromolecules, substances for enzyme assays, organelles, cells, for example mammalian cells, yeast cells, algae cells or organisms such as bacteria, viruses, prions, etc. Typically, the droplets have a diameter in the range of 1-120 μm, which can be large (or small), and constitute a volume that can be in the range of nanoliters to femtoliters.

[0004] Active elements integrated on microfluidic devices can be used to control individual droplets, a technique already described by the inventors in, for example, US Pat. No. 6,399,363, which allows on-chip extraction of the contents of microdroplets by incorporating them in a continuous flow. Other general background prior art regarding microdroplets can be found in patents / applications in the name of RainDance Technologies Inc., for example, US Pat. No. 6,399,363. Single cell or particle sorting is known from the prior art, for example, US Pat. No. 6,399,363, US Pat. No. 6,399,385, non-patent document 1, non-patent document 2, non-patent document 3.

[0005] While these techniques are useful, they can be expanded. For example, a class of vexing problems involves diagnosing biological behavior in very rare biological entities or events, such as those associated with drug resistance. Another challenge is ensuring monoclonality, i.e., drug regulatory agencies often require that biopharmaceuticals and other chemical or biochemical entities be cultured from a single viable entity, or a specific combination of entities. This is because, for example, culturing a cell population arising from a single cell, or from multiple substantially identical viable cells, ensures that monoclonality occurs with a high probability across the cell population. Yet another challenge involves selecting a group of cells for fermentation that has a high production yield. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO International Publication No. 2009 / 050512 [Patent Document 2] International Publication No. 2008 / 063227 [Patent Document 3] US Patent Application Publication No. 2008 / 0053205 [Non-patent literature]

[0007] [Non-Patent Document 1] “Encapsulation of single cells on a microfluidic device integrating droplet generation with fluorescence-activated droplet sorting”, Biomed Microdevices 2013, 15(3):553-60 [Non-Patent Document 2] “Analyzing Cancer at Single Cell Resolution with Droplet Technology”, RainDance Technologies [Non-Patent Document 3] URL: http: / / raindancetech.com / rdt / wp-content / uploads / 2012 / 05 / poster_analyzing_cancer_with_droplet_technology.pdf Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need for techniques that can be used to address these issues. [Means for solving the problem]

[0009] According to a first aspect of the present invention, there is provided a method of providing droplets containing one or more entities, the method comprising the steps of providing a plurality of entities in a fluid, preparing droplets from the fluid, determining whether the droplets contain one or more of the plurality of entities or whether the droplets do not contain the entities, sorting the droplets in response to a result of the determination, and dispensing the sorted droplets into a storage container, the dispensing step comprising, after the sorting step, selecting a fluid flow path for the fluid containing the sorted droplets, and expelling the sorted droplets from the selected path.

[0010] The inventors have realized that the probability of obtaining a single entity or a defined single combination of entities in a single droplet, e.g., a picodroplet (whose volume is less than about 1000 or several thousand picoliters), can be high using embodiments of this method. Each of the preparation, determination / sorting, and dispensing steps of the method has a certain probability of obtaining one or more target entities in a single droplet. In this method, by combining these steps, a high probability of obtaining a single target entity or a single target combination of said entities in a single droplet can be obtained with more than 99.9%, particularly more than 99.99%. For example, this can be achieved with three stages of selection: encapsulation by Poisson statistics (described below), sorting at the junction, and sorting / selection at the time of dispensing, each with, for example, more than 90% selection. In a similar manner, embodiments of this method facilitate the extraction of very rare target droplets from a large population.

[0011] The inventors note that where reference is made throughout this specification to a single (target) combination of entities, this may include a defined number of substantially identical entities (e.g., a defined number of cell types), or a defined range of numbers of substantially identical entities, or a combination of a defined number of a first entity (e.g., a defined number of a first cell type) and a defined number of a second or further entity (e.g., a defined number of second or further cell types) (within a specified or range). Thus, the one or more target entities in a droplet may be, for example, a defined number (one or more) of entities of type A, a defined number (one or more) of entities of type A and a defined number (one or more) of entities of type B, a defined number (one or more) of entities each of three or more different types.

[0012] In a preferred embodiment, the method further includes identifying sorted droplets for dispensing, extracting the sorted droplets from a first fluid flow path of the fluid by transferring the sorted droplets from the first fluid flow path into a second fluid flow path, and ejecting the sorted droplets from the second fluid flow path into a reservoir by applying pressure to the second fluid flow path.

[0013] In a preferred embodiment of the method, the transferring step includes applying pressure to the sorted droplets to dispense them while in a portion of the first fluid flow path, and then transferring the sorted droplets from the first fluid flow path into the second fluid flow path and ejecting the sorted droplets. In an embodiment, the (water-in-oil) emulsion flows along the first fluid flow path, through a shared region of the flow path, and then may continue along the first fluid or be diverted into the second flow path by positive and / or negative pressure (suction) applied to either the flow path or the shared flow path. Thus, in an embodiment, the dispensing step may employ a branched or T-junction channel arrangement with a shared region of flow, optionally but preferably in conjunction with a droplet selection / sorting mechanism to select the path.

[0014] This approach can, in some embodiments, perform a further sorting step of droplets already previously sorted. The droplets can thereby be detected and, if appropriate, analyzed for their constituents before the second sorting step. The detection and / or analysis of the sorted droplets can thereby be performed in the first fluid flow path and in a shared flow path of the path to which pressure can be applied. Alternatively, or in addition, the droplets can be detected and / or analyzed before the shared flow path. In a preferred embodiment, the second fluid flow path from which the liquid can be discharged can comprise a first and a second (or further) ejection channel. The pressure in the second ejection channel can be reduced (e.g., by using a pump connected to the second ejection channel). The droplets can then be dispensed through the first ejection channel by applying pressure to the sorted droplets while they are in the first fluid flow path in response to the detection and / or analysis of the sorted droplets, as outlined above. This can ensure that the sorted droplets are not extracted into the second ejection channel using the pump. If the droplet is to be dispensed from the second output channel, e.g., if the droplet is not of interest, no pressure may be applied, causing the pump to aspirate the droplet into the second output channel. Analysis of the droplet to determine whether it is of interest may include analysis of a defined number of target entities in the droplet, and / or properties of one or more entities in the droplet (e.g., fluorescence levels, conductivity, and other physical or chemical properties). One embodiment of such a channel network is further described below.

[0015] In another preferred embodiment of the method, the dispensing step includes transferring the sorted droplets from the first fluid flow path to the second fluid flow path and then applying pressure to the second fluid flow path to eject the droplets. This is preferred because the flow of other droplets in the first fluid flow path cannot be interrupted since pressure is applied only to the second fluid flow path. However, alternatively, in this type of dispensing, when the droplets are not desired, a "decoupling unit" as described below can be used to transfer the undesired droplets from the first fluid flow path to a second waste path, and the droplets are dispensed from the first flow path, for example by closing a valve in the first flow path and applying pressure to the emulsion to eject the droplets from a nozzle or the like at the end of the first flow path.

[0016] Embodiments of devices used to transfer droplets from a first fluid flow path to a second fluid flow path are described further below, and may employ, for example, a rotation unit, a translation unit, or a decoupling unit as described later. Whether a droplet should be transferred from a first fluid flow path to a second fluid flow path may be determined before the droplet enters the transfer unit and / or while the droplet is in the transfer unit. The decision as to whether a droplet should be transferred from a first fluid flow path to a second fluid flow path may further depend on a determined number of target entities and / or properties (e.g., fluorescence levels, conductivity, and other physical or chemical properties) of one or more entities in the droplet.

[0017] In a preferred embodiment of the method, the extracting step comprises extracting sorted droplets only when they contain a defined number of said entities and / or when they contain said entities having a defined property, whereby the defined number of entities may be within a defined range. The defined property may be, for example, a physical property (e.g., a fluorescence level or other physical property) and / or a chemical property.

[0018] This may be preferable, for example, as the yield of droplets containing one or more target entities to be discharged into a reservoir for further analysis and / or culturing may be increased. As outlined above, entities that are target entities may thereby include entities having defined properties, such as, but not limited to, physical and / or chemical properties, and / or droplets containing a defined number of entities.

[0019] The number of entities in the droplet may be determined, for example, optically. The number of entities may alternatively or additionally be determined by analyzing a physical property (e.g., fluorescence level) and / or a chemical property of the droplet and / or the entity(ies), which may correspond to the number of entities contained in the droplet. The physical and / or chemical property may be determined using standard techniques.

[0020] A variety of methods and devices for preparing droplets from a fluid can be used, examples include, but are not limited to, T-junctions, Y-junctions, flow focusing devices, and others.

[0021] Whether a droplet contains one or more entities, in particular one or more target entities, or no entities at all may be determined based on one or more of the droplet's electrical, optical, thermal, acoustic, mechanical, temporal, spatial, and other properties, e.g., other physical or chemical properties.

[0022] Techniques for sorting droplets include, but are not limited to, magnetic field based droplet sorting, dielectrophoresis, acoustic wave droplet sorting, and others.

[0023] The extraction and / or identification steps may allow for further determination as to whether a single droplet contains one or more target entities, thereby increasing the probability of obtaining a single target entity or a single target combination of entities in a single droplet.

[0024] Method embodiments described herein are particularly suitable for example for increasing monoclonality assurance of viable cells in droplets, as further described below.

[0025] In a preferred embodiment of the method, the ejecting step comprises ejecting the sorted droplets at a defined location depending on the determination and / or properties of the entities. The sorted droplets may thereby be ejected, for example, into a well of a microtiter plate. The particular well of the microtiter plate into which the sorted droplets should be dispensed is thereby selected depending on a previously determined number of entities in the droplet (which may be a defined number of entities of one type or each of multiple types of entities) and / or depending on properties of one or more entities, for example physical properties (e.g. fluorescence level or other physical properties) and / or chemical properties.

[0026] In a preferred embodiment of the methods described herein, the droplets have a volume of 50-1,000 picoliters, preferably 300-700 picoliters (fused droplets can be larger). Such volumes are preferred because they facilitate the inclusion of a single entity, or a small number of entities, for example, cells or biomolecules, while keeping fluid consumable costs to a minimum, and in addition, smaller droplets facilitate faster processing. However, larger droplets can also be employed.

[0027] For example, it may be preferred to cultivate and / or maintain microbial cultures or cells. Thus, in a preferred embodiment, the method further comprises a step of incubating the droplet to cultivate and / or maintain the one or more entities. Since the probability for obtaining a single target entity or a single target combination of entities in a single droplet is, in embodiments, greater than 99.997%, the high monoclonality guarantee allows for obtaining a large number of, e.g., viable cells, when culturing one or more entities produced using the method embodiments described herein.

[0028] The droplets may be sent to an incubator prior to the determination as to whether the droplets contain one or more entities (or no entities at all) and / or may be sent to an incubator after the determination and sorting step. Additionally or alternatively, the droplets may be incubated after the extraction step.

[0029] In a further preferred embodiment, the method further comprises performing a stability test on one or more entities during incubation. For example, performing a stability test on one or more cells in a single droplet allows for the selection of viable cells that do not reduce, alter or die during the stability test of an important analyte or biomolecule. This allows for example to obtain even higher monoclonality assurance of viable cells or high producers or important analytes or the like.

[0030] In a preferred embodiment of the method, the step of transferring the sorted droplets from the first fluid flow path to the second fluid flow path includes the step of decoupling the sorted droplets from the first fluid flow path in a decoupler configured to separate the sorted droplets from the first fluid flow path and direct the separated droplets to the second fluid flow path.

[0031] In this manner, a single droplet may be separated from a fluid containing one or more droplets. The droplet may then be translated from one fluid flow path to another, from which it may then be dispensed, for example, from the microfluidic chip in which the method is implemented. It may be particularly desirable to determine whether a droplet contains a viable single entity (or a specific combination of only viable entities or only a plurality of viable entities), for example, a viable single cell. This may allow for simply isolating droplets containing a viable single entity (or a specific combination of only viable entities or only a plurality of viable entities) from the fluid. As a result, the monoclonality guarantee that each droplet results in a single droplet containing a viable single entity (or a specific combination of only viable entities or only a plurality of viable entities) may be further increased.

[0032] Expelling the transferred droplets, for example, from the microfluidic chip, may be particularly preferred as one or more entities contained in a single droplet may be further analyzed off-chip.

[0033] In a preferred embodiment of the method, the ejecting step includes heating a transport fluid (such as, but not limited to, oil) through which the selected droplets are transported, which allows the droplets to be ejected in a manner similar to how fluid is ejected from an inkjet printer.

[0034] In a preferred embodiment, the method further comprises injecting culture medium fluid into the second fluid flow path and expelling the sorted droplets in the culture medium fluid, which may be particularly preferred as, for example, a population may be cultured from one or more entities contained in a single expelled droplet, and the expelled droplet may be expelled, for example, into a microtiter well in the culture medium fluid for culturing the population, for example a population of cells or a population of biomolecules.

[0035] In a further preferred embodiment, the ejecting step includes ejecting the identified droplet via pressurized fluid ejection. This may be particularly preferred as droplets containing one or more entities, particularly one or more viable entities, may be dispensed at a faster rate and / or in a controlled manner. In this regard, separating droplets containing one or more viable entities from a first fluid flow path of a fluid carrying multiple droplets and sending the droplets to a second flow path may be particularly important, as this allows for pressurized fluid ejection of a single droplet from the second flow path without affecting the flow of other droplets in the first fluid flow path. This may achieve controlled ejection of, for example, one or more viable cells or biomolecules in a single droplet.

[0036] The methods and systems described herein may be particularly suitable for cells and / or reagents, such as, but not limited to, biomolecules, since, as outlined above, for example, the culture of a cell population may be required under legal regulations to be derived from a single viable cell (or only a large number of viable cells).

[0037] By implementing the methods and systems described herein, consumable costs, particularly the consumable costs of the fluid containing the entity, and / or the length of time required to deliver the lead agent may be particularly reduced.

[0038] In a preferred embodiment, the method further comprises providing a second plurality of entities in a second fluid, preparing a second droplet from the second fluid, and fusing the first droplet prepared from the first fluid with the second droplet prepared from the second fluid to obtain a fused droplet, wherein the extracting comprises extracting the fused droplet by transferring the fused droplet from the first fluid flow path into the second fluid flow path. This may allow obtaining a fused droplet containing the first and second cells, cells and reagents, and other combinations. The step of fusing the droplets may be performed by charging one or both of the first and second droplets using electrocoalescence, by passive or mechanical fusion, for example by forcing the droplets to gather by forming a shape or other configuration in the flow channel, by employing chemically facilitated fusion, and / or by using other techniques.

[0039] Thus, fusing the first and second droplets may, for example, allow one to investigate how a single cell (or a number of cells) reacts with a reagent. Furthermore, stability studies may be performed on single cells (or a number of cells) that have been exposed to a reagent, or interactions between single cell pairs (or combinations of cells) may be analyzed.

[0040] For example, to fuse a single cell(s) with a reagent, it may be desirable to first determine whether a first droplet contains a single entity (or multiple entities). Thus, in a preferred embodiment, the determination and / or sorting is performed prior to the fusion of the droplets.

[0041] In a further preferred embodiment, the method further comprises the steps of determining whether the second droplet contains one or more entities of the second plurality of entities or whether the second droplet does not contain one of the entities of the second plurality of entities, and sorting the second droplet depending on the result of the determination. This may for example ensure that the first droplet is fused with the second droplet only if the second droplet contains one or more target entities. Preferably, fusion is performed only for the first droplet and the second droplet that have been determined to contain one or more target entities of the first and second plurality of entities, respectively.

[0042] Thus, embodiments of the method allow for the analysis of a single pair of cells, or a single pair of cells and reagents, such as, for example, a biomolecule, or a single target combination of one or more cells and one or more reagents contained in a single droplet.

[0043] Thus, in a preferred embodiment of the method, the fused droplet contains a single pair of two cells, or a single pair of a cell and a reagent, or a single combination of one or more cells, or a single combination of one or more cells and one or more reagents, in particular where the reagent is a biomolecule. It will be understood that other combinations of single pairs of two entities contained in a single droplet may be analyzed and / or further processed using the method embodiments described herein.

[0044] One of skill in the art will appreciate that the methods may also be used to combine multiple entities / reagents.

[0045] In a further preferred embodiment of the method, the sorting step includes detecting the droplets at multiple locations in the fluid flow path at different times and determining the velocity of the droplets in the fluid flow path to time the sorting. Method embodiments can improve the control of the sorting step since the specific location of the droplets in the fluid flow path can be determined and / or predicted. Thus, for example, a pair of sensors, such as optical gate sensors, can be used to estimate the movement speed of the droplets. The position of the droplets at the time of merging can then be predicted. This can be used, for example, to check that the droplets are in the correct channel or oriented / sorted by checking that they are present at the expected time and place.

[0046] In a related aspect of the invention, a method is provided for providing droplets containing a single pair of two biological entities, the method comprising the steps of providing a first plurality of biological entities in a first fluid and a second plurality of biological entities in a second fluid, preparing a first droplet from the first fluid and preparing a second droplet from the second fluid, determining whether the first droplet contains a single entity of the first plurality of biological entities and whether the second droplet contains a single entity of the second plurality of biological entities, respectively, and fusing the first and second droplets that have been determined to contain said single entities. Thus, the probability for obtaining a single pair of two entities in a single droplet may be increased, since in this example, for both the first and second droplets, respectively, the determining step is performed before fusing the droplets.

[0047] The preferred embodiment of the first aspect of the present invention described above applies equally here. Thus, for example, the fused droplets can then be extracted from the first fluid flow path of the fluid by transferring the sorted droplets from the first fluid flow path into the second fluid flow path. Preferably, the first and second droplets can each undergo all the steps of the first aspect of the present invention described above and its preferred embodiments. The probability of obtaining a fused droplet containing a single pair of entities from the first and second multiple biological entities, respectively, can thereby be significantly increased.

[0048] The step of fusing the droplets may be performed by electrocoalescence, by charging one or both of the first and second droplets by electrostatic attraction to fuse the droplets, by passive or mechanical fusion, by chemically assisted fusion, or by other techniques.

[0049] In a related aspect of the invention, there is provided a method of metabolizing and / or analyzing a biological entity contained in a droplet, the biological entity comprising a single cell, or a single pair of two cells, or a single pair of a cell and a reagent, the method comprising providing the biological entity contained in the droplet using the method of any one of the aspects and embodiments described above.

[0050] The step of metabolizing (culturing) may include, for example, increasing the number of the entity, in the case of bacteria, or may include metabolizing an entity, such as a mammalian cell, for example, to produce a product.

[0051] Because the method embodiments described herein result in a very high degree of monoclonality assurance, they are particularly suitable for culturing, for example, a single cell (or biomolecule, or another entity) contained in a single droplet, since the cells in a cell population cultured from a single cell have a very high probability of all being substantially identical to one another. Similarly, analyzing a single cell in a single droplet allows one to be sure that a measurement is performed on the cell of interest. This cannot be guaranteed for a large number of cells contained in a single droplet, for example, because mutations result in a statistical distribution of cells (or other entities) that are not all substantially identical.

[0052] In a further aspect of the invention, a microfluidic system for providing droplets containing one or more entities is provided, the system comprising a droplet formation device for preparing droplets from a fluid containing a plurality of entities, an analyzer for determining whether said droplets contain one or more of the plurality of entities, a droplet sorting device for sorting said droplets depending on the result of the determination, and a decoupler for decoupling the sorted droplets from a fluid flow path of a fluid in the microfluidic system.

[0053] In a preferred embodiment, the microfluidic system further comprises a further analyzer configured to identify the decoupled droplets (or, e.g., the sorted droplets transferred from the first fluid flow path to the second fluid flow path via a decoupler), and a droplet dispensing unit configured to eject the identified droplets, e.g., into a storage container (e.g., a microtiter plate).

[0054] Preferably, the analyzer is configured to determine whether the droplet contains a single target entity of the plurality of entities and / or whether the droplet contains a single target combination of entities of the plurality of entities.

[0055] Thus, embodiments of the microfluidic system allow for obtaining a single entity, e.g., a single pair of single cells or biological entities, or a single target combination of entities, in a single droplet. The droplet formation device, the analyzer and droplet sorting device, and the decoupler each allow for obtaining one or more target entities in a single droplet with a certain probability. The combination probability for obtaining a single entity or a single combination of entities in a single droplet using embodiments of the microfluidic system may be greater than 99.9%, in particular greater than 99.99%. The decoupler may thereby comprise an analyzer for further determination as to whether the single droplet contains a single entity.

[0056] In a preferred embodiment of the microfluidic system, the decoupler comprises a separation unit for separating the sorted droplets from a fluid flow path and a guide for directing the separated droplets into a second fluid flow path of the microfluidic system. This may be particularly preferred since a single entity (or a single combination of entities) contained in a single droplet may then be separated and dispensed and / or analysed separately from other droplets or fluids.

[0057] In a further preferred embodiment of the microfluidic system, the guide comprises a rotation unit, the rotation unit comprising a droplet storage unit for storing the separated droplets, the rotation unit being configured to be rotated to transport the separated droplets stored in the droplet storage unit into a second fluid flow path.

[0058] After a droplet containing an entity (or entities) of interest is detected, the droplet may be deposited in a droplet storage unit and the rotation unit may be rotated to send the droplet into the second fluid-flow path, hi some embodiments, the rotation unit completes the first and second fluid-flow paths.

[0059] In some other embodiments, branching flow channels may be used for similar purposes, more specifically for selecting / dispensing droplets.

[0060] In some embodiments, the droplet storage units may be channels completing the first and second fluid flow paths, respectively.

[0061] In a further preferred embodiment of the microfluidic system, the guide comprises a translation unit, the translation unit comprising a droplet storage unit for storing the separated droplets, the translation unit being configured to be moved in a translation direction to transport the separated droplets stored in the droplet storage unit into a second fluid flow path.

[0062] Similar to the rotation unit, after a droplet containing an entity (or entities) of interest is detected, the droplet may be deposited in a droplet storage unit of the translation unit, and the translation unit may be moved to send the droplet into the second fluid-flow path, hi some embodiments, the translation unit completes the first and second fluid-flow paths.

[0063] In some embodiments, the droplet storage units may be channels completing the first and second fluid flow paths, respectively.

[0064] In a preferred embodiment, the guide may be configured to combine an input channel with two or more output channels, whereby the channels of the guide may be moved in a swinging motion, allowing the input fluid flow path to be connected with multiple output fluid flow paths.

[0065] In a preferred embodiment, the microfluidic system further comprises an incubator for incubating the droplets to culture and / or maintain one or more entities. As outlined above, the probability for obtaining a single target entity or a single target combination of entities (which may be multiple substantially identical entities) in a single droplet is, in embodiments, greater than 99.99%, such that a high monoclonality guarantee allows for obtaining a large number of, e.g., viable entities (e.g., cells, biomolecules, or other entities) when culturing one or more entities using embodiments of the microfluidic systems described herein.

[0066] The incubator may be placed in front of the analyzer in the fluid flow direction and / or behind the analyzer and droplet storage device in the fluid flow direction. Additionally or alternatively, the incubator may be placed behind a decoupler in the fluid flow direction in the microfluidic system.

[0067] In a preferred embodiment of the microfluidic system, the incubator comprises a stability test unit for performing a stability test on one or more entities during incubation. Performing a stability test on one or more entities, e.g., cell(s), in a single droplet allows for sorting out viable entities that do not reduce, alter or die during the stability test of a critical analyte or biomolecule. This allows for obtaining even higher monoclonal assurance of viable entities, such as, but not limited to, viable cells, biomolecules or other entities, and / or cells that produce high levels of a critical analyte or the like.

[0068] In a preferred embodiment, the microfluidic system further comprises a dispensing unit for dispensing the decoupled droplets, whereby the decoupled droplets may be dispensed, for example, into individual wells of a microtiter plate, where one or more entities contained in the single droplet (or droplets) can be stored and then removed for further processing or analysis, or for subsequent use, for example, in fermentation or similar processes.

[0069] In a further preferred embodiment of the microfluidic system, the dispensing unit comprises a reservoir for storing culture medium fluid, and the dispensing unit is configured to dispense the decoupled droplets into the culture medium fluid. This may be particularly preferred, since the steps of dispensing and depositing a single droplet containing one or more entities in the culture medium fluid may be combined into a single step. Droplets may be prepared from the culture medium fluid containing the entities or entity-containing droplets and then dispensed, for example, into individual wells of a microtiter plate.

[0070] In a preferred embodiment of the microfluidic system, the dispensing unit comprises a pressure boost (or decompression) unit for dispensing droplets via a pressurized fluid discharge. The change in pressure can be caused by many techniques, including but not limited to piezoelectric, thermal, and pressurized gas methods. Discharging droplets via a pressurized fluid discharge may be particularly preferred, since the time to dispense droplets from the microfluidic system may be significantly reduced, and / or the discharge via different discharges may be controlled, as further described below. By separating or decoupling the droplets of interest to be dispensed from the microfluidic system from the first fluid flow path, the droplets to be dispensed are only exposed to the high pressure unit. Droplet analysis and sorting in the analyzer and droplet sorting device may be difficult to achieve if the droplets are exposed to the high pressure unit, so it may be preferred to only expose the decoupled droplets to the high pressure unit.

[0071] In a preferred embodiment of the microfluidic system, the droplet forming device includes a plurality of droplet forming devices, and the microfluidic system further comprises a droplet fusion device for fusing a plurality of droplets prepared using the plurality of droplet forming devices. Thus, embodiments of the microfluidic system allow for the creation of an assay of, for example, a single pair of cells, or a single pair of cells and reagents, or a single combination of specific entities contained in a single fused droplet.

[0072] The droplet fusion device may be placed in front of the analyzer and droplet sorting device in the fluid flow direction in the microfluidic system, and preferably, the droplet fusion device may be placed behind the analyzer and droplet sorting device in the fluid flow direction, as this allows for the fusion of only droplets that have been determined to contain one or more target entities of the first and second pluralities of entities, respectively.

[0073] The droplet fusion device may alternatively be placed behind a decoupler, the advantage of which is that there may be a relatively high probability that two droplets each contain the entity or entities of interest before the droplets are fused.

[0074] The droplet fusion device may include an electric field generator for generating an electric field to fuse the droplets by electrocoalescence. Alternatively, or in addition, the droplet fusion device may include one or more charging devices for charging the droplets. The fusion of the droplets is thereby facilitated via electrostatic attraction of the droplets. Alternatively, or in addition, droplet fusion may be achieved by implementing a droplet fusion device with a channel geometry that allows passive droplet fusion, for example, via a channel construction in which a droplet may collide with another droplet and thereby fuse with the other droplet.

[0075] In a preferred embodiment of the microfluidic system, the analyzer comprises one or more of a fluorescence detector, a scattered light detector, an acoustic wave generating and detecting unit, and a magnetic activated cell sorting device. The contents of the droplets may be determined via one or more of these devices, and the droplets may be sorted in the droplet sorting device according to this determination. It will be understood that one or more particular methods or devices for analyzing the contents of the droplets may be preferred over other methods or devices depending on the target contents to be detected in the droplets.

[0076] In a further preferred embodiment of the microfluidic system, the analyzer and / or droplet sorting device comprises a plurality of sensors for detecting the droplets at different locations (at different times) of the microfluidic system and determining the velocity of the droplets in the microfluidic system, and the droplet sorting device is configured to sort the droplets depending on the result of the velocity determination. This allows for improved sorting of droplets, as the location of the droplets in the fluid flow path of the microfluidic system can be determined and / or predicted.

[0077] In a further aspect of the invention, a droplet sorting or dispensing device is provided comprising a substrate having a set of one or more microfluidic input channels, a set of microfluidic output channels, and a sorting element movable between the input and output channels, the sorting element comprising a set of microfluidic channels for connecting the input channels to the output channels, the sorting element being movable between a first position where at least a first of said input channels is connected to allow fluid flow from a first of said input channels to a first of said output channels, and a second position within said channels where a length of fluid carrying one or more droplets is moved such that said length of fluid flows into a second of said output channels.

[0078] Thus, embodiments of the droplet sorting / dispensing device allow for the extraction of droplets from a first fluid flow path by transferring the droplets from the first fluid flow path into a second fluid flow path. Thus, the droplet sorting device may be used to connect and / or complete microfluidic channels of a microfluidic device. As outlined above, this may be particularly preferred as the droplets may be decoupled from the first fluid flow path of a microfluidic system and dispensed from the system, for example, by pressurized fluid ejection from the second fluid flow path.

[0079] In a preferred embodiment of the droplet sorting / dispensing device, the sorting element is rotatable between a first position and a second position, such that fluid lengths from the first and second input channels are either swapped so that they flow into the respective first and second output channels, or swapped so that they flow into the respective second and first output channels. Thus, a previous determination of the droplet's constituents may enable the droplet to be sorted by extracting a droplet of interest (or alternatively a droplet of no interest) from the first fluid flow path and directing it to the second fluid flow path based on the determination.

[0080] Thus, embodiments of the droplet sorting / dispensing device enable the extraction of droplets from a fluid flow path within a microfluidic system without disturbing the fluid flow path.

[0081] In a related aspect of the invention, a method of sorting or dispensing droplets is provided that includes providing first and second microfluidic streams, at least one of the streams containing droplets, and rotating a turntable containing a portion of the streams to selectively direct droplets into one of the first and second outgoing microfluidic streams. Thus, embodiments of the method allow for sorting droplets based, for example, on a previous determination of properties of the droplets and / or their contents.

[0082] In one related aspect of the invention, a device for sorting or dispensing droplets in a microfluidic stream is provided that includes first and second input microfluidic streams, at least one of the streams containing droplets, first and second output microfluidic streams, a rotating turntable containing a portion of the stream between the input and output streams, and a controller for controlling rotation of the turntable to selectively direct droplets into the first and second output microfluidic streams.

[0083] After the droplets enter, for example, one of the portions of the rotating turntable stream from the first input microfluidic stream, the turntable can be rotated to allow the droplets to exit at the second output microfluidic stream. If it is desirable to maintain the droplets within the channel based on a previous analysis of the droplets and / or their contents, the turntable is not rotated to allow the droplets to flow, for example, from the first input microfluidic stream to the first output microfluidic stream via the portion of the rotating turntable stream.

[0084] In a related aspect of the invention, a cell, biomolecule, and entity sorting or dispensing device for transferring a picodroplet from a first fluid flow path to a second fluid flow path is provided, the sorting device comprising a decoupler for decoupling the picodroplet from the first fluid flow path and a guide for directing the decoupled picodroplet to the second fluid flow path, the guide comprising a rotation unit configured to be rotated to transport the decoupled picodroplet to the second fluid flow path and / or a translation unit configured to be moved in a translational direction to transport the decoupled picodroplet to the second fluid flow path. A determination as to whether a droplet should be decoupled and directed from the first fluid flow path to the second fluid flow path may be made before the droplet enters the sorting device. Alternatively, or in addition, this determination may be made when the droplet is in the sorting device.

[0085] In a preferred embodiment, the cell, biomolecule and entity sorting device further comprises an xy-motion platform comprising one or more microtiter plates, and / or one or more glass slides, and / or one or more arrays or other formats in which one or more of the picodroplets can be dispensed at one or more addressable locations. Thus, droplets may be dispensed into one or more individual wells of a microtiter plate, and / or one or more glass slides, and / or one or more arrays or other formats according to a predefined pattern, and the droplets and their contents may be analyzed and / or further processed, for example to culture a population.

[0086] Embodiments of the droplet sorting / dispensing device and / or the cell, biomolecule, and entity sorting / dispensing device described herein may be incorporated into a droplet dispenser, in particular a picodroplet dispenser, configured to selectively dispense selected (sorted) droplets from a microfluidic channel into a reservoir.

[0087] Additionally, embodiments of the droplet sorting / dispensing device and / or embodiments of the cell, biomolecule and entity sorting / dispensing device and / or embodiments of the picodroplet dispenser described herein may be implemented within a microfluidic device.

[0088] cartridge In a further aspect, the invention provides a microfluidic cartridge for automated sorting and dispensing of sorted microfluidic droplets, the cartridge comprising a sort input channel coupled to a sorting region, the sorting region comprising two or more sort output channels coupled to the input channel and a droplet director for selectively directing droplets from the sort input channel to a selected one of the sort output channels, and a droplet dispenser coupled to one of the sort output channels for dispensing the selected droplets from the cartridge for collection in a reservoir, the droplet dispenser comprising a droplet ejection mechanism for ejecting droplets from an emulsion stream into the dispenser output channel for collection in the reservoir.

[0089] An embodiment of such a cartridge is 6 or 10 9 This results in a disposable / consumable device that can be used to solve a number of important problems, such as identifying and recovering targets that may only be present in one of the droplets, e.g., drug-resistant bacteria. Broadly speaking, this can be done in principle by identifying signals from droplets of interest in a common reservoir, but in some preferred embodiments in separate distinguishable reservoirs, and then recovering these droplets for further analysis. This latter approach allows cells or other entities with individually distinguished properties to be removed, for example, from a multi-well plate and then analyzed or used, where effectively separate wells of such a multi-well reservoir can be used to recover individual entities with selected properties.

[0090] Complementarily, embodiments of the cartridge may not be used directly in an assay, but rather in a "stability testing mode" to identify altered (e.g., negative) responses in a population of droplets (e.g., cells) that are all conceptually identical. As an example, in this mode of operation the cartridge may be used to identify individual instances where, for example, production of a substance by cells has been switched off, for example to troubleshoot a drop in yield in a fermentation batch.

[0091] In a preferred embodiment, the cartridge comprises an incubation area or chamber in which the droplets can be held, preferably at a controlled temperature, to allow incubation of the droplet contents prior to controlled release. In an embodiment, this area is configured to hold the droplets at a higher density than when dispersed in the emulsion, which can be achieved by holding the droplets (typically "floating") in the upper part of the chamber and providing a valve or similar at the top of the chamber to provide controlled release of the droplets, but in use the cartridge is oriented so that the top of the incubation chamber faces upwards. Excess carrier medium (oil) may be released to waste. Preferably, but not essentially, the incubation area is upstream of the sorting area.

[0092] Those skilled in the art will appreciate that the emulsion (which may be a double emulsion) is typically a water-in-oil emulsion. The cartridge is preferably configured to interface with a microdroplet processing system, i.e., in embodiments, the cartridge may be attached to and released from a microdroplet processing system that provides sensing and control functions. In embodiments, the microdroplet processing system may provide incubation chambers for droplet sorting and temperature control for droplet property sensing, and may also provide relative motion between the dispenser release channel and the reservoir well to collect the droplets.

[0093] In a typical embodiment, the cartridge generally has the form of a flat plate carrying the microfluidic channels, one or more holding areas, valves, and the like. It is preferably substantially optically transparent and can be fabricated from a variety of plastic materials, such as polydimethylsiloxane (PDMS) or cyclic olefin polymers or copolymers (COP or COC). The cartridge can then be mounted vertically or at a suitable angle into the system / instrument, with the dispenser discharge channel oriented downward toward the multiwell or microtiter plate. The instrument can then move the cartridge and / or plate to orient the discharge channel into a selected well.

[0094] In a preferred embodiment, the cartridge includes multiple fluid connections that can be made automatically when the cartridge is inserted into the instrument. Emulsion generation can be performed on-cartridge or off-cartridge. For example, in an embodiment, the cartridge includes reservoirs along one edge (so that they are oriented correctly when the cartridge is vertical) that can hold oil and water media (water and culture medium) for droplet generation on a droplet-on-chip.

[0095] Embodiments of the microfluidic cartridge have been described previously and are directed to identifying a very small number of extremely rare droplets that contain biological entities of interest from a much larger population. The droplet selector may operate one or two orders of magnitude faster than the droplet dispenser, e.g., the dispenser may operate at 1-10 Hz while the selector may operate at 100-1,000 Hz. However, due to the statistics of the target entities, the dispenser may not operate continuously. Thus, in some preferred implementations of the cartridge, one or more emulsion flow buffer regions are provided between the sorting and dispensing regions of the cartridge to control the emulsion flow rate to the droplet dispenser. In embodiments, such buffer regions may comprise a chamber somewhat similar to the incubation region already described, where the droplets can accumulate while excess oil is siphoned or released to waste. For example, the droplets may be allowed to float to the top of the chamber from where they can be released in a controlled manner by a valve or the like.

[0096] If a buffer region is included, in embodiments this may comprise a length of microfluidic channel, for example in a serpentine configuration, configured such that the order of the droplets is maintained. In this manner, the identity of the droplets with droplet properties measured upstream of the buffer may be maintained as the order or sequence of the droplets is maintained. However, in other arrangements, a wider channel or chamber may be employed, which has the advantage of being easier to accommodate larger volumes. In this case, the droplet properties may be measured after they exit the buffer region (in order). In both cases, preferred embodiments link one or more properties of the droplet to an identifiable property of the droplet, typically its location, so that when the droplet is dispensed into the well / reservoir, the properties of the droplet in the well / reservoir are known.

[0097] Thus, in preferred embodiments of these and other methods / apparatuses described herein, the contents of a dispensed droplet in an identified well / reservoir are known and stored for later use. In this manner, droplet contents having desired / target properties (e.g., cells, proteins, antibodies, reagents, or analytes) may be removed after droplet dispensing by removing the contents of a well / reservoir known (recorded) to have contents with the desired / target properties.

[0098] In some preferred embodiments, the dispenser discharge channel includes a mechanism for facilitating the release of the droplet, or the contents of the droplet, from the emulsion. As previously mentioned, the contents of the droplet typically include water and biological entities, such as cells, often in culture medium. The purpose of the cartridge is to extract those (rare) biological entities with specific properties or characteristics, by collecting the target droplet in one or more reservoirs, which may already contain, for example, 20-50 μl of water / culture medium, in the wells of a microtiter plate. By comparison, the ejected slug of emulsion may have a volume of 50-300 nl (depending in part on the channel width), while the droplet may have a volume on the order of 50 pl or more. Indeed, it has been found that when a slug of emulsion is ejected from the dispenser discharge channel, the water from the droplet, and its contents, may end up floating on the surface of the oil (depending in part on the oil density and whether it is fluorinated). Thus, embodiments of the droplet ejection mechanism preferably include a system for extracting droplets from the emulsion and / or breaking up the droplets, which may be accomplished mechanically, for example, by shaping, e.g., narrowing, the nozzle of the dispenser discharge channel and / or by providing a mesh in the discharge channel, and / or electrically, for example, by providing a pair of electrodes adjacent the discharge of the dispenser discharge channel to which a voltage can be applied to generate an electric field to break up the emulsion / droplets, and / or a chemical mechanism may be employed, for example, by adding a flow of a demulsifier, such as perfluorooctanol, into the dispenser discharge channel.

[0099] In some preferred embodiments, the microfluidic cartridge further comprises a plurality of sensors for detecting the droplets at different locations (at different times) of the microfluidic cartridge to determine a velocity of the droplets within the microfluidic cartridge, and the droplet director is configured to direct the droplets depending on the result of the velocity determination. This may be particularly advantageous as the location of the droplets within the cartridge may be determined and / or predicted, further improving control of sorting the droplets within a sorting region.

[0100] In some preferred embodiments, the droplet ejection mechanism includes a mechanism for increasing the pressure of the emulsion stream to direct droplets into the dispenser ejection channel. In some embodiments, this can be accomplished by configuring the dispenser ejection to have a main channel and a side channel (in the form of a T-junction, as described below), and then applying a pressure pulse to force droplets into the side channel (dispenser ejection channel) for ejection. In one embodiment, such an arrangement can include a pair of valves, such as pinch valves, along the length of the emulsion stream along with the side channel (dispenser ejection channel). In this way, the valves can be closed to separate the streams, and pressure can then be applied to the separated slug of emulsion to force the emulsion, including the droplets, into the dispenser ejection channel. In another approach, the slug of emulsion can be transferred from a first channel / stream to a second channel / stream, where a pressure pulse can be applied to eject the slug and its associated droplets. Those skilled in the art will recognize that other configurations are possible. A suitable pressure pulse may be applied mechanically, for example using a piezoelectric transducer. Alternatively, the pressure pulse may be generated electrically (in a manner similar to an inkjet printer), for example using an electric heating element and bubble expansion. In some preferred embodiments, an elevated pressure pulse is applied to eject a slug of emulsion along with its droplets, although those skilled in the art will appreciate that in principle the arrangements we describe below could be adapted so that a lower pressure pulse could instead be used to eject droplets from the emulsion stream into the dispenser discharge channel.

[0101] In some preferred implementations, it is advantageous to physically separate the droplet ejection mechanism from the system used to sense / detect the presence of the ejecting droplet. In an embodiment, this is achieved by using an instrument housing the cartridge (microdroplet processing system) to sense one or both of the position and velocity of the droplets selected by the sorting system for ejection. Sensing can be performed upstream of the droplet ejection mechanism so that the position of the droplets can be predicted when they reach the ejection mechanism. In principle, the droplet velocity does not need to be measured if the emulsion flow rate can be controlled accurately enough, but in practice, it is advantageous to measure the velocity. It is further advantageous to control the channel size tolerance to reduce channel size variations and therefore droplet velocity variations. Imaging the droplets at a separate location to the droplet ejection mechanism in this manner simplifies the cartridge / instrument and increases its effectiveness.

[0102] As already mentioned, in some preferred embodiments, droplets are dispensed into individual wells of a microtiter plate or the like, which comprises a plurality of separate wells. The instrument (microdroplet processing system) may then comprise a control system for selectively directing dispensed droplets to a reservoir. In embodiments, either the cartridge or the multi-well plate is mounted on an XY movable stage so that the cartridge and plate can be moved relative to each other to direct the dispenser discharge channel to a selected reservoir. As already mentioned, a well may hold one or more droplets, depending on the application / assay / operation mode of the instrument.

[0103] In some preferred implementations, when one or more characteristics of a droplet are queried by the instrument for sorting, corresponding data is stored for selected target droplets having the desired characteristics. The data is then associated with information identifying the particular well or reservoir into which the droplet is dispensed. In this manner, droplets having a particular characteristic may then be removed from the reservoir for later analysis, and / or a multi-well reservoir may be used to extract a set of droplets having a particular characteristic or range of characteristics. Sorting may be based, for example, on a comparison of the measured value to a threshold value, but it is possible that the selected droplets have a range of values ​​of a parameter, which may then be correlated with some other characteristic that measures the activity of the biological entity in the droplet. It is therefore beneficial to be able to link a measure of one or more parameters to a droplet in a particular well of a reservoir. Examples of properties that may be sensed by the instrument include (but are not limited to) optical properties such as one or more of fluorescence, scattering, luminescence, absorbance, electrical or magnetic properties such as electrical conductivity, one or more derived or inferred properties such as whether a cell is live or dead (which may be inferred from optical measurements). Those skilled in the art will appreciate that although it is convenient in embodiments to sense one or more properties of individual droplets as they are sorted, in principle the sensed data stored in association with the reservoir identification data may additionally or alternatively be sensed after sorting by a separate arrangement of sensors. In both cases, the combined cartridge and instrument embodiment facilitates tracking of individual dispensed droplets for later analysis or use.

[0104] As previously described, uses made for cartridge embodiments include direct droplet assays and biological entity stability / viability assays. In some applications, assurance of monoclonality is important, i.e., it may be desirable for individual reservoirs / wells containing biological entities such as cells to contain only a single such entity. This is important, for example, when antibodies are produced from cell lines, i.e., for regulatory purposes, it may be important to be able to substantially guarantee that an antibody is derived from a single clone of cells, for example, when the antibody is being used to make a drug. For these applications, an incubation chamber is not usually necessary, and therefore a cartridge version without such a chamber may be employed. Alternatively, the incubation chamber may be bypassed, or in another version, a more general cartridge may be realized in which droplets may pass through the incubation chamber as previously described without incubation.

[0105] One way that embodiments of the microfluidic cartridge can be used for monoclonality assurance is to use a droplet sorter to select droplets containing only a single cell and discard empty droplets and droplets containing more than one cell. The number of cells in a droplet can be determined optically, for example, by detecting optical absorption, scattered light, fluorescence, or (in some preferred embodiments) by visualizing and counting individual cells within the droplet. Such an approach is facilitated by generating droplets with a strong statistical bias toward the formation of empty droplets. Typically, droplet generation systems employ flow focusing to generate droplets containing a large number of biological entities that follow Poisson statistics. Monoclonality assurance is facilitated by operating such systems such that less than half of the droplets contain a biological entity, preferably less than 30%, 20%, or 10%, for example, the system may be operated such that only about 5% of the droplets contain a single entity / single cell.

[0106] These techniques together can provide a substantial guarantee of monoclonality, i.e., only one single cell per well / reservoir. However, in embodiments, further guarantees are desirable, which may be provided by adding a second sorting step following the first step. Even though in theory the first step should only allow droplets containing a single biological entity / single cell to enter the selected release channel. In embodiments, this second sorting step may be effectively combined with the droplet dispenser, i.e., the dispenser may in embodiments be configured to itself select droplets having a defined number (or numerical range) of biological entities per droplet. Thus, the droplet ejection mechanism may be selective, depending on the confirmation that the droplet contains only a single biological entity, or more generally, a defined number or numerical range of biological entities. Conveniently, the detection of the number / numerical range of entities per droplet may be made by a sensor used to sense the velocity / position of the selected droplet, as already described. In this way, an extremely high degree of monoclonal assurance can be achieved.

[0107] Although the inventors have described a particular application of monoclonality assurance, the techniques described above may be specifically adapted to target a defined number of biological entities per droplet rather than a single entity per droplet. By way of example, when working with droplets containing, for example, bacteria rather than mammalian cells, a numerical range may be more appropriate, which may define tens of organisms per droplet, e.g., 25-35 entities per droplet.

[0108] In yet a further application of the technology, microfluidic cartridges of the general type described above may be provided with a "fusion assay" or functional assay mode of operation.

[0109] To accommodate this mode of operation, the microfluidic cartridge includes a droplet fusion region with at least two input channels and at least one output channel, which serves as the input to the sorting region (the incubation region, if present, can be either before or after the sorting region). Those skilled in the art will appreciate that there are many techniques for fusing microdroplets, including physical fusion techniques and techniques employing electric or other fields to induce droplet fusion (some examples are described in our previous application, U.S. Patent No. 5,399,333, which is incorporated herein by reference). Broadly speaking, the droplet fusion region is used to combine biological entities upstream of the sorting region such that fused droplets may be selected based on potentially rare interactions between the combined entities. As previously described, the two (or more) emulsions containing the droplets to be fused may be generated on or off the cartridge. In some preferred embodiments, such a system may be used to identify very rare cases, potentially down to one in a million or one in a billion, when one biological entity is resistant to the other. Thus, examples may include T cells resistant to infection by HIV or bacteria resistant to phagocytes, and one of skill in the art will readily appreciate that embodiments of the system may be used to identify other similar situations. Again, as one of skill in the art will recognize, cell sorting may be based on any of a variety of physical properties, typically optical properties, and one approach involves a determination of whether an entity within a droplet is live or dead.

[0110] Although the inventors have described a sorting region with two release channels, in principle, three or more release channels may be provided, for example, to sort a mixed population of entities into groups or categories (sorting into multiple channels may be achieved, for example, by applying an electric field using electrode pairs to selectively orient droplets). For example, in a population of cells intended for fermentation, the population may be sorted into groups of high, medium, and low production efficiency, and it may be that, for example, cells with medium production efficiency are then more desirable than those with high production efficiency because they are more robust in large-scale fermentation systems. It will therefore be appreciated that in such an arrangement, one or more of the release channels may be employed to collect the contents of selected droplets for further processing / analysis.

[0111] In cartridge embodiments, in use, broadly speaking, the sample and oil are loaded into separate sterilized reservoirs at one edge of the cartridge (the cartridge is mounted vertically or at a suitable angle) and an emulsion is generated. The emulsion is pumped to an incubation chamber where the droplets float upwards and excess oil is discarded. The incubation chamber is then heated, for example, to 37°C for a period of several minutes to several hours, and then cooled, for example, to about 8°C to limit metabolism. The droplet population is then moved (by a pump) to a sorting area where a target droplet is selected and moved from there to a holding area, again to remove excess oil (the excess can be siphoned off or discarded), and then the droplets are released one by one from the holding area (additional oil is added to facilitate this process) and moved to a dispensing area where a droplet ejection mechanism ejects the selected droplet into a common or separate reservoir for droplets.

[0112] The inventors have described cartridges that may have multiple droplet processing regions, particularly for droplet generation, droplet incubation, droplet sorting, droplet retention ("buffering"), and droplet dispensing. In embodiments, the cartridge is a modular cartridge, having a base or substrate to which separate modules performing some or all of these functions may be attached as desired. This may be facilitated by having a standard interface or set of interfaces between the modules in such an arrangement.

[0113] In a relevant aspect of the invention, a microfluidic system for providing one or more target entities in droplets is provided, the microfluidic system comprising a first detector for detecting whether the droplet contains the one or more target entities and one or both of the properties of the one or more target entities, the microfluidic system further comprising a sorting device for sorting the droplets depending on the result of the detection, and a droplet dispensing unit for dispensing the sorted droplets at one or more target locations, the microfluidic system configured to correlate the target locations with the properties of the one or more target entities detected by the first detector.

[0114] This allows the droplets to be dispensed into individual wells of a microtiter plate or onto another medium. Because the droplets can be dispensed based on the detection results at the first detector, the droplet at a particular target location (which may be, for example, a well of a microtiter plate) can be correlated to properties of the droplet as a whole and / or as its components (if any). These properties may be, but are not limited to, one or more of fluorescence levels, scattered emission levels, absorbance levels, conductivity levels, and other chemical and / or geographic properties.

[0115] Thus, for example, the method / system embodiments we have described, in accordance with both this and other aspects of the invention, may be used to determine which droplets / reservoirs (e.g., which wells of a multi-well plate) have which properties. This may be used later to identify, and more specifically, to deliver a sample of, a cell or biological entity in a particular manner depending on what assay or test is being performed by the microfluidic system (because target entities / droplets having the identified properties are retained in the identified wells, and in embodiments, a log record is kept indicating which droplets / cells / entities are dispensed into which reservoirs / wells).

[0116] Alternatively, sorted and analyzed droplets may be dispensed into a single reservoir.

[0117] It may be preferable to perform one or more further analyses of the droplet and / or its components before a decision can be made as to which target location the droplet may be dispensed.

[0118] Thus, in a preferred embodiment, the microfluidic system further comprises a second detector for detecting said properties of the one or more target entities, and the droplet dispensing unit is further configured to correlate the target position with said properties of the one or more target entities detected by the second detector.

[0119] These one or more further analyses may thereby increase, for example, the probability of identifying one or more target entities in the droplet, in particular a particular numerical value of a target entity and / or one or more target entities having a particular characteristic (e.g. a particular fluorescence level, which may be within a particular range of fluorescence levels).

[0120] In a further preferred embodiment of the microfluidic system, the droplet dispensing unit comprises a second sorting device for transferring the droplets from a first fluid flow path to a second fluid flow path, the second sorting device comprising a decoupler for decoupling the droplets from the first fluid flow path and a guide for guiding the decoupled droplets to the second fluid flow path, the guide comprising a rotation unit configured to be rotated to transport the decoupled droplets to the second fluid flow path and / or a translation unit configured to be moved in a translation direction to transport the decoupled droplets to the second fluid flow path.

[0121] The second sorting device may be used to separate droplets of interest based on the pre-analysis. Thus, the probability that a dispensed droplet has one or more target entities in the droplet, particularly a particular number of target entities and / or one or more target entities with particular properties, may be increased by separating the droplet from the first fluid flow path.

[0122] In a further preferred embodiment of the microfluidic system, the droplet dispensing unit comprises a microfluidic channel junction having an input channel and a plurality of output channels, and the droplet dispensing unit is configured to control to which of the plurality of output channels the droplet is directed based on the detection result via the first and / or second detector.

[0123] Thus, preferably, droplets not of interest may be discarded, and other droplets may be sorted according to the characteristics of their components (if any) while the droplets are being dispensed from the microfluidic system based on the results of previous analysis in the first and / or second detectors.

[0124] It will be understood that one or more additional detectors and / or analyzers may be utilized within the microfluidic system, and the dispensing of droplets from the microfluidic system may be correlated with one or more of these additional analyses in the one or more detectors and / or analyzers.

[0125] In a preferred embodiment of the microfluidic system, the droplet dispensing unit comprises a pressure modulation unit for varying the pressure in a fluid flow path of the droplet dispensing unit to control to which of the multiple ejection channels the droplets are directed, e.g. by varying the pressure between different ejection channels, the droplets can be directed towards and dispensed from a specific ejection channel depending on a previous analysis or detection result of the droplet.

[0126] Alternatively, or in addition, controlling the ejection channel from which the droplets are dispensed may be controlled by one or more of controlling the temperature of the droplets using a heating device, using piezoelectric electrodes, using acoustic actuators, and utilizing other physical and / or mechanical properties of the droplets.

[0127] In a further preferred embodiment of the microfluidic system, a first of the ejection channels is connected to the input channel generally in the fluid flow direction of the input channel and a second of the ejection channels branches off from the input channel, the droplet dispensing unit further comprises a pump connected to the second ejection channel, the pump configured to suck up the droplets from the input channel, and the pressure modulation unit configured to increase the pressure in the input channel in response to the detection in the first and / or second detectors to expel the droplets via the first ejection channel.

[0128] After a droplet of interest is detected, pressure may be increased in the input channel, causing the droplet to be sent to the first output channel instead of being pumped into the second output channel.

[0129] Alternatively, it will be appreciated that the pressure in the input channel may generally be greater than a threshold such that droplets are sent to the first output channel without a pressure change in the input channel. After a droplet of interest is detected, the pressure in the input channel may be reduced such that the target droplet is pumped into the second output channel.

[0130] In a preferred embodiment, the microfluidic system further comprises one or more droplet holding areas disposed between the sorting device and the droplet dispensing unit, the holding areas configured to control the flow of the sorted droplets into the droplet dispensing unit. This embodiment allows for more precise and accurate control of the flow of droplets into the droplet dispensing unit.

[0131] In a further preferred embodiment, the microfluidic system further comprises a plurality of sensors for detecting the droplets at different locations (at different times) of the microfluidic system to determine the velocity of the droplets in the microfluidic system, and the sorting device is configured to sort the droplets depending on the result of the velocity determination. This may be particularly advantageous as the location of the droplets in the microfluidic system may be determined and / or predicted, allowing for improved control of the step of sorting the droplets in the sorting device.

[0132] In a further related aspect of the invention, a microfluidic chip is provided comprising a droplet generation region for generating microdroplets containing one or more biological entities, a droplet incubator and / or storage region for incubating and / or storing said biological entities, a droplet sorting region for sorting said microdroplets based on one or more properties of said biological entities, and a droplet dispensing region for dispensing the sorted microdroplets, wherein said droplet generation region, said droplet incubator and / or storage region, said droplet sorting region, and said droplet dispensing region are integrated onto a single chip design of said microfluidic chip.

[0133] Microfluidic chips can ensure sterility, ease of use, and provide significant cost savings since the generator, incubator, sorting chip, and dispenser are integrated into a single microfluidic chip.

[0134] In a preferred embodiment, the microfluidic chip comprises a microfluidic flow path diversion unit for altering the microfluidic flow paths of the microdroplets between the regions, and a control unit comprising a first processor for controlling the microfluidic flow path diversion unit. This may be preferred as a particular part or parts of the microfluidic chip, e.g., an incubation region, may be omitted and therefore not used to process a particular droplet or droplets.

[0135] Various valves may be provided on the microfluidic chip, which allow for controlling the flow or rate of flow of microdroplets through different regions of the microfluidic chip.

[0136] In a preferred embodiment, the microfluidic chip further comprises a heating device for locally heating the droplets in the droplet incubator and / or storage area (and / or in other specific areas), and further comprises a second processor for controlling said heating device. The first and second processors may be a single processor. Thus, the embodiment may advantageously allow for heating the droplets and their components only in specific locations or areas of the microfluidic chip, thus avoiding heating of the droplets in other areas of the microfluidic chip.

[0137] In a further preferred embodiment, the microfluidic chip further comprises a cooling device for locally cooling the droplets in the droplet incubator and / or storage area (and / or in other specific areas), and further comprises a third processor for controlling said cooling device. The first, second and / or third processor may be a single processor. Thus, the embodiment may advantageously allow for cooling the droplets and their components only in specific locations or areas of the microfluidic chip, and thus cooling of the droplets in other areas of the microfluidic chip may be avoided.

[0138] Embodiments capable of heating (and optionally cooling) may allow for example polymerase chain reaction to occur in one or more specific regions of the microfluidic chip.

[0139] The preferred embodiments described above with respect to the microfluidic systems, droplet sorting or dispensing devices, droplet dispensers, microfluidic devices, microfluidic cartridges, microdroplet processing systems, and instruments for microdroplet-based processing of biological entities may also be implemented as preferred embodiments in microfluidic chips.

[0140] Multimode Operation In another aspect, the invention provides an apparatus for microfluidic droplet-based processing of biological entities, the apparatus comprising a droplet generation system for generating one or more water-in-oil emulsions of droplets comprising the biological entities, a droplet processing system for processing the droplets, and a droplet dispensing system for dispensing the processed droplets into one or more reservoirs, the droplet processing system being configurable for a number of different operational modes, including at least a monoclonal operational mode and an assay operational mode.

[0141] In an embodiment, the droplet processing system comprises at least a droplet sorting function / unit, and the monoclonal operation mode comprises sorting droplets based on the occupancy of the droplets by biological entities. The assay operation mode comprises incubating the droplets, preferably but not essentially, in a droplet incubation unit prior to sorting. Sorting may then be based on the product of the biological entities upon incubation. The droplet processing system may further comprise at least one droplet fusion unit, and the operation mode may then comprise a fusion assay mode, where sorting is based on the outcome of the combination of the biological entities in one set of droplets with substances, e.g., other biological entities, in a second set of droplets. In an embodiment, the droplet processing system is embodied in one or more interchangeable or modular cartridges, i.e., for example, a different cartridge may be provided for each of the different operation modes, or alternatively, a modular cartridge may be employed in which different droplet processing functions, such as sorting functions, incubation functions, dispensing functions, and the like, may be selectively assembled on a cartridge substrate.

[0142] More specifically, the monoclonal mode of operation may involve encapsulating biological entities, typically cells, in droplets, preferably in a manner biased towards the majority of empty droplets. Droplets identified in the sorting unit as being occupied by a single cell are then selectively directed to the droplet dispensing unit, where they are processed and dispensed. In embodiments, a single droplet may be dispensed into an individual well or reservoir, although dispensing two or more droplets into a reservoir or well may be acceptable. For example, a well / reservoir may hold more droplets if the entity or cell is to be reinjected for further processing.

[0143] In the assay mode, it may be acceptable to encapsulate multiple entities / cells per droplet, for example, 2 or 3 cells per droplet. In embodiments, these are then sent to an incubation chamber where they are warmed and metabolized to produce a product, such as a protein or the like. Typically, the product reacts with one or more reagents in the droplet to generate a measurable signal, for example a fluorescent signal, which can then be used to sort the droplets, for example to collect and dispense the best producers. Optionally, a sorting unit may be provided before the incubation area, in particular to remove empty droplets before incubation. This helps to reduce the volume of the incubation chamber, which is particularly beneficial when droplet generation / entity encapsulation results in a large percentage of empty droplets, and depending on the process used, more than 90% of the droplets may be empty.

[0144] Another assay mode, which may be referred to as a stability assay mode, may employ a similar incubation, selection, and dispensing process, but aims to identify a subgroup of the initial population. Such a subgroup may be one that typically exhibits optimal production of the protein, for example for drug production. Optimal production does not necessarily have to be maximal production, but may be, for example, stable production (production that is maintained over a period of time rather than decaying over time), or production that is maintained over a (wide) range of environmental conditions. Thus, such stability assay embodiments may be employed to identify entities that are stable producers, for example, to select producers for a production process, such as a fermentation process, or for quality control of a production process. In an embodiment, the entities may be encapsulated with one or more reagents in a droplet, then incubated, followed by looking for a measurable signal, such as a fluorescent signal, which is then used to distinguish a target subpopulation of entities, followed by dispensing and collection in one or more reservoirs.

[0145] In a further embodiment, in a fusion mode of operation, a typical procedure involves generating two sets of droplets, at least one of which encapsulates a biological entity, which may be pre-generated and provided in respective reservoirs, or may be generated, for example, on-cartridge. The fusion assay may then include droplet fusion, incubation, sorting, and dispensing. Optionally, pre-sorting may be employed before incubation as previously described, and / or after droplet fusion. For example, the fusion assay may be employed for toxicological studies, environmental studies, metagenomic studies, drug uptake studies, or the like. For example, in the latter case, a drug may be provided in a set of microdroplets at a known concentration, and the concentration of the drug may then be measured after incubation (at a remote location or by taking an aliquot) to determine a change in the concentration of the drug from which drug uptake may be inferred.

[0146] For each of the operating modes described above, in embodiments the dispensing unit may also provide an additional selection step, i.e. the droplet dispensing system may comprise a combined stage of droplet sorting and dispensing.

[0147] These and other aspects of the present invention will now be further described, by way of example only, with reference to the accompanying drawings in which: [Brief description of the drawings]

[0148] [Figure 1] 1 is a schematic diagram illustrating droplet detection, sorting, and dispensing according to an embodiment of the present invention. [Diagram 2] 1A-1C are schematic diagrams illustrating further examples of droplet detection, sorting, and dispensing according to embodiments of the present invention. [Diagram 3] 1A-1C are schematic diagrams illustrating further examples of droplet detection, sorting, and dispensing according to embodiments of the present invention. [Figure 4a] FIG. 1 is a schematic diagram illustrating a microfluidic cartridge according to an embodiment of the present invention. [Figure 4b] FIG. 1 is a schematic diagram illustrating a microfluidic cartridge according to an embodiment of the present invention. [Figure 5a] FIG. 2 is a schematic diagram illustrating a decoupler and a control unit according to an embodiment of the present invention. [Figure 5b] FIG. 2 is a schematic diagram illustrating a decoupler and a control unit according to an embodiment of the present invention. [Figure 6] FIG. 2 is a schematic diagram illustrating fluid flow and a decoupler according to an embodiment of the present invention. [Figure 7a] FIG. 2 is a schematic diagram illustrating a decoupler according to an embodiment of the present invention. [Figure 7b] FIG. 2 is a schematic diagram illustrating a decoupler according to an embodiment of the present invention. [Figure 7c] FIG. 2 is a schematic diagram illustrating a decoupler according to an embodiment of the present invention. [Figure 8] FIG. 1 is a schematic diagram illustrating a dispenser according to an embodiment of the present invention. [Figure 9]FIG. 1 illustrates an exemplary droplet generation and sorting system for use in embodiments of the present invention. [Figure 10a] FIG. 2 is a schematic diagram illustrating a further microfluidic cartridge according to an embodiment of the present invention. [Figure 10b] FIG. 2 is a schematic diagram illustrating a further microfluidic cartridge according to an embodiment of the present invention. [Figure 11a] FIG. 2 illustrates an exemplary control system for controlling a droplet processing system according to one embodiment of the present invention. [Figure 11b] FIG. 2 illustrates an exemplary control system for controlling a droplet processing system according to one embodiment of the present invention. [Figure 11c] FIG. 2 illustrates an exemplary control system for controlling a droplet processing system according to one embodiment of the present invention. [Figure 11d] FIG. 2 illustrates an exemplary control system for controlling a droplet processing system according to one embodiment of the present invention. [Figure 12] FIG. 1 illustrates an exemplary physical configuration of a droplet processing system according to an embodiment of the present invention. [Figure 13] FIG. 1 is a schematic diagram showing the phases for cell analysis. [Figure 14a] FIG. 1 is a schematic diagram of a method for determining the contents of a droplet. [Figure 14b] FIG. 1 is a schematic diagram of a method for determining the contents of a droplet. [Figure 15] FIG. 1 shows monoclonal antibodies in picodroplets by hybridoma cells. [Figure 16a] FIG. 1 shows Chinese Hamster Ovary Cell Populations. [Figure 16b] FIG. 1 shows Chinese Hamster Ovary Cell Populations. [Figure 17] FIG. 1 shows the percentage of viable CHO-S cells versus culture time for different picodroplet sizes. [Figure 18a] FIG. 1 is a schematic diagram illustrating a microfluidic chip, according to an embodiment of the present invention. [Figure 18b]FIG. 2 is a schematic block diagram illustrating a control system according to an embodiment of the present invention. [Figure 19a] FIG. 1 is a schematic diagram showing detection of a single cell within a picodroplet using an embodiment of the present invention. [Figure 19b] FIG. 1 is a schematic diagram showing detection of a single cell within a picodroplet using an embodiment of the present invention. [Figure 19c] FIG. 1 is a schematic diagram showing detection of a single cell within a picodroplet using an embodiment of the present invention. [Figure 20a] FIG. 1 shows a scatter plot of red and green fluorescence from picodroplets over time. [Figure 20b] FIG. 1 shows a scatter plot of red and green fluorescence from picodroplets over time. [Figure 20c] FIG. 1 shows a scatter plot of red and green fluorescence from picodroplets over time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0149] 1 is a schematic diagram illustrating a first example of droplet detection, sorting, and dispensing according to embodiments described herein, which in this example is referred to as a monoclonality guaranteed mode.

[0150] In this example, a plurality of cells are provided in a fluid, in this example water, optionally including culture medium. In a first step, individual droplets are formed from the fluid. As outlined above, this can be achieved, for example, by using T-junctions, Y-junctions, flow focusing devices, and other devices. The droplets that are generated are transported in a fluid, in this example oil.

[0151] The individual droplets, which may or may not contain one or more cells, are then guided through a microfluidic device within the oil emulsion.

[0152] In this example, the picodroplet cell suspension, i.e., droplets in an oil emulsion, are directed toward a detection and sorting device. Whether a single droplet contains one or more cells can be detected in an analyzer based on one or more of the droplet's electrical, optical, thermal, acoustic, mechanical, temporal, spatial, and other physical properties. Based on the analysis in the analyzer, i.e., the determination as to whether a single droplet contains one or more target cells, the droplet can be sorted in a droplet sorting device. In this example, picodroplets that do not contain one or more cells are discarded. Furthermore, in this example, droplets containing a single cell of interest are directed toward a decoupler of the microfluidic system.

[0153] A droplet containing one or more cells of interest is then extracted from the first fluid flow path and transferred into the second fluid flow path. In this example, the target droplet is extracted from the first fluid flow path in the culture medium fluid. The droplet containing the target cell is thereby incorporated into the culture medium fluid, which is then dispensed into the microtiter plate via pressurized fluid ejection. A pressure source is attached in this example to the flow path through which the culture medium fluid is injected. The picodroplet may thereby be diluted. In this example, a robotic xy translation stage is provided to dispense the droplet into different wells of the microtiter plate.

[0154] Droplet detection, sorting, and cell dispensing are performed in this example in a temperature controlled environment.

[0155] In this example, picodroplets that contain single cells and are not to be disposed in a microtiter plate are diverted in the first fluid flow path and discarded.

[0156] As outlined above, the probability of finding a single cell in a single droplet disposed within a microtiter plate can be greater than 99.997%.

[0157] In the example of FIG. 1, an automated plate stacker interface may be provided to stack various microtiter plates one on top of the other.

[0158] In some preferred implementations of the system of FIG. 1, and also of the systems described below, an imaging device such as a camera is provided at or near the point where the droplets are dispensed (into a microtiter plate). The imaging device can image the droplets to determine whether they contain only a single cell (or other biological entity), or alternatively, two or more cells, or no cells at all. In other approaches, optical detection of scattering from the droplets can be performed for this purpose. Such optical systems can be used as a tool to monitor monoclonality, more specifically in conjunction with a control system, to identify and log when a well does not contain only a single cell (or vice versa), for example, the system can then flag an error for the identified well.

[0159] 2 is a schematic diagram showing a further example of droplet detection, sorting and dispensing according to embodiments described herein. Two different modes of operation are displayed: a direct assay mode and a stability test mode.

[0160] In this example, cells are prepared in a fluid along with assay reagents and then individual droplets are formed from the fluid as outlined in the example shown in FIG.

[0161] The droplets prepared from the fluid containing the cells and the assay reagents are then introduced into an incubator. The incubator may be used to culture and / or maintain the cells in the droplets. As outlined above, the incubator may include a stability test unit that allows stability tests to be performed on the cells during incubation. Performing stability tests on the cells in a single droplet allows the analyzer and droplet sorting device to select only viable cells for the detection and sorting steps, which are not degraded or dead during the stability test.

[0162] Further steps of determining the contents of the droplets, sorting the droplets based on the determination, and potentially extracting the droplets of interest in a decoupler are performed as outlined with respect to the schematic diagram of FIG. 1.

[0163] 3 is a schematic diagram illustrating a further example of droplet detection, sorting, and dispensing according to embodiments described herein, which in this example is referred to as a picodroplet fusion assay mode.

[0164] In this example, a picodroplet fusion assay mode is illustrated. A first cell type A is prepared in a first fluid. Then, separate droplets are formed from this first fluid. A second cell type B is prepared in a second fluid, from which separate droplets are formed. The droplets prepared from the first fluid as well as from the second fluid are guided towards a fusion device (electrodes in FIG. 3). In this example, two droplets from the first and second fluids, respectively, are prepared by electrocoalescence. The fused droplets can be further processed as shown in the schematic diagrams of FIG. 1 and / or FIG. 2.

[0165] As outlined above, the droplet fusion device may, for example, be placed behind the analyzer and droplet sorting device in the fluid flow direction of the microfluidic system. Such a configuration may allow for droplet fusion in the droplet fusion device, in this example, only for droplets determined to contain cells of interest for culturing and / or further analysis and processing.

[0166] Reference is now made to FIG. 4a, which shows a block diagram of one embodiment of a microdroplet processing system / instrument 400 incorporating a (disposable) microfluidic cartridge 410 according to one embodiment of the present invention. The instrument 400 has a reservoir 402 that holds an aqueous medium, typically including culture medium, in which biological entities such as mammalian cells, bacteria, or the like are held for processing. A set of reservoirs 404a-d hold oils for forming emulsions at various stages of the system. The reservoirs 402, 404 are shown as part of the instrument and not as part of the cartridge, although in other embodiments that may be part of the cartridge (as shown later in FIG. 10a), there may be multiple aqueous reservoirs 402 depending on the function of the cartridge, for example, whether it is to be used for fusion or functional assays. Fluids from the reservoirs 402, 404 are pumped by respective pumps in FIG. 4a, shown by a fluid drive module 406, which again may be integrated with the cartridge. As will be shown later with reference to FIG. 12, a set of fluid flow conduits 408 connect to respective ports on the cartridge when the cartridge is inserted into the instrument.

[0167] The illustrated cartridge 410 includes a droplet generation region 412, a droplet incubation / storage region 414, a droplet sorting region 416, a flow buffer region 418, and a droplet dispenser 420. In cartridge embodiments, each of these regions is modular, and an emulsion flow processing configuration may be selected by selecting modules to attach to a cartridge base or holder such that the selected modules mate with each other to perform the desired function for the particular cartridge.

[0168] In FIG. 4a, the smaller circular area represents the input / output ports to the droplet processing area which may represent on-cartridge or off-cartridge connections depending on the cartridge configuration, the larger circular area represents the optical droplet sensing area, and the circle with a cross represents the valve.

[0169] In an embodiment, the droplet generation module or region 412 of the cartridge includes an aqueous sample input 422 and an oil input 424 for generating an emulsion. These liquid streams are directed to a flow focusing junction 426 where an emulsion is generated and directed to the region's output channel 428. By way of example, the product oil may have a flow rate of 1400 μl per hour, the sample, e.g., a cell suspension, may have a flow rate of 1000 μl per hour, and the water-in-oil emulsion in the output channel may have a flow rate of 2400 μl per hour with 700 picoliter droplets at 1000 Hz, to name a few illustrative numbers.

[0170] The emulsion (which may alternatively be made off-cartridge and added to a reservoir) is then sent to the incubation / storage area 414, in this example. This comprises a storage chamber 430, in which the individual droplets 432 float upwards, and excess oil flows (via a valve) through a channel 434 to be disposed of into a reservoir 436, from which the oil can potentially be recycled. In an embodiment, the oil comprises a fluorous oil, which is particularly suitable for scavenging oxygen, for example enabling the promotion of growth of biological entities, for example cells, in the droplets while stored in the chamber 430. Thus, the chamber 430 may be filled with fluorous oil above a threshold value (e.g., 50% of the volume of the chamber 430, or more), which may provide sufficient oxygen for the entities in the droplets over a certain period of time. In an embodiment, the instrument in which the cartridge is placed comprises a heating device, such as a heater plate, and / or a cooling device, such as a Peltier effect element, adjacent to the incubation chamber 430, preferably together with a temperature sensor for temperature control. In this manner, the chamber may be heated for a controlled period of time to incubate the droplet contents and then cooled to substantially prevent incubation. The incubation chamber has a discharge channel 438 with an optional side channel 440 to receive spacing oil for spacing the droplets in the emulsion in the discharge as it is dispensed out of the chamber 430. Continuing with the previous numerical example, around 200,000 droplets are stored in a volume of 0.22 ml, and just under 2 trillion droplets are stored in a volume of around 2.2 ml (assuming 67% fill), so around 2.2 ml of oil may be wasted (because they displaced the oil that had already filled the chamber). The output channel 438 from the incubation area may have a flow rate of around 350 μl per hour and spacing oil may be added at port 440 or in the subsequent sorting area (see below) at a flow rate of around 8,000-11,000 μl per hour.

[0171] The sorting region 416 is coupled to an output channel 438 of the incubation region, as an input channel 442 in this embodiment, and a pair of output channels 444, 446, with the channel 444 containing the desired droplets with a load of the selected target analyte (one or more entities). The channel 446 leads to a waste reservoir 436. Optionally, an input 448 is provided for adding spacing oil to the flow into the sorting region. The sorting region includes a region 450 where the droplet contents are interrogated (optically) by an instrument holding a cartridge, which then drives an electrode 452 to selectively direct the droplets into either channel 444 or channel 446 (connections to electrode 452 are not shown in FIG. 4a for simplicity). Continuing with the previous numerical example, the mark:space ratio of the droplets in the input emulsion to the sorting region 416 may be on the order of 1:10 to 1:20, and the droplets may enter the sorting region at a rate of several hundred Hz. The flow rate in channel 444 may be on the order of 5,000 μl per hour, containing between 1,000 and 10,000 droplets, and the flow rate of the emulsion in channel 446 may be substantially the same, but the emulsion may contain on the order of 190,000 to 2,000,000 droplets (per hour).

[0172] The sorting region's "target" discharge channel 444 is coupled to an input channel 454 of the space / holding or "flow buffer" region 418. It comprises a chamber 456, typically somewhat smaller than the incubation chamber 430, in which the droplets are temporarily held (e.g., suspended at the top of the chamber) before being released into the release channel 458. In an embodiment, the oil comprises a fluorous oil, which is particularly suitable for scavenging oxygen, for example enabling the promotion of growth of biological entities, such as cells, in the droplets while stored in the chamber 456. Thus, the chamber 456 may be filled with fluorous oil above a threshold value (e.g., 50% of the volume of the chamber 456, or more), which may provide sufficient oxygen for the entities in the droplets over a certain period of time. Release from the chamber 456 is facilitated by adding spacing oil via an input channel 460. Similar to the incubation region, the flow buffer region also comprises a discharge channel 462 to a waste reservoir 436. Using a numerical example, a 10 ml volume chamber 456 can hold 10,000 droplets. The output flow rate in channel 458 can be on the order of 4 μl per hour, spacing oil in channel 460 can flow at 5,000 μl per hour, and waste in channel 462 can flow at a variety of flow rates, for example, between 10 and 5,000 μl per hour.

[0173] In the illustrated embodiment, the outlet channel 458 of the flow buffer region 418 provides an input via channel 464 to the droplet dispenser region 420. In the illustrated embodiment of the dispenser, channel 466 provides a second input to the dispenser that contains an eluent, i.e., an oil used for / to extract and eject the target droplets.

[0174] The dispenser has a first outlet channel 468 which flows back to waste, and a second outlet channel 470 with an outlet 472 for the dispenser to expel selected droplets for collection in a reservoir. A further channel 476 is coupled to an input 478, which in this example receives compressed air at a pressure in the range of 0-2.5 bar, for example. In this way, a pressure pulse can be applied to the emulsion flowing in the outlet channel 470 (after closing valve 477) to expel a slug of emulsion 480 containing selected target droplets 482 for collection in a multi-well reservoir 484. In the illustrated embodiment, the dispenser 420 has a rotary valve 474 which can be used to selectively direct droplets into a fluid stream linking the dispenser outlet channel 470 or into a fluid stream linking the waste channel 468.

[0175] Optionally, the dispenser 420 may include one or more droplet sensing regions for 486. These may be used to identify when droplets are present in the stream and control the timing of droplet ejection accordingly to eject the emulsion slug 480. Additionally or alternatively, one or more of these sensing regions may be used to sense the contents of the target droplet, which in embodiments may be used to apply a further level of selection to the ejected droplet, for example to provide further assurance of monoclonality of the contents of the droplet 482, or to provide some further selectivity to the droplet contents or a number or range of numbers of entities within the droplet 482.

[0176] In an embodiment, the multi-well plate 484 may comprise a number of wells 488 in which an aqueous (culture) medium 490 may be prepared as appropriate. In an embodiment, the multi-well plate 484 is moved, for example by an XY stage (not shown), so that each well receives only one slug of emulsion containing only a single droplet. Furthermore, in an embodiment, the instrument controlling the system of FIG. 4a senses any property of the droplets, or more specifically the contents of the droplets, in the sorter (or later in the dispenser if the droplets are not easily tracked as they pass through the buffer region). Thus, a log may be generated and stored, defining one or more properties, e.g., fluorescence or the like, of each droplet in each well. In this way, a small number or individual single entities having one or more properties of interest may be selected for further study or use, e.g., for analysis, antibody production, fermentation, or the like.

[0177] Various sensors may be used, for example, in regions 414, 416, and 420 of the microdroplet processing system / apparatus 400, which allow for detection of droplets. These sensors may be optical and / or electrical sensors. Electrical sensors may use the fact that if cells are present in the droplet, the insulating cells affect the electrical properties of the droplet, and thus capacitance measurements allow for detection as to whether a droplet containing a cell is present. Using capacitance measurements, the size of the droplet may further be determined. Those skilled in the art will be familiar with alternative methods and techniques for detecting droplets and / or droplet components.

[0178] The sensor may be used to count droplets, and by providing multiple sensors in sequence, the velocity of droplets flowing through the processing system / instrument may be determined. This may, for example, allow for activating droplet sorting at the appropriate time. This may make the droplet processing system or microfluidic chip more efficient, as units enabling droplet sorting may only need to be activated at certain times.

[0179] In some examples, a further sensor is provided behind the sorting junction in the flow direction. The further sensor can be used to double-check whether the droplets have been sorted into the correct flow path / channel by comparing the detection result of the droplets by the further sensor with the detection data from the sensor upstream from the sorting junction and based on the determined velocity data. If the droplets have not been sorted correctly, further actions can be performed downstream from the sorting junction by further sorting the previously sorted droplets between multiple flow paths / channels.

[0180] Reference is now made to FIG. 4b, which shows another embodiment of a microdroplet processing system / instrument 400′, in which elements similar to those previously described are indicated by similar reference numbers. The arrangement of FIG. 4b may be implemented partially or even completely separate from the cartridge previously described, for example on a linked module within an instrument. One preferred implementation of the arrangement of FIG. 4b is as a “semi-modular” cartridge, with off-board droplet generation and off-board droplet sorting, but on-board droplet incubation / storage and dispensing. As previously, the circle with a cross indicated a valve, in a variation of FIG. 4b, two flow buffer regions are illustrated. Those skilled in the art will appreciate that several different systems can be assembled with different functions according to requirements.

[0181] Some of the optical systems used in preferred embodiments of the instrument will now be described with reference to FIG. 4b, with the understanding that similar optical systems may be used with the arrangement of FIG. 4a, and that not all of the described systems may be required depending on the application. Thus, in the arrangement of FIG. 4b, a first optical sensing area 491 is used by the optical system to determine droplet size, and a second optical sensing area 492 is used for optical diagnostic purposes. A third optical sensing area 493 may be used for optical cell detection and sorting (e.g., by imaging cells within the droplet), and a fourth optical sensing area 494 may be used for post-sorting diagnostics. Optical sensing area 495 may also be used for diagnostic purposes, optical sensing area 496 may be used for timing purposes (e.g., to establish droplet position / velocity / time relative to the dispenser), and droplet optical sensing area 497 may be used for droplet content (cell) detection, e.g., for use in (selective) dispensing. Dispensing area 420' in the device of FIG. 4b may comprise a dispenser of the type described below with reference to FIG.

[0182] FIG. 5a shows a schematic diagram of a decoupler 500 according to an embodiment that may be described herein, whereby droplets may be extracted from a first fluid flow path (channel) 502 by transferring the droplets from the first fluid flow path to a second fluid flow path (channel) 504.

[0183] In this example, individual droplets are directed towards a decoupler in a first channel 502 in a water-in-oil emulsion. After the droplet of interest enters the decoupler, a rotation unit 506 of the decoupler can be rotated such that the droplet of interest is transferred from the first channel into a second channel 504 of the device. Oil flowing in the second channel can then direct the droplet to an outlet 508 of the second channel. After the droplet enters the second channel 504 of the device, it is decoupled from the first channel and then pressure is preferably applied to the second channel to expel the selected droplet from the outlet 508.

[0184] Decouplers may be used, for example, on microfluidic chips to separate a bolus of fluid (a slug of emulsion) containing a picodroplet or other bolus of interest (e.g., cells, bacteria, and others) and translate this bolus of fluid from a first fluid flow path to a second fluid flow path, from which the droplet can then be dispensed from the microfluidic chip.

[0185] In this example, the decoupler with the rotation unit allows for accurate capture and separation of the fluid body without causing significant disruption to the first fluid flow path in the microfluidic channel network. The decoupler further allows for decoupling of the off-chip dispensing mechanism, ensuring accurate and rapid removal of the separated fluid body from the chip; in this example, the rotation unit of the decoupler can operate at speeds up to 5 Hz or more (limited in embodiments by the speed of the step movement of the microtiter plate).

[0186] A decoupler such as that shown in Figure 5a may form part of a droplet dispenser. In one approach, as shown, droplets selected for ejection may be transferred from channel 502 to channel 504, and then pressure, such as air pressure, may be applied to channel 504 to eject the droplets (within the slug of emulsion). If a droplet is unwanted / should not be ejected, it may be left in channel 502 and discarded. Alternatively, channel 504 may be discarded and unwanted droplets may be transferred from channel 503 to channel 504, and then the ejecting droplets may be ejected from an opening in channel 502, again by air pressure or the like.

[0187] 5b shows a block diagram of an exemplary control unit for a decoupler. The control unit 500 comprises a detector 502, which in this example can be used to detect the contents of a droplet and its physical properties. The detector 502, as well as the other units of the control unit 500, are connected to each other via a bus 504.

[0188] The processor 506 is configured to process signals obtained from the detector 502. An operating system / software 508 may be included in the control unit 500 and processed in the processor 506 and applied to signals obtained via the detector 502, in this example to determine the components of cells in the droplet and their physical properties. The operating system / software 508 may be stored in the memory 512.

[0189] Depending on the result of the signal provided from detector 502 to processor 506, a signal may be sent to decoupler 510. The signal, in this example, is used to control a rotation unit of decoupler 510, thereby maintaining the droplet in the first fluid flow path or transferring the droplet from the first fluid flow path to the second fluid flow path.

[0190] For example, information regarding the components of the droplets analyzed, the number of droplets transferred from the first fluid flow path to the second fluid flow path, and other information may be stored in a memory that may be integral to memory 512.

[0191] 6 is a schematic diagram showing an exemplary fluid flow and decoupler according to an embodiment described herein. As can be seen, a sample of interest is provided in a container, e.g., a fluid that can be stored in a microfluidic device. Picodroplets can be prepared from the fluid in general as outlined above. In this example, additional containers are provided for eluents and diluents. An oil reservoir can allow an oil flow to direct droplets containing one or more entities through the device.

[0192] In this example, the eluent container, the diluent (oil) container, and the sample fluid container are each connected to a different input of the decoupler by a microfluidic channel. After the droplets enter the decoupler input, they may be guided through the decoupler channel network, in this example, to a rotation unit. The droplets may then be transferred from one channel to the other by rotating the rotation unit.

[0193] In this example, a rotary solenoid is used to rotate the rotary unit, which may be controlled as outlined above with respect to Figure 5b.

[0194] After it is determined that the droplets will exit a particular outlet of the decoupler, the droplets may be dispensed from the decoupler or the microfluidic device or chip. In this example, an xy stage is provided to dispense the droplets at a desired location in the xy plane, for example, into a microtiter plate or other format. Additionally, a pneumatic flow restrictor may be provided to more precisely control the flow rate when dispensing the droplets from the device. A thermocouple, in this example, is connected to the channel between the outlet of the decoupler and the pneumatic flow restrictor. One or more nozzles may be provided at the outlet of the decoupler. In this example, an additional high-speed solenoid valve is provided to control the dispensing of the droplets.

[0195] In the exemplary device of FIG. 6, a high pressure unit is provided that allows droplets to be dispensed from the device via pressurized fluid discharge. The high pressure unit in this example comprises a compressed gas container, various pressure regulators for adjusting the pressure in the channel network, and a T-junction connecting the compressed gas container and regulators with a pressure sensor. It will be appreciated that alternative configurations may allow for controlling the pressure in the channel network to discharge droplets via pressurized fluid discharge.

[0196] In this example, a waste container is connected to the discharge of the droplet sorting device: droplets containing entities of no interest or no entities at all can be discarded.

[0197] It will be appreciated that any of the inputs of the decoupler shown in Figure 6 may be used as outputs, and vice versa.

[0198] FIG. 7 is a schematic diagram illustrating a decoupler according to an embodiment described herein.

[0199] 7a is a schematic top view of an exemplary decoupler. As can be seen, various inputs are provided on the decoupler. The inputs are connected via channels to a rotation unit of the decoupler. The rotation unit is further connected via a network of channels to an output of the decoupler from which droplets can be dispensed from the device.

[0200] Figure 7b is a schematic side view of a rotating unit of a decoupler. It will be understood that the dimensions of the rotating unit are merely exemplary. The rotating unit may comprise an opening (D in Figure 7b) through which droplets enter and / or exit the rotating unit.

[0201] 7c is a schematic perspective view of a decoupler with a rotating unit. In this example, four inputs and one output are provided. However, as outlined above, depending on the specific requirements of the device, each of these inputs may also be used as an output, and the output may equally be used as an input.

[0202] 8, which shows an exemplary embodiment of a droplet dispenser 800. It comprises an input channel 802 with a shared channel (flow) portion 808 and a first output channel 804 and a second output channel 806. Channel 804 comprises a nozzle 810 at the output to dispense a slug of emulsion 812 containing droplets 814. Channel 806 goes to waste in an embodiment under suction. The dispenser also has an air input 816 for a pressurized air source. Input channel 802 receives an oil supply 818 with droplets 820 spaced apart. Input channel 802 comprises a feed valve 822, waste output channel 806 comprises a shutoff valve 824, and air supply input 816 comprises an air valve 826. In the illustrated example, area 828 defines the optical droplet detection area. The separation 830 between the air input 816 and the channel 806 defines the length of the shared flow path between the two discharge channels 804 , 806 and defines the length of the discharged slug of emulsion 812 .

[0203] When oil without droplets or oil with unwanted droplets is passing through the dispenser, feed valve 822 opens, shutoff valve 824 opens, and the input fluid stream passes along the shared channel area 808 and is drawn into channel 806 to waste. When a droplet to dispense is detected in channel 808, valves 822 and 824 are shut off and air valve 826 is opened to eject a slug of emulsion 812 containing droplets 814 from channel 808 along discharge channel 804 and out via nozzle insert 810. A corresponding procedure may be performed when a droplet to dispense is predicted to be present in channel 808 in a system where droplet detection is performed upstream.

[0204] In embodiments, the nozzle may be shaped to break up slugs, or more specifically droplets, of emulsion and / or may include a mesh for a similar purpose, so that when the slugs / droplets are dispensed into a well containing an aqueous medium, the droplet contents are liberated into the aqueous medium of the well rather than floating to the top. Optionally, the nozzle 810 may be equipped with other means for similar purposes, for example one or more electrodes for generating an electric field, and / or additional channels for carrying a demulsifier.

[0205] A flow control unit may optionally be provided to control the rate at which droplets flow into the droplet dispenser 800. The flow control unit may be coupled to a holding chamber in front of the droplet dispenser 800, which allows droplets to be delivered to the droplet dispenser at a defined flow rate.

[0206] In some embodiments, channel 806 is wider than channel 804, which results in a bias of droplets towards waste channel 806. This may be particularly useful in embodiments where the microfluidic system or chip in which droplet dispenser 800 is incorporated is used in a vertical orientation, and aqueous droplets have a tendency to flow towards the top of the system or chip. Thus, in embodiments where the system or microfluidic chip is used vertically, channel 806 is preferably located on the upper side of droplet dispenser 800.

[0207] FIG. 9 illustrates an exemplary droplet generation and sorting system for use in embodiments of the present invention. Thus, FIG. 9 illustrates one example of a system that may be implemented on a cartridge to implement the droplet generation and sorting functions previously described. After the components of the droplet (if any) have been analyzed, for example as outlined with respect to FIG. 14a and FIG. 14b, the droplets may be sorted based on this analysis, in this example by applying an electric field using electrodes (shown in black in the inset). Droplet sorting is based in this example on the dipole moment of the droplet, whereby the droplets are subjected to different electric forces depending on their components.

[0208] 10a shows a perspective view of a droplet processing cartridge 1000 according to one embodiment of the invention in which a sample / oil reservoir is assembled within the cartridge. As illustrated, the cartridge includes an area 1002 for actuating the cartridge's valves, electrical connections 1004 for the sorting area, and a dispensing nozzle 1006 (hidden in the perspective view shown).

[0209] FIG. 10b shows a top view of a modular cartridge 1050 according to one embodiment of the invention. The illustrated modular implementation comprises a droplet generation module 1052, a droplet incubation / storage module 1054, a droplet sorting module 1056, a droplet / holding (flow buffer) area 1058, and a droplet dispensing area 1060. The figure also shows on-cartridge connections 1062 ("manifold channels") between the modules, as well as the placement of exemplary valves 1064 and datum location bins 1066. In an embodiment, the channels 1062 are defined in a base plate of the cartridge on which a gasket is provided, and then the modular droplet processing areas 1052-1060 are implemented by mounting a separate plate on top of the gasket, preferably located by datum pins 1066.

[0210] Reference is now made to Figure 11, which illustrates an embodiment of a control system for a microdroplet processing instrument according to one embodiment of the present invention. The control system may be implemented on a general purpose or special purpose computer equipped with various sensors and actuators, as described further below.

[0211] Thus, referring to FIG. 11a, this shows a droplet generation control system 1100 comprising a control computer system 1102 coupled to sensors for the sample 1104 and the oil 1106, and for controlling a valve 1108 that controls the emulsion generation. The controller 1102 is also coupled to an intelligent camera 1110, i.e. a camera with on-board processing capabilities. However, it will be understood that alternatively the processing shown in the camera could be implemented by the controller 1102, and vice versa, in principle the functions of the controller 1102 could be implemented on the camera 1110. The intelligent camera comprises an image capture system 1112 that controls lighting 1114, e.g. light emitting diode (LED) lighting. The image capture module 1112 also comprises a camera (not shown) for capturing images. The captured images are processed by a droplet size control procedure 1116 that implements a control loop 1118 for controlling pumps 1120, 1122 to control the flow rates of the sample and oil and thus the emulsion formation.

[0212] 11b shows a temperature control system 1130, in this example using the same controller 1102, coupled in this example to a PID (Proportional Integral Derivative) controller 1132. This may implement one or multiple control loops, each loop comprising a temperature sensor 1134 and a temperature control device, for example a heating device (not shown) and / or a cooling device such as a Peltier effect element 1136. Other sensors / controls may also be implemented as appropriate.

[0213] In some embodiments, a heating / cooling unit may be provided that allows heating and cooling of the entire system or the microfluidic chip. Heating and cooling may be performed locally in some areas, e.g., in specific areas of the microfluidic chip only. For example, heating and cooling may be performed at the incubator of the system or the microfluidic chip. In some embodiments, the temperature of the system or the microfluidic chip is set to 8-10° C. The heating / cooling unit may then be used to raise the temperature, e.g., to 37° C. in the incubator, and the temperature may then be lowered to a temperature of 8-10° C., e.g., to stop biological activity of cells or other entities stored in the droplets. Thus, the embodiments described herein may generate a polymerase chain reaction (PCR) of biological entities, which generally consists of repeated cycles of heating and cooling. The droplets may then be sorted after the heating / cooling cycles.

[0214] 11c illustrates a droplet sorting control system 1140 that also includes an intelligent camera 1142 (although again, a dumb camera may alternatively be used). In the illustrated example, the sorting system includes a droplet flash illumination device 1144, a photomultiplier tube 1146 for cell detection, and an optional further imaging device 1148 for cell detection confirmation, preferably all operating under the control of a cell detection algorithm 1150. The sorting control system also includes a signal generator 1152 and optional amplifier 1154, which sends control signals to the sorting electrodes previously described.

[0215] 11d shows a droplet dispensing control system 1160, which in the illustrated example comprises a further intelligent camera 1162 (but again, this could be a dumb camera), with processing performed by controller 1102. In the illustrated example, camera 1162 controls droplet illumination, in particular a flash device 1164, implements an image capture procedure 1166 from an imaging device (not shown), and detects droplet contents, e.g., cells, using a cell detection algorithm 1168. A control procedure 1170 controls a compressed air source 1172, appropriate valves 1174 (such as valve 477 in FIG. 4a), and an XY stage 1176 holding a multiwell plate 484.

[0216] FIG. 12 illustrates an exemplary physical configuration of a droplet processing instrument 1200, where similar elements to those previously described are indicated by similar reference numbers. FIG. 12 illustrates the arrangement of intelligent cameras 1202, 1204, 1206 for droplet generation, sorting, and dispensing, respectively. These have respective (LED) lights 1114, 1144, 1164 under the control of an LED controller 1208. The system also illustrates a cartridge 410 and an incubator heater plate 1210 for the cartridges, as well as valve actuators 1212 for droplet generation and incubation. A multi-well plate 484 is mounted on an XY stage 1214. FIG. 12 further illustrates a sample reservoir 402 and oil reservoirs and pumps 404a-d for supplying oil for droplet generation, sorting, spacing, and dispensing. The instrument also includes an internal oil reservoir 1216 for supplying these reservoirs, and a waste oil reservoir 436. The instrument includes a control system as previously described, as well as a user interface 1218.

[0217] FIG. 13 is a schematic diagram showing the phases for cell analysis. Devices and methods known to those skilled in the art are implemented in phases A, B, and D. Antigens are selected and produced in phase A according to standard techniques. Immunization and isolation of B cells, in this example, may be performed in phase B, thereby obtaining a fluid containing cells to be sorted and / or analyzed by the embodiments described herein. In phase C, single cells may be obtained in a single droplet using the embodiments of the devices and methods described herein. Viable cells in a single picodroplet may be provided, on the basis of which further experiments, in this example transcriptomics and sequencing experiments, may be performed in phase D.

[0218] 14a and 14b are schematic diagrams of a method for determining the contents of a droplet. In this example, two fluorescent dyes, AF647 and AF488, are placed in a picodroplet. In this example, when hybridoma cells are present in the picodroplet, products of the hybridoma cells, such as proteins or antibodies, can bind to each of the AF647 and AF488 fluorescent dyes (each of which has an associated antibody that binds to a different respective portion of the hybridoma cell product). The binding thereby causes fluorescence resonance energy transfer (FRET) to occur between the AF647 and AF488 fluorescent dyes, resulting in a change in fluorescence. Thus, a signal, in this example an optical signal, is generated in the event of an interaction, allowing selective detection of the product of a biological entity, in this example a cell.

[0219] FIG. 14b shows fluorescence detection based on the presence of one or more hybridoma cells in this example picodroplets. After a signal above a threshold is detected, hybridoma cells are identified within the picodroplets. The background of about 0.5V in this example is due to empty picodroplets being fed into the analyzer. Droplets resulting in a signal above the threshold, about 1.1V in this example, may therefore be collected for further sorting and / or analysis and / or culturing. Droplets generating a signal below this threshold may be discarded if desired.

[0220] It will be appreciated that the fluorescence of the donor and acceptor may be optimized for a range of FRET assays and depending on the particular cell or cells or other entities to be detected in the droplets.

[0221] Fluorescence detection as shown in Figures 14a and 14b may be used, for example, in a droplet sorting device and / or a decoupler as outlined above.

[0222] Figure 15 shows monoclonal antibody secretion in picodroplets by hybridoma cells. Measurements were performed using a FRET-based ELISA.

[0223] The graph is a plot of the concentration of antibody produced versus the duration of culture. The dark bars represent bulk cultures formed from standard test tubes incorporating multiple cells. The red bars represent samples in which a single cell was placed in a picodroplet (the volume was approximately 700 pl). The inset in Figure 15 shows a graph of fluorescence intensity versus concentration.

[0224] As can be seen, the number of antibodies produced is comparable between cultures formed from bulk cultures and cultures formed from single cells over a period of one hour or more, and it will be appreciated that the concentration of a single cell in a picodroplet at t=0 is negligibly small. However, as outlined above, it may be advantageous to perform cell culture from a single cell, as this ensures high monoclonality across the cell population. This indicates that the concentration of cells cultured from cells in picodroplets prepared according to the embodiments described herein may be comparable or even higher than when cultured from multiple cells in a standard test tube, thereby achieving a higher monoclonality guarantee for a population cultured from a single entity.

[0225] Figures 16a to 16c show visual images of a population of Chinese Hamster Ovary (CHO-S) cells. In this example, the picodroplets have an average diameter of about 84 μm and an average volume of about 300 pl. In Figure 16a, the cells can be seen under bright field. To determine if the cells are viable, a fluorescent dye, in this example DRAQ-7, can be prepared (Figure 16b). The merged image is shown in Figure 16c, and viable cells show green fluorescence in Figure 16c.

[0226] The percentage of viable (i.e., living) CHO-S cells versus culture time is shown in FIG. 16d for the various techniques. The blue bars show control cultures. The green bars represent cell cultures obtained via picodroplet preparation techniques according to embodiments described herein. The purple bars show analysis results for populations cultured from droplets sorted by FACS, and the pink bars represent populations sorted and dispensed using FACS.

[0227] As can be seen, the percentage of viable cells cultured from samples prepared using the embodiments described herein is significantly higher than that obtained via FACS techniques. This result demonstrates that encapsulation of the cell(s) in picodroplets has no measurable effect on cell viability, and that droplet preparation, sorting, and dispensing according to the embodiments described above are advantageous.

[0228] 17 shows the percentage of viable CHO-S cells versus culture time for different picodroplet sizes, in this example 300 pl, 500 pl, and 700 pl. The results of this analysis may allow one to determine whether the size of the picodroplets can be reduced to increase production rates.

[0229] In general, it can be seen that the percentage of viable cells is similar for all picodroplet sizes investigated. However, after some incubation time, e.g., 9 hours, picodroplets with a volume of 700pl contained the highest percentage of viable cells. This is to be expected since the larger droplet volume provides a longer period of time for the cells to be provided with nutrients, allowing them to survive for a longer period of time.

[0230] FIG. 18a shows an image of a microfluidic chip 180 according to an embodiment described herein.

[0231] Microfluidic chip 180 integrates various modules of a system as described throughout this specification into a single chip design. Microfluidic chip 180, in this example, includes a droplet generation region 412, a droplet incubator / storage region 414, a droplet sorting region 416, and a droplet dispensing region 420. In this example, microfluidic chip 180 includes a storage chamber 456 and valves 182 that allow droplets to be held within chamber 456 for a controllable period of time (e.g., seconds to minutes) and thus delay the transport of droplets throughout microfluidic chip 180 when desired.

[0232] The holding chamber 456 of the microfluidic chip 180 is preferably located as close as technically possible to the droplet dispensing region 420. This can facilitate control of droplet transport and dispensing, which can be particularly difficult because water droplets may have a different flow rate compared to oil. The closer the holding chamber 456 is to the droplet dispensing region 420 (or droplet dispensing unit), the more controllable the droplet dispensing will be, as the time it takes for droplets to be transported between the holding chamber 456 and the droplet dispensing region 420 can be reduced. This can be particularly important in chip designs that operate vertically, whereby droplets float above the chip.

[0233] In an embodiment of the microfluidic chip 180, a control sequence is used that allows for separating different workflows of different parts and regions of the microfluidic chip 180. For example, droplets may be sent directly from the droplet generation region 412 to the droplet sorting region 416 and then arrive at the droplet dispensing region 420. In this example, the droplet incubation / storage region 414 between the droplet generation region 412 and the droplet sorting region 416 is omitted. It will be understood that different part / region(s) of the microfluidic chip 180 may be omitted.

[0234] Figure 18b shows a schematic block diagram of a control system 1800 for operating the microfluidic chip shown in Figure 18a. It will be understood that the control system 1800 can be equally used to operate the microfluidic systems, droplet sorting or dispensing devices, droplet dispensers, microfluidic devices, microfluidic cartridges, microfluidic processing systems, and instruments for microdroplet-based processing of biological entities described above. Thus, the control system 1800 can be used in systems where one or more of the droplet generation device / unit, droplet storage / incubation device / unit, droplet sorting device / unit, and droplet dispensing unit / droplet dispenser are not integrated into a single chip design.

[0235] Broadly speaking, the control system 1800 comprises a suitably programmed general-purpose processor 1802. The control system 1800 further comprises a working memory 1804, a permanent program memory 1806, and a data store 1808, all linked by a common data and controller 1810. In this example, a user interface 1812 is also provided for configuring the system. The control system 1800 also comprises an output 1814 connected to one or more of a display, memory, printer, data store, and network 1816, for example, to display, store, print, or distribute data correlating one or more properties of a droplet (or one or more properties of one or more entities in the droplet) with, for example, placement in a microtiter plate. Those skilled in the art will appreciate that other forms of storage / output may be employed in addition or alternatively.

[0236] In this example, working memory 1804 is used to hold (which may be temporary), process, and manipulate droplet entity data, droplet size data, droplet fluorescence data, droplet velocity data, and other physical and / or chemical data / properties related to the droplets and / or their one or more entities.

[0237] Permanent program memory 1806, in this example, may include (optional) user interface code, operating system code, data communications control code for controlling the interface to the output, droplet delivery code for controlling the delivery of droplets from the droplet processing system / microfluidic chip, e.g., from a reservoir, droplet preparation code for controlling the preparation of droplets, e.g., from a solution, e.g., containing a biological entity, droplet flow rate code for controlling the flow rate of droplets in a water-in-oil emulsion within the droplet processing system / microfluidic chip, droplet flow path code for controlling the flow path of droplets in one or more portions of the droplet processing system / microfluidic chip (e.g., one area or processing step, e.g., incubation, may be omitted and droplets may be delivered directly from one area / processing unit to another), droplet fusion code for controlling the fusion of droplets, droplet entity determination code for controlling the determination / identification of one or more entities in the droplets, detection of fluorescence of the droplets and processing thereof, the operating system code (which may be platform independent) including droplet fluorescence detection code for controlling a droplet processing system / microfluidic chip to detect droplets in a droplet processing system / microfluidic chip, droplet velocity determination code for controlling determination of a velocity of a droplet in a droplet sorting unit / region, droplet decoupling code for controlling decoupling of droplets from a first fluid-flow path to a second fluid-flow path, droplet selection code for controlling selection of droplets based on previously acquired data of one or more properties of the droplets and / or their entities, droplet ejection / dispensing code for controlling ejection / dispensing of droplets by, for example, adjusting pressure in the droplet processing system / microfluidic chip at one or more regions of the one or more flow paths, droplet incubation code for controlling incubation of droplets, droplet stability testing code for controlling stability tests performed on one or more droplets, and droplet holding code for controlling holding of droplets in a holding chamber.

[0238] These codes are loaded and implemented by the processor 1802 to implement corresponding functions for the control system 1800 .

[0239] Some or all of this code may be provided on a carrier medium, illustrated by a removable storage medium 1818, for example a CD-ROM.

[0240] Data store 1808, in this example, stores droplet entity data that provides information about one or more entities stored in the droplet (and whether the droplet contains an entity, e.g., a biological entity), droplet size data that provides information about the size of the droplet, droplet fluorescence data that provides information about the fluorescent properties of the droplet, and droplet velocity data that provides information about the velocity of the droplet at a particular location and / or region within the droplet processing system / microfluidic chip at a given time. One skilled in the art will appreciate that other physical and / or chemical properties of the droplet and / or its entities may be stored in data store 1808.

[0241] The present invention further provides processor control code for implementing the above described systems and methods, for example on a general purpose computer system or a digital signal processor (DSP). The code is provided on a non-transitory physical data carrier, such as a disk, a CD-ROM, a DVD-ROM, a program memory, such as a non-volatile memory (e.g. flash memory) or a read-only memory (firmware). The code (and / or data) for implementing an embodiment of the present invention may include source, object or executable code in a conventional programming language (interpreted or compiled), such as C, or assembly code, or code for a hardware description language. Those skilled in the art will appreciate that such code and / or data may be distributed among several coupled components that communicate with each other.

[0242] Next, we describe some exemplary control sequences for use with embodiments of the chip. Control of the microfluidic chip (cartridge) can be achieved through software control of the instrument. In embodiments, a sequence of hardware control commands or scripts is sent to a hardware / software control interpreter, which translates this information into commands specific to the hardware components.

[0243] After loading the sample (e.g., a sample of cells or other biological or assay-relevant material) onto the chip, the chip is loaded into the instrument. Various modes of operation are possible, which are implemented by selection of suitable command control sequences (control scripts). Two example instrument operation modes are outlined below: (1) guaranteed monoclonality mode and (2) direct assay mode.

[0244] An example of a chip control sequence for the guaranteed clonality mode sequence is as follows:

[0245] Typical initialization steps are sample loading and chip priming (channels and cavities of the chip are filled with oil to avoid air bubbles).The steps then proceed as follows: 1. Picodroplet generation and sample encapsulation 2. Optical detection of monoclonality (e.g., based on imaging and / or light scattering) 3. Picodroplet sorting (based on optical signals) 4. Picodroplet collection 5. Picodroplet dispensing (preferably including optical detection / confirmation of monoclonality as previously described).

[0246] After the droplets are dispensed into the slug of emulsion, the slug enters a well (of a microtiter plate) that typically already contains water. There, through physical action and / or other means, e.g., using an electric field, the cells are released from the droplet into the water and collected in the well, and the oil separates from the water. Once system operation is complete, the system / chip is preferably cleaned.

[0247] An example chip control sequence for the direct assay mode may have the same initialization and payload recovery steps as described above for the guaranteed monoclonality mode. In addition, the direct assay mode may include the following steps: 1. Picodroplet generation and sample encapsulation 2. Incubation (e.g., at 37°C) 3. Optical detection of monoclonality (e.g., based on imaging and / or light scattering) 4. Optical detection for assays (e.g., fluorescence and / or luminescence) 5. Picodroplet sorting (based on optical signals) 6. Picodroplet Recovery (Assay Positive) 7. Picodroplet dispensing (preferably including optical detection / confirmation of monoclonality as previously described).

[0248] 19a-c show the detection of a single cell within a picodroplet using embodiments described herein. In this example, the amount of antibody produced is determined.

[0249] The x-axis of the graphs in Figures 19a-c shows green fluorescence intensity in arbitrary units, and the y-axis shows red / green fluorescence ratio intensity in arbitrary units. The droplets are exposed to green light. In this example, the more antibodies produced from the Chinese Hamster Ovary cells, the greater the green-to-red shift present in the fluorescence signal as a result of higher FRET intensity. Those skilled in the art are familiar with the observation of a green-red shift as a result of increasing numbers of antibodies. The methods and systems described herein show that using the embodiments described herein, a significant number of antibodies can be produced from Chinese Hamster Ovary cells after 2 hours, where the cluster labeled "A" represents a population of antibodies. Cluster "B" represents a picodroplet population that does not contain any cells or does not contain any antibodies.

[0250] The method may then be implemented, for example, to sort only droplets that exhibit red and green fluorescence intensities that fall within a particular range. This is illustrated in Figures 20a-c, which show a time course of scatter plots of red and green fluorescence from picodroplets. The red square defines a gating set for picodroplet sorting, and only picodroplets that exhibit red fluorescence that falls within a particular range and green fluorescence that falls within a particular range may be sorted. It will be appreciated that the shape, size, and arrangement of the red square that defines the gating set for picodroplet sorting may be selected according to the particular properties (e.g., fluorescent properties) that the droplets (and their entities) are to possess.

[0251] In a preferred embodiment, we have described the technique as applied to treating droplets of a water-in-oil emulsion containing biological entities. However, in principle, non-biological entities, such as organic or inorganic entities, can be treated in a similar manner. Similarly, the technique we have described is also in principle applicable to treating droplets of oil in a water-in-oil emulsion.

[0252] No doubt many other effective alternatives will occur to those skilled in the art, and it will be understood that the invention is not limited to the described embodiments, but encompasses modifications apparent to those skilled in the art that are within the spirit and scope of the appended claims. [Explanation of symbols]

[0253] 180 Microfluidic Chips 182 Valve 400 Microdroplet Processing Systems / Instruments 400' Microdroplet Processing System / Instrument 402 Storage Container 404a-d A set of storage vessels 406 Fluid Drive Module 408 A set of fluid flow lines 410 (Disposable) Microfluidic Cartridges 412 Droplet generation area 414 Droplet Incubation / Storage Area 416 Droplet Sorting Region 418 Flow Buffer Area 420 Droplet dispenser, Droplet dispensing area 420' Dispensing Area 422 Aqueous sample input section 424 Oil injection section 426 Flow focusing junction 428 Emission Channel 430 Storage Chamber 432 Droplets 434 Channel 436 Storage Container 438 Emission Channel 440 Side Channel 442 Input Channel 444, 446 Emission Channel 448 Injection section 450 Areas 452 Electrode 454 Input Channel 456 Chamber 458 Emission Channel 460 Input Channels 462 Emission Channel 464 Channels 466 Channels 468 Emission Channel 470 Secondary Emission Channel 472 Release section 474 Rotary Valve 476 Channels 477 Valve 478 Injection section 480 Emulsion 482 Target Droplet 484 Multi-well storage vessel 488 wells 490 Aqueous (culture) media 491 First Optical Sensing Region 492 Second Optical Sensing Region 493 Third Optical Sensing Region 494 Fourth Optical Sensing Region 495, 496, 497 optical sensing area 500 Decoupler 500 Control Unit 502 First fluid flow path (channel) 502 Detector 504 Second Fluid Flow Channel 504 Bus 506 Rotating Unit 506 Processor 508 Release section 508 Operating Systems / Software 510 Decoupler 512 Memory 800 Droplet Dispenser 802 Input Channel 804 First Emission Channel 806 Second Emission Channel 808 Shared Channel (Flow) Part 810 Nozzle 812 Emulsion 814 Droplets 816 Air Inlet 818 Oil supply 820 Droplets 822 Feed valve 824 Shut-off valve 826 Air valve 828 Area 830 Separation 1000 Droplet Processing Cartridge 1002 Area 1004 Electrical connections 1006 Dispensing nozzle 1050 Modular Cartridge 1052 Droplet Generation Module 1054 Droplet Incubation / Storage Module 1056 Droplet Sorting Module 1058 Droplet / Retention (Flow Buffer) Area 1060 Droplet Dispensing Area 1062 Channels 1064 Valve 1066 Datum Placement Bin 1100 Droplet generation control system 1102 Control computer system, controller 1104 Sample 1106 Oil 1108 Valve 1110 Intelligent Camera 1112 Image Capture System 1114 Lighting 1116 Droplet size control procedure 1118 Control Loop 1120, 1122 Pump 1130 Temperature Control System 1132 PID (Proportional Integral Derivative) Controller 1134 Temperature Sensor 1136 Peltier effect element 1140 Droplet sorting control system 1142 Intelligent Camera 1144 Droplet Flash Illumination Device 1146 Photomultiplier tube 1148 Imaging Device 1150 Cell Detection Algorithm 1152 Signal Generator 1154 Amplifier 1160 Droplet Dispensing Control System 1162 Intelligent Camera 1164 Flash Device 1166 Image Capture Procedure 1168 Cell Detection Algorithm 1170 Control Procedure 1172 Compressed Air Source 1174 Valve 1176 XY Stage 1200 Droplet Processing Equipment 1202, 1204, 1206 Intelligent Camera 1208 LED Controller 1210 Heater Plate 1212 Valve Actuator 1214 XY Stage 1216 Internal oil container 1218 User Interface 1800 Control System 1802 General Purpose Processor 1804 Working Memory 1806 Permanent Program Memory 1808 Datastore 1810 Common Data and Controller 1812 User Interface 1814 Output 1816 Network 1818 Removable Storage Media

Claims

1. 1. A method of providing a droplet containing one or more entities, comprising the steps of: Providing a plurality of entities in a fluid; preparing droplets from the fluid; determining whether the droplet contains one or more of the plurality of entities, or whether the droplet does not contain the entity; sorting the droplets in response to the determination; dispensing the sorted droplets into a reservoir; and the dispensing step comprises, after the sorting step, selecting a fluid flow path for a fluid containing the sorted droplets; ejecting the sorted droplets from the selected paths; A method comprising:

2. The step of dispensing comprises: identifying the sorted droplets for dispensing; extracting the sorted droplets from a first fluid flow path of the fluid by transferring the sorted droplets from the first fluid flow path into a second fluid flow path; expelling the sorted droplets from the second fluid flow path into the reservoir by applying pressure to the second fluid flow path; The method of claim 1 , comprising:

3. 3. The method of claim 1 or 2, wherein the step of transferring comprises applying pressure to the sorted droplets to dispense them while in a portion of the first fluid flow path, thereafter transferring the sorted droplets from the first fluid flow path into a second fluid flow path, and ejecting the sorted droplets.

4. 3. The method of claim 1 or 2, wherein the step of dispensing comprises transferring the sorted droplets from the first fluid flow path to the second fluid flow path and then applying pressure to the second fluid flow path to eject the droplets.

5. 5. The method of claim 1, 2, 3 or 4, wherein the step of extracting comprises extracting the sorted droplets only when the sorted droplets contain a defined number of the entities and / or when the sorted droplets contain the entities having defined properties, and in particular the probability that the ejected droplets contain the single target entity or a single target combination of the entities is greater than 99.9%, in particular greater than 99.99%, more particularly greater than 99.997%, based on the preparation, determination, sorting and dispensing.

6. The method of claim 1 , wherein the step of ejecting comprises ejecting the selected droplet at a location determined in response to the determination and / or a property of the entity.

7. 7. The method of any one of claims 1 to 6, further comprising a step of incubating the droplet to culture and / or maintain the one or more entities, in particular performing a stability test on the one or more entities during incubation.

8. 8. The method of claim 1, wherein the step of transferring the sorted droplets from the first fluid flow path to the second fluid flow path comprises the step of decoupling the sorted droplets from the first fluid flow path in a decoupler, the decoupler being configured to separate the sorted droplets from the first fluid flow path and direct the separated droplets to the second fluid flow path.

9. The method of claim 1 , wherein the step of ejecting comprises heating a fluid in which the sorted droplets are transported.

10. 10. The method of claim 1, further comprising the step of injecting a culture medium fluid into the second fluid flow path and expelling the sorted droplets in the culture medium fluid.

11. placing a second plurality of entities in a second fluid; Preparing a second droplet from a second fluid; and fusing the first droplet prepared from the first fluid with the second droplet prepared from the second fluid to obtain a fused droplet; 11. The method of claim 1, wherein the extracting step comprises extracting the fused droplets by transferring the fused droplets from the first fluid flow path into the second fluid flow path.

12. The method of claim 11 , wherein the determining and / or selecting is performed prior to the fusion.

13. determining whether the second droplet contains one or more entities of the second plurality of entities, or whether the second droplet does not contain the entities of the second plurality of entities; sorting the second droplet in response to the determination; 13. The method of claim 11 or 12, further comprising:

14. 14. The method of claim 13, wherein the step of fusing is performed only for the first and second droplets that are determined to contain a single target entity of a first and second plurality of entities, respectively, and / or the first and second droplets that are determined to contain a single target combination of the first and second plurality of entities, respectively.

15. 15. The method of any one of claims 11 to 14, wherein the fused droplets contain a single pair of two cells, or a single pair of a cell and a reagent, or a single combination of one or more cells, or a single combination of one or more cells and one or more reagents, in particular the reagents are biomolecules.

16. 16. The method of claim 1 , wherein the sorting step comprises detecting the droplets at multiple locations in the fluid flow path at different times and determining a velocity of the droplets in the fluid flow path to time the sorting.

17. 1. A method for preparing a droplet containing a single pair of two biological entities, comprising: providing a first plurality of biological entities in a first fluid and a second plurality of biological entities in a second fluid; preparing a first droplet from the first fluid and a second droplet from the second fluid; determining whether the first droplet contains a single entity of the first plurality of biological entities and whether the second droplet contains a single entity of the second plurality of biological entities; and fusing the first droplet and the second droplet, each of which has been determined to contain the single entity.

18. 20. The method of claim 17, wherein the step of fusing is performed by electrocoalescence, by charging one or both of the first and second droplets by electrostatic attraction to cause the droplets to fuse, by physical shrinkage, or by physical collision.

19. 20. A method for metabolizing and / or analyzing a biological entity contained in a droplet, the biological entity comprising a single cell, or a single pair of two cells, or a single pair of a cell and a reagent, the method comprising the step of supplying the biological entity contained in the droplet using a method according to any one of claims 1 to 18.

20. 1. A microfluidic system for dispensing droplets containing one or more entities, comprising: a droplet formation device for preparing droplets from a fluid containing a plurality of entities; an analyzer for determining whether the droplet contains one or more of the plurality of entities; a droplet sorting device for sorting said droplets in response to said determination; a decoupler for decoupling the sorted droplets from a fluid flow path of the fluid in the microfluidic system; A microfluidic system comprising:

21. 21. The microfluidic system of claim 20, wherein the decoupler comprises a separation unit for separating the sorted droplets from the fluid flow path and a guide for directing the separated droplets into a second fluid flow path of the microfluidic system.

22. 22. The microfluidic system of claim 21 , wherein the guide comprises a rotation unit, the rotation unit comprising a droplet storage unit for storing the separated droplets, the rotation unit configured to be rotated to transport the separated droplets stored in the droplet storage unit into the second fluid flow path.

23. 22. The microfluidic system of claim 21 , wherein the guide comprises a translation unit, the translation unit comprising a droplet storage unit for storing the separated droplets, the translation unit configured to be moved in a translational direction to transport the separated droplets stored in the droplet storage unit into the second fluid flow path.

24. 24. The microfluidic system of any one of claims 20 to 23, further comprising an incubator for incubating the droplet to culture and / or maintain the one or more entities.

25. 25. The microfluidic system of claim 24, wherein the incubator comprises a stability testing unit for performing a stability test on the one or more entities during the incubation.

26. 26. The microfluidic system of claim 20, further comprising a dispensing unit for dispensing the decoupled droplets.

27. 27. The microfluidic system of claim 26, wherein the dispensing unit comprises a reservoir for storing a culture medium fluid, the dispensing unit configured to dispense the decoupled droplets into the culture medium fluid.

28. 28. The microfluidic system of claim 26 or 27, wherein the dispensing unit comprises a high pressure unit for dispensing the droplets via pressurized fluid ejection.

29. 29. The microfluidic system of any one of claims 20 to 28, wherein the droplet forming device comprises a plurality of droplet forming devices, and the microfluidic system further comprises a droplet fusion device for fusing a plurality of droplets prepared using the plurality of droplet forming devices.

30. 30. The microfluidic system of claim 29, wherein the droplet fusion device comprises an electric field generator for generating an electric field to fuse the droplets by electrocoalescence.

31. 30. The microfluidic system of claim 29, wherein the droplet fusion device comprises one or more charging devices for charging droplets and fusing the droplets by electrostatic attraction.

32. 32. The microfluidic system of any one of claims 20 to 31, wherein the analyzer comprises one or more of a fluorescence detector, a scattered light detector, an imaging detector, an acoustic wave generation and detection unit, and a magnetic activated cell sorting device.

33. 33. The microfluidic system of any one of claims 20 to 32, wherein the analyzer and / or droplet sorting device comprises a plurality of sensors for detecting the droplets at different locations and times in the microfluidic system to determine a velocity of the droplets within the microfluidic system, and the droplet sorting device is configured to sort the droplets depending on the results of the velocity determination.

34. 1. A droplet sorting or dispensing device comprising: a set of one or more microfluidic input channels; a set of microfluidic emission channels; and a substrate having a movable sorting element between the input channel and the discharge channel; the sorting element comprises a set of microfluidic channels for connecting the input channel to the output channel; A droplet sorting or dispensing device, wherein the sorting element is movable between a first position where at least a first input channel is connected to allow fluid flow from the first input channel to a first output channel, and a second position where the sorting element is within the channel and a length of fluid carrying one or more droplets is moved such that that length of fluid flows into a second output channel.

35. 35. The droplet sorting or dispensing device of claim 34, wherein the sorting element is rotatable between a first position and a second position, whereby fluid lengths from the first and second input channels are either swapped so that they flow into the respective first and second output channels, or swapped so that they flow into the respective second and first output channels.

36. 1. A method for sorting or dispensing droplets, comprising: providing first and second microfluidic streams, at least one of the first and second microfluidic streams containing droplets; rotating a turntable containing a portion of the stream to selectively direct droplets into one of the first and second emitted microfluidic streams; A method comprising:

37. 1. A device for sorting or dispensing droplets within a microfluidic stream, comprising: first and second input microfluidic streams, at least one of the first and second input microfluidic streams containing droplets; first and second output microfluidic streams; a rotating turntable that contains a portion of the flow between the input and output streams; a controller for controlling rotation of the turntable to selectively direct the droplets into the first and second emitted microfluidic streams; A device comprising:

38. A cell, biomolecule, and entity sorting or dispensing device for transferring picodroplets from a first fluid flow path to a second fluid flow path, the device comprising: a decoupler for decoupling the picodroplet from the first fluid flow path; a guide for directing the decoupled picodroplets into the second fluid flow path; and Equipped with The guide is a rotation unit configured to rotate the decoupled picodroplets to transport them into the second fluid flow path; and / or A cell, biomolecule, and entity sorting or dispensing device comprising a translation unit configured to be moved in a translational direction to transport the decoupled picodroplet to the second fluid flow path.

39. The cell, biomolecule and entity sorting or dispensing device of claim 38, further comprising one or more microtiter plates, and / or one or more glass slides, and / or an x-y motion platform comprising one or more arrays or other formats in which one or more of the picodroplets can be dispensed at one or more addressable locations.

40. A droplet sorting or dispensing device as described in claim 34 or 35, or a cell, biomolecule and entity sorting or dispensing device as described in claim 38 or 39, and a droplet dispenser comprising a microfluidic channel orifice for dispensing droplets selected by the droplet sorting or dispensing device from a microfluidic channel to a storage container.

41. 41. A microfluidic device comprising a droplet sorting / dispensing device according to claim 34 or 35, or a sorting device according to claim 38 or 39, or a droplet dispenser according to claim 40.

42. 1. A microfluidic cartridge for automated sorting and dispensing of sorted microfluidic droplets, comprising: a sort input channel coupled to a sorting region, the sorting region comprising two or more sort outlet channels coupled to the sort input channel and a droplet director configured to selectively direct droplets from the sort input channel to a selected one of the sort outlet channels; a droplet dispenser coupled to one of the sorting discharge channels for dispensing selected droplets from the microfluidic cartridge for collection in a reservoir, the droplet dispenser comprising a droplet ejection mechanism for ejecting droplets from an emulsion stream into the dispenser ejection channel for collection in the reservoir; A microfluidic cartridge comprising:

43. 43. The microfluidic cartridge of claim 42, further comprising an emulsion flow buffer region between the sorting region and the dispense region to control emulsion flow rate to the droplet dispenser.

44. 44. The microfluidic cartridge of claim 42 or 43, further comprising a droplet incubation area having a droplet input, a droplet holding area for holding droplets for incubation, and a release coupled to the sorting input channel.

45. 45. The microfluidic cartridge of claim 42, 43, or 44, wherein the droplet dispenser output channel comprises a mechanism for facilitating release of the droplet, or contents of the droplet, from the emulsion.

46. 46. ​​The microfluidic cartridge of any one of claims 42 to 45, wherein the droplet dispenser is configured to select droplets containing a defined number or range of biological entities per droplet.

47. 47. The microfluidic cartridge of any one of claims 42 to 46, further comprising a plurality of sensors for detecting the droplets at different locations and times in the microfluidic cartridge to determine a velocity of the droplets within the microfluidic cartridge, wherein a droplet director is configured to direct the droplets depending on the results of the velocity determination.

48. 48. The microfluidic cartridge of any one of claims 42 to 47, wherein the droplet ejection mechanism comprises a mechanism for increasing pressure of the emulsion stream to direct droplets into the dispenser outlet channel.

49. 49. The microfluidic cartridge of claim 48, wherein the droplet ejection mechanism is configured to selectively modulate the pressure to select droplets in the emulsion stream for ejection.

50. 50. A microdroplet processing system comprising the microfluidic cartridge of claim 49, further comprising a sensor for sensing one or both of a velocity and a position of the selected droplet, and a controller for modulating the pressure in response to the velocity / position.

51. 50. A microdroplet processing system comprising a microfluidic cartridge according to any one of claims 42 to 49, wherein the microdroplet processing system further comprises a plurality of the reservoirs and a control system for selectively directing dispensed droplets to the reservoirs.

52. a droplet sensing system specifically associated with said sorting region for sensing one or more characteristics of droplets for sorting and sorted into a discharge channel; 52. The microdroplet processing system of claim 51 , wherein the control system is configured to store the droplet data related to the one or more sensed characteristics such that droplet data for individual droplets is retrievable to retrieve data for individual droplets in identified reservoirs.

53. 1. A microfluidic system for delivering one or more target entities into a droplet, comprising: whether the droplet contains the one or more target entities; and A characteristic of said one or more target entities a first detector for detecting one or both of The microfluidic system comprises: a sorting device for sorting the droplets depending on the result of the detection; A microfluidic system further comprising a droplet dispensing unit for dispensing the sorted droplets at one or more target locations, the microfluidic system configured to correlate the target locations with the characteristics of the one or more target entities detected by the first detector.

54. 54. The microfluidic system of claim 53, further comprising a second detector for detecting the characteristic of the one or more target entities, the droplet dispensing unit further configured to correlate the target location with the characteristic of the one or more target entities detected by the second detector.

55. The droplet dispensing unit comprises a second sorting device for transferring the droplets from a first fluid flow path to a second fluid flow path, the second sorting device comprising: a decoupler for decoupling the droplet from the first fluid flow path; a guide for directing the decoupled droplet into the second fluid flow path; The guide comprises: a rotation unit configured to rotate the decoupled droplets to transport them into the second fluid flow path; and / or 55. The microfluidic system of claim 53 or 54, comprising a translation unit configured to be moved in a translational direction to transport the decoupled droplets to the second fluid flow path.

56. 56. The microfluidic system of claim 53, 54, or 55, wherein the droplet dispensing unit comprises a microfluidic channel junction having an input channel and a plurality of output channels, and the droplet dispensing unit is configured to control which of the plurality of output channels the droplet is directed to based on detection results via a first and / or second detector.

57. 57. The microfluidic system of claim 56, wherein the droplet dispensing unit comprises a pressure modulation unit for varying pressure within a fluid flow path of the droplet dispensing unit to control to which of the plurality of ejection channels the droplet is directed.

58. 58. The microfluidic system of claim 57, wherein a first outlet channel is connected to the input channel generally in a fluid flow direction of the input channel and a second outlet channel branches off from the input channel, the droplet dispensing unit further comprising a pump connected to the second outlet channel, the pump configured to pump the droplet from the input channel, and the pressure modulation unit configured to increase pressure in the input channel in response to the detection in the first and / or second detectors to expel the droplet via the first outlet channel.

59. 59. The microfluidic system of any one of claims 53 to 58, further comprising one or more droplet holding areas disposed between the sorting device and the droplet dispensing unit, the holding areas configured to control a flow of the sorted droplets to the droplet dispensing unit.

60. 60. The microfluidic system of any one of claims 53 to 59, further comprising a plurality of sensors for detecting the droplets at different locations and times in the microfluidic system to determine a velocity of the droplets within the microfluidic system, the sorting device being configured to sort the droplets depending on the results of the velocity determination.

61. 1. An apparatus for microfluidic droplet-based processing of biological entities, comprising: a droplet generation system for generating one or more water-in-oil emulsions of droplets containing the biological entity; a droplet processing system for processing the droplets; a droplet dispensing system for dispensing the processed droplets into one or more reservoirs; Equipped with The instrument, wherein the droplet processing system is configurable for a number of different operational modes including at least a monoclonal operational mode and an assay operational mode.

62. 62. The apparatus of claim 61, wherein the droplet processing system comprises at least one droplet sorting unit, and the monoclonal mode of operation comprises sorting droplets based on occupancy of the droplets by the biological entities, and the assay mode of operation comprises incubating the droplets and sorting droplets based on a product of the biological entities during the incubation.

63. 63. An apparatus according to claim 61 or 62, wherein the droplet processing system comprises one or more replaceable or modular droplet processing cartridges.

64. 64. The apparatus of claim 63, wherein the one or more replaceable or modular droplet processing cartridges further comprise at least one droplet fusion unit, and the operational modes include a droplet fusion assay operational mode.

65. A microfluidic chip comprising: a droplet generation region for generating microdroplets containing one or more biological entities; a droplet incubator and / or storage area for incubating and / or storing said biological entities; a droplet sorting region for sorting the microdroplets based on one or more properties of the biological entities; a droplet dispensing area for dispensing the selected microdroplets; Equipped with A microfluidic chip, wherein the droplet generation region, the droplet incubator and / or storage region, the droplet sorting region, and the droplet dispensing region are integrated onto a single chip design of the microfluidic chip.

66. a microfluidic flow path diversion unit for changing a microfluidic flow path of the microdroplet between the regions; a control unit including a first processor for controlling the microfluidic flow path diversion unit; 66. The microfluidic chip of claim 65, further comprising:

67. 67. The microfluidic chip of claim 65 or 66, further comprising a heating device for locally heating droplets in the droplet incubator and / or storage area, and further comprising a second processor for controlling the heating device.

68. 67. The microfluidic chip of claim 65 or 66, further comprising a cooling device for locally cooling droplets in the droplet incubator and / or storage area, and further comprising a third processor for controlling the cooling device.

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