Improvements in or relating to a dispensing device

Abstract

A chip for manipulation of a plurality of micro-entities, the chip comprising: a microfluidic space configured to accommodate a plurality of micro-entities in a substantially planar array; wherein the height of the microfluidic space in the plane perpendicular to the array is sized to accommodate a single layer of micro-entities; wherein the microfluidic space contains a carrier fluid in which the array of micro- entities can be localised and manipulated using a force; wherein the microfluidic space has an exit orifice that extends a distance in the plane of the array that is at least double the height of the microfluidic space so that two or more micro-entities in parallel can be moved through the exit orifice simultaneously under the force; and a dispense region configured to provide a flow of carrier fluid substantially non-parallel to the exit orifice so that the micro-entities are moved by the carrier fluid through the dispense region and out of the chip.

Description

[0001] IMPROVEMENTS IN OR RELATING TO A DISPENSING DEVICE

[0002] The present invention relates to improvements in or relating to a chip on which a plurality of micro-entities can be tracked, manipulated and analysed. Movement of the micro-entities on the chip may be actuated magnetically, electrokinetically or even acoustically. For example, the present invention may be implemented with an electrowetting-on-dielectric (EWOD) or optoelectrowetting-on-dielectric (oEWOD) chip and the micro-entities may be a plurality of microdroplets.

[0003] Devices for manipulating micro-entities, especially microdroplets are well known in the art. One technique for the manipulation of droplets involves the use of electrowetting- on-dielectric (EWOD) or optoelectrowetting-on-dielectric (oEWOD) technique.

[0004] During the loading process of microdroplets onto the EWOD or oEWOD chip, a large number of droplets are injected into the active area of the chip and undergo a selection process to retain, for example, those that contain content. In some loading scenarios, the majority of droplets are not retained but must be rejected from the chip and delivered out into a waste container. However, previously known devices were not efficient for quick removal of unwanted droplets. The process of placing unwanted droplets into an aperture in the chip had limitations in that droplets would not spontaneously exit the chip through an aperture. Furthermore, applying pressure to an exit aperture in order to remove the droplets from the chip would induce a flow in the body of the chip, which risks dislodging droplets that are under EWOD or oEWOD control. There is, as such, a maximum flow, which can be tolerated within the chip, and so a maximum rate at which unwanted droplets can be cleared.

[0005] Thus, there is a requirement to provide a device that is able to facilitate the quick removal of droplets from the chip. Ideally, the device should be capable of removing the microdroplets at least as quickly as they are injected. In addition, there is also a need to minimise or reduce the use of a large amount of expensive carrier fluids to facilitate the removal of droplets from the chip. Therefore, it is desirable to provide a device that enables the efficient ejection of multiple microdroplets from the chip.

[0006] It is against this background that the present invention has arisen.

[0007] According to the present invention there is provided a chip for manipulation of a plurality of micro-entities, the chip comprising: a microfluidic space configured to accommodate a plurality of micro-entities in a substantially planar array; wherein the height of the microfluidic space in the plane perpendicular to the array is sized to accommodate a single layer of micro-entities; wherein the microfluidic space contains a carrier fluid in which the array of micro-entities can be localised and manipulated using a force; wherein the microfluidic space has an exit orifice that extends a distance in the plane of the array that is at least double the height of the microfluidic space so that two or more micro-entities in parallel can be moved through the exit orifice simultaneously under the force; and a dispense region configured to provide a flow of carrier fluid substantially non-parallel to the exit orifice so that the micro-entities are moved by the carrier fluid through the dispense region and out of the chip.

[0008] Furthermore, according to the present invention, there is provided a chip for manipulation of a plurality of micro-entities, the chip comprising: a microfluidic space containing a carrier fluid in which an array of micro-entities can be localised and manipulated using a force; wherein the microfluidic space has an exit orifice sized to accommodate more than two micro-entities in parallel moved under the force; a dispense region configured to provide a flow of carrier fluid substantially non-parallel to the exit orifice so that the micro-entities are moved by the carrier fluid through the dispense region and out of the chip.

[0009] In some embodiments, the chip can be an electrowetting, EWOD or oEWOD chip. Alternatively, the chip can be an optical tweezer device, or an opto-electronic tweezer (OET) device, or a dielectrophoresis (DEP) device. Electrowetting, EWOD, oEWOD, Optical tweezers, opto-electronic tweezers and dielectrophoresis (DEP), and the like, are differing techniques which can be utilised as appropriate for the particular application of interest.

[0010] In some embodiments, the micro-entities can part of the contents of a micro-object such as a sequestration pen. The micro-entity may be temporarily held or contained within the micro-object e.g. within the sequestration pen. The micro-entities may move in or out of the micro-object e.g. in and out of a sequestration pen using a force such as EWOD or oEWOD forces.

[0011] According to another aspect of the present invention, there is provided an EWOD or oEWOD chip for manipulation of a plurality of microdroplets, the chip comprising: a microfluidic space containing a carrier fluid in which an array of microdroplets can be localised and manipulated using EWOD or oEWOD; wherein the microfluidic space has an exit orifice sized to accommodate more than two microdroplets in parallel moved under EWOD or oEWOD; a dispense region configured to provide a flow of carrier fluid substantially non-parallel to the exit orifice so that the microdroplets are moved by the carrier fluid through the dispense region and out of the chip.

[0012] The device as disclosed herein enables the efficient removal of unwanted microdroplets and / or micro-entities from a microfluidic device. By moving several tens or hundreds of microdroplets and / or micro-entities simultaneously to the dispense region, the unwanted microdroplets can be quickly removed from the chip. This increases the capacity within the chip for manipulating microdroplets and / or microentities of interest. Moreover, moving multiple microdroplets and / or micro-entities out of the chip simultaneously reduces or prevents a backlog of unwanted microdroplets and / or micro-entities within the chip and thus, it can minimise the loading time of microdroplets into the chip. This can be advantageous as it enables experiments to be quickly and efficiently carried out on the chip and reduces experimental downtime for the user. Hence, the chip of the present invention provides a simplified and cost- effective approach for dispensing multiple microdroplets and / or micro-entities out of the chip.

[0013] The microfluidic space of the chip contains a carrier fluid in which an array of microdroplets and / or micro-entities can be localised and manipulated using EWOD or oEWOD. The micro-entities may be contained within a microdroplet that encapsulates one or more micro-entity and moves with that which it encapsulates. In some instances, at least a subset of microdroplets and / or a subset of micro-entities within the array may be held stationary in the microfluidic space of the chip. In some embodiments, the carrier fluid within the microfluidic space is zero or near zero velocity. Typically, the flow velocity in the microfluidic space is less than 100 microns per second. This enables EWOD or oEWOD control and manipulation of microdroplets and / or micro-entities without interference from the flow of the carrier fluid.

[0014] In some embodiments, the dispense region may be shaped and configured in order to minimise the flow of carrier fluid into the microfluidic space.

[0015] For efficient EWOD or oEWOD operations to take place on microdroplets and / or micro-entities in the array, it is important that the carrier fluid within the microfluidic space be minimised or maintained at a velocity of zero or near zero. In the case where the carrier fluid velocity within the microfluidic space is too high, the motion may displace micro-entities from their holding features as the fluid drag overcomes the holding forces. The shape and configuration of the dispense region can be configured to prevent the flow of carrier fluid from the dispense region flowing into the microfluidic space. Carrier fluid flow at a high velocity rate can interfere with the oEWOD or EWOD forces applied to microdroplets and therefore, this may disrupt or disturb the microdroplets being held by EWOD or oEWOD within the array. By minimising or eliminating the flow of carrier fluid into the microfluidic space, the user is able to apply EWOD or oEWOD manipulation of microdroplets and / or micro-entities with greater control and efficiency within the microfluidic space.

[0016] As used herein and unless otherwise disclosed elsewhere, EWOD or oEWOD operations or manipulation of microdroplets and / or micro-entities may include one or more of the following: moving, holding, merging, splitting or discarding at least a subset of microdroplets and / or micro-entities in an array and / or an entire array of microdroplets and / or micro-entities. In some embodiments, EWOD or oEWOD operation may include augmenting at least a subset of microdroplets and / or microentities in the array with further microdroplets and / or micro-entities.

[0017] The chip of the present invention is configured to be capable of the manipulation of tens, hundreds, thousands and / or millions of microdroplets and / or micro-entities, simultaneously using a force such as an (opto)electrowetting force. The micro-entities may be contained within a microdroplet that encapsulates one or more micro-entity and moves with that which it encapsulates. Alternatively, the micro-entities may be contained within a micro-object, which is a stationary physical construct, such as a sequestration pen.

[0018] In some embodiments, the dispense region can be shaped and configured in order to maximise the rate at which micro-entities, either independently or encapsulated within microdroplets, can be moved through the dispense region and out of the chip.

[0019] The shape and configuration of the dispense region allow for a clean and efficient transition of micro-entities from the microfluidic space to the exit orifice and into the dispense region for clearance out of the chip under flow. The transition of microentities, either independently or encapsulated in microdroplets, from the microfluidic space into the exit orifice takes place under an appropriate force which may be EWOD or oEWOD, for example where the micro-entities are encapsulated in microdroplets or via DEP, magnetic or electrokinetic control which may be more appropriate in examples where the micro-entities are not microdroplets or are not encapsulated in microdroplets.

[0020] In the dispense region, a carrier fluid is provided via one or more carrier inlet(s). The carrier inlets are shaped and configured to enable the carrier fluid flow to be directed towards an outlet of the dispense region. The carrier fluid provided within the dispense region is at a velocity that is more than zero i.e. at a higher velocity than the velocity of the carrier fluid provided within the microfluidic space. For example, the carrier fluid may have a centreline velocity of at least 50pm / s, typically 500pm / s, as high as 2000pm / s and in an extreme case up to 2cm / s. Micro-entities are moved by the high- velocity carrier fluid flow in the direction towards an outlet of the dispense region and out of the chip.

[0021] The removal of a plurality of microdroplets and / or micro-entities out of the chip can typically take between milliseconds and minutes once it has entered the dispense region of the chip. It is important to be able to manage ingress and egress from the chip at similar rates because, following loading, whilst some fraction of the input microentities are retained, a high proportion may be sent directly to waste and it is important that the waste dispense structure is capable of managing this level of throughput in order to avoid the dispense circuit being the rate limiting feature of the device, which is not an optimum condition. Input can be expected within the range of around 100- 1000 entities / second and the dispense structure should be capable of dispensing at a similar rate.

[0022] In some embodiments, the carrier fluid can be oil, HFE 7500 and / or media. In particular, the carrier fluid can be a fluorocarbon fluid such as FC40, HFE7700, HFE 7100, Opteon SF10, Opteon SF20. Alternatively, the carrier fluid can be a mineral oil, paraffin oil, any suitable hydrocarbon oils, a vegetable oil or fat; or any other suitable electrically-insulating liquid. The chip as disclosed herein can be further advantageous because less carrier fluid e.g. oil needs to be consumed compared to other known chips in the art in order to move microdroplets out of the chip. Moreover, traditional devices tend to use large amounts of carrier fluid oil to remove droplets from the chip, which often requires an oil reservoir to supply the oil and thus, making it less practical for use. In contrast, the chip of the present invention uses much less valuable resources such as the oil carrier fluid and thus, many workflows using the chip of the present invention are more practical, sustainable and cost-effective.

[0023] In some embodiments the carrier fluid may be water or a water-based medium such as a buffer, electrolyte solution or cell media and the micro-entities may be dispersed within this water based medium. In some embodiments the carrier fluid may be a gas.

[0024] In some embodiments, the exit orifice may be sized to accommodate at least 5 microdroplets in parallel. In some embodiments, the exit orifice may be sized to accommodate at least 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more microdroplets in parallel.

[0025] The wide structure of the exit orifice allows for several parallel trains of micro-entities to enter the structure unimpeded. Preferably, there can be between 1 to 50 parallel trains of microdroplets entering the exit orifice at the same time. This configuration provides an exit orifice with a droplet-offloading capacity at least as high as the capacity of the inlet loading structures for the chip. It allows micro-entities to be supplied from a highly parallel storage array without a time-consuming rearrangement to enable sequential removal. This makes chip re-use more practical.

[0026] The exit orifice may comprise a single gap which makes optimal use of the space and provides simplicity of manufacture and alignment. Alternatively, the exit orifice may be configured from a series of gaps, a sieve, a semi-permeable membrane, such as a pillar, to enable the microdroplet and / or the micro-entity to enter the dispense region of the chip.

[0027] In some embodiments, the dispense region may be configured to provide a flow of carrier fluid substantially at an angle of between 45° and 80° to the exit orifice. In other embodiments, the angle can be anything between 0° and 180° although the extremes of this range are less effective than the range of 45° to 80°. In some embodiments, the dispense region may be configured to provide a flow of carrier fluid substantially at an angle of between 60 to 70 degrees to the exit orifice. This ensures that the directionality of the flow of the carrier fluid is directed towards the outlet of the dispense region. Preferably, the optimised angle is between 60 to 70 degrees. The optimised angle ensures that all microdroplets and / or micro-entities within the dispense region moved by the carrier fluid flow out of the chip via the outlet. Thus, no microdroplets are left in the dispense region. The chip according to the present invention as disclosed herein may further comprise a plurality of carrier fluid inlets for introducing the carrier fluid into the dispense region.

[0028] In some embodiments, the chip may comprise two symmetrically positioned carrier fluid inlets on either side of an outlet provided in the dispense region. This may enable easier manufacturing of the dispense region of the chip.

[0029] In some embodiments, the movement of microdroplets and / or micro-entities through the dispense region and out of the chip is flow-driven via a carrier fluid at a high velocity.

[0030] Typically, the carrier fluid flow in the dispense region has a flow rate of between 5 to 100 pL / min, or it can be more than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 pL / min. In some embodiments, the flow rate of the carrier fluid in the dispense region can be less than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 or 5 pL / min.

[0031] In some embodiments, the dispense region may comprise a cavity with a depth of between 100pm to 200pm, or it may be more than 100, 110, 120, 130, 140, 150, 160, 170, 180 or 190 pm. In some embodiments, the dispense region may comprise a cavity with a depth of less than 200, 190, 180, 170, 160, 150, 140, 130, 120 or 110 pm.

[0032] In some embodiments, the movement of microdroplets and / or micro-entities through the dispense region and out of the chip can be buoyancy-driven. In this instance, the microdroplets and / or micro-entities can be driven to the edge of the cavity and float, under their buoyancy in the carrier fluid flow, into the cavity and out towards the outlet of the dispense region. This embodiment of the invention has an advantage in that the buoyant force acts to propel the microdroplets and / or micro-entities out of the chip more quickly than flow alone.

[0033] Furthermore, the movement of microdroplets and / or micro-entities through the dispense region and out of the chip can be buoyancy-driven requiring less oil to be consumed in order to purge microdroplets and / or micro-entities from the chip.

[0034] In some embodiments, the force can be, but is not limited to, an optically-mediated or a non-optically-mediated force. In some embodiments, the force can be dielectrophoresis (DEP), EWOD and / or oEWOD. The microdroplet which may contain one or more micro-entities can be moved through the exit orifice and into the dispense region of the chip. In some embodiments, empty microdroplets i.e. microdroplets that do not contain a micro-entity can be moved through the exit orifice using a force, such as DEP, EWOD or oEWOD (as appropriate) and into the dispense region of the chip.

[0035] In some embodiments, the sequestration pen may contain one or more micro-entities. In this instance, a plurality of sequestration pens is located within the microfluidic space. The contents of the sequestration pen i.e. the micro-entity or entities contained within the sequestration pen, can be moved out of the pen and through the exit orifice and into the dispense region under a force, such as an optically-mediated force or non-optically-mediated force.

[0036] The micro-entity or micro-entities can be one or more of the following: a microdroplet; a cell, a part of a cell and / or a bead such as a microbead, an organelle, an organism, a liposome or a biosynthetic structure, a micro-capsule, a gel bead, a tentagel bead or a vesicle. Where more than one micro-entity is provided, the micro-entities may be of the same type or differing types. Multiple micro-entities may be agglomerated together in any appropriate manner including, but not limited to a micro-entity adhered to the surface of another micro-entity such as a cell attached to the surface of a bead; a micro-entity encapsulated in another micro-entity such as a cell, organelle or vesicle contained within a microdroplet.

[0037] Provided herein are methods for selecting cell(s) for investigating secretion(s) from a single cell(s) or cell-cell interactions. For example, the methods may comprise targeted cell-cell interactions where two or more selected cells are brought together. It may be desirable to analyse any kind of cell using the methods of the present invention, but the cells may be of the same type, for example they are B cells or T cells (lymphocytes). The cell(s) may be natural or it may be artificial. The cell(s) may be microcells. The methods of the invention may be cell free and use part(s) of a cell(s), for example nuclei and / or mitohbrichondria. The cell may be a cell from a human or animal, optionally a mammal, a plant cell, insect cell, fungal cell, bacterial cell, ameobal cell, a yeast, macrophage or hybridoma, and are selected from, but are not limited to: CHO, Jurkat, CAMA, HeLa, B-cell, T-cell, MCF-7, MDAMB-231 , E. coli and Salmonella. The cell may be a cell-fusion such as a hybridoma. The cells may be taken from a cell culture, for example a culture of stem cells, pluripotent cells, genetically engineered cells and the like.

[0038] If the micro-entity e.g. a cell is derived from a sample, this may be any human, animal, environmental (natural, contrived or modified), or food sample containing at least one micro-entity type e.g. cell type. The sample may be selected from: stool, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humour, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, faecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mammary secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alternatively, the sample may come from a tissue sample.

[0039] The cell may be isolated from a patient or individual. The chip of the present invention as described herein may be used to screen such cells and return them to the patient (autologous cell transfer). The cells may be isolated from one individual and selected to be administered to a patient (allogenic cell transfer).

[0040] For some embodiments, the panel of microdroplets containing at least one cell contain cells of the same type, for example lymphocytes such as T cells. Therefore, the cells may be pre-selected prior to their inclusion into microdroplets. However, some contamination may occur with any biological cells wherein cells of a different type may also be included within the microdroplets, for example, B cells when T cells are the desired type.

[0041] For some embodiments, there may be a diverse population of cell types included in the panel of microdroplets, such as if an environmental sample is being screened with unknown bacterial cells present.

[0042] The cell may be a human or mammalian cell. The cell may be any suitable type from any tissue type, such as from organ or tissue of the body.

[0043] The cell may be an immune system cell. Such cells include monocytes, macrophages, osteoclasts, neutrophils (polymorphonuclear leukocytes) dendritic cells, microglial cells, mast cells, T cells (including helper T cells, regulatory T cells, cytotoxic T cells and natural killer T cells), B cells, natural killer cells and hematopoietic stem cells.

[0044] The cell can be a CHO cell or it can be a Jurkat cell. In some examples, Chinese hamster ovary (CHO) cells are modified to produce an immuno-therapeutic drug (e.g. a TCR) and then emulsified into microdroplets and loaded onto the microfluidic platform. Empty or multi-occupancy microdroplets are discarded. The remaining microdroplets containing single CHO cells are incubated on-chip to promote the production of the immunotherapeutic drug. The CHO containing droplets may then be split to obtain multiple doses of the drug produced by each cell. T cells and target tumour cells are separately emulsified and loaded into arrays onto the microfluidic platform, adjusting the cell occupancy of each microdroplet as desired. The T cell and tumour cell arrays are then merged. A second merge operation is used to add a dose of immunotherapeutic drug, tracking which CHO cell each dose came from. The resulting assay is incubated and T-cell killing behaviour is monitored using the detection system by detecting caspase 3 / 7 fluorescence, a fluorescent marker of apoptosis. CHO cells that produced effective doses of the test drug can be dispensed from the device into a well plate.

[0045] The cell may be a pluripotent or stem cell, isolated or prepared via culturing techniques. The pluripotent stem cells may be reprogrammed mature cell types.

[0046] The cell may be genetically engineered prior to encapsulation into the microdroplet. The cell may be genetically engineered after encapsulation in the microdroplet.

[0047] Genetic engineering of the cell may be by any suitable method, including transduction (viral gene transfer), gene editing (using a nuclease such as zinc finger nucleases, TALEN, CRISPR / Cas9 base and prime editing) non-viral gene delivery (such as nanoparticle delivery), gene knock down, gene knock in and gene manipulation using RNA, for example, such as gene silencing or activation, or optogenetics. The genetic engineering generally involves the introduction of a genetic element into the cell, by any suitable means.

[0048] In some embodiments, the biological and / or chemical entity is any one or more of the following entities: an antibody; an antigen; a receptor; a substrate; an enzyme; a ligand; a nucleic acid; a cell; a part of a cell; an extracellular vesicle; a liposome; a polymer; a chemical; a drug; a FRET reporter; a chemiluminescent material; a sample of tissue; a virus or bacteriophage; a cytokine; and / or a protein. According to another aspect of the present invention, there is provided an EWOD or oEWOD chip according to the previous aspect, comprising a plurality of dispense regions.

[0049] In some embodiments, the microfluidic space may comprise a single exit orifice leading into a plurality of dispense regions. In some embodiments, the exit orifice may be divided into multiple sub-regions. The exit orifice can be sized to accommodate more than two microdroplets in parallel moving into the plurality of dispense regions under EWOD or oEWOD control.

[0050] It will be apparent and / or known to the skilled person in the art that the number of dispense regions provided in one chip can be adjusted for example, by increasing or decreasing the number of dispense regions provided on the chip in order to ensure that there is sufficient capacity to remove microdroplets out of the chip at an efficient rate.

[0051] According to an aspect of the present invention, there is provided a species screened by the chip, apparatus or method as disclosed herein.

[0052] According to an aspect of the present invention, there is provided a species selected by the chip, apparatus or method as disclosed herein.

[0053] According to an aspect of the present invention, there is provided a species isolated by the chip, apparatus or method as disclosed herein.

[0054] According to an aspect of the present invention, there is provided a species made by the chip, apparatus or method as disclosed herein.

[0055] The species may be chemical, biochemical, or biological in nature and may be of any size. The micro-entity is a species of micro dimension.

[0056] For example, the present invention may provide an agonist / antagonist to a micro-entity as identified by the screening, selection and / or isolation method disclosed herein.

[0057] The present invention may provide an agonist / antagonist to a micro-entity as identified by the screening, selection and / or isolation method disclosed herein, for use in therapy.

[0058] According to an aspect of the present invention, there is provided a use of the chip, apparatus, method or species as disclosed herein. According to an aspect of the present invention, there is provided a use of the chip, apparatus, method or species as disclosed herein in therapy.

[0059] The present invention may provide for a use of the chip, apparatus, method or species as disclosed herein in making a product. The product made may be chemical, biochemical, or biological in nature.

[0060] The use may be peptide synthesis.

[0061] The use may be synthetic biology.

[0062] The use may be cell line engineering or development.

[0063] The use may be cell therapy.

[0064] The use may be drug discovery.

[0065] The use may be antibody discovery.

[0066] According to an aspect of the present invention, there is provided a use of the chip, apparatus, method or species as disclosed herein in analysis.

[0067] The analysis may be physical, chemical, or biological.

[0068] The use may be sub-cellular imaging.

[0069] The use may be high content imaging.

[0070] The use may be diagnostics.

[0071] The use may be a biological assay.

[0072] The biological assay may be high throughput screening.

[0073] The biological assay may be ELISA.

[0074] The use may be cell secretion.

[0075] The use may be QC safety profiling.

[0076] The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawings, in which: Figure 1 shows a chip, such as an EWOD or oEWOD chip, according to the present invention;

[0077] Figure 2 shows the movement of microdroplets within the EWOD or oEWOD chip according to the present invention;

[0078] Figure 3 shows an alternative embodiment of the EWOD or oEWOD chip according to the present invention;

[0079] Figure 4 provides a side view of the EWOD or oEWOD chip according to the present invention;

[0080] Figure 5 shows an embodiment of the chip containing a plurality of dispense regions; and

[0081] Figure 6A and 6B show an alternative embodiment of the chip containing a plurality of dispense regions.

[0082] Referring to Figure 1 , there is shown a chip 10, such as an EWOD or oEWOD chip, comprising a microfluidic space 12 containing a carrier fluid in which an array of microdroplets and / or an array of micro-entities, such as cells, can be localised and manipulated using a force, for example EWOD or oEWOD forces (as appropriate). The micro-entities may be contained within a microdroplet that encapsulates one or more micro-entity and moves with that which it encapsulates. Alternatively, the microentity can be encapsulated within the micro-object. For example, a cell can be contained within a microdroplet and / or it can be contained within a sequestration pen.

[0083] The microfluidic space 12 has an exit orifice 14 that is sized to accommodate more than two microdroplets in parallel moved under EWOD or oEWOD. In some instances, five or more microdroplets in parallel can be moved through the exit orifice 14 under EWOD or oEWOD. The chip 10 further comprises a dispense region 16, which is configured to provide a flow of carrier fluid substantially non-parallel to the exit office 14. The microdroplets within the dispense region 16 are released from EWOD or oEWOD control. The high flow velocity of the carrier fluid provided within the dispense region 16 is configured to move the plurality of microdroplets through the dispense region 16 and out of the chip 10. As shown in Figure 1 , a carrier inlet channel 26 can be provided on either side of the dispense region 16. The carrier inlet channel 26 comprises an inlet port 25 and is configured to introduce a carrier fluid flow into the dispense region 16.

[0084] Referring to Figure 2, there is shown an EWOD or oEWOD chip 10 comprising a microfluidic space 12, an exit orifice 14 and a dispense region 16. The microfluidic space receives a plurality of microdroplets 18. The microdroplets 18 can be arranged in an array within the microfluidic space 12 as shown in Figure 2. At least a subset of the microdroplets 18 may undergo one or more of the following EWOD or oEWOD operations or manipulation including: holding, merging, splitting or discarding at least a subset of microdroplets in an array and / or an entire array of microdroplets. In some cases, a subset of microdroplets in the array or an entire array of microdroplets may be held stationary under EWOD or oEWOD forces. In some cases, EWOD or oEWOD operation may include sequential merging of microdroplets. For example, to augment at least a subset of microdroplets in the array with further microdroplets that may contain micro-entities or other species. In addition, the carrier fluid flow within the microfluidic space 12 of the chip 10 is substantially at zero velocity.

[0085] At least a subset of microdroplets 18 within an array in the microfluidic space 12 may be selected and moved towards the exit orifice 14, as indicated by the arrow 20 in Figure 2. The exit orifice 14 has a width that is sufficiently wide in the vertical and / or horizontal direction to accommodate two or more microdroplets 18 moving through the exit orifice 14 simultaneously under EWOD or oEWOD. Preferably, the exit orifice 14 has a sufficient width to accommodate up to one hundred microdroplets 18 in parallel. For example, the exit orifice 14 can accommodate between 2 to 5, 2 to 10, 2 to 20, 2 to 50 or 2 to 60, 2 to 75, 2 to 80 or 2 to 90 microdroplets 18 in parallel.

[0086] Typically, the exit orifice 14 can accommodate 30 microdroplets in parallel. In some instances, the exit orifice 14 has a sufficient width to accommodate at least 10 microdroplets in parallel. At the junction 22 between the exit orifice and the dispense region 16, the microdroplets 18 may be released from EWOD or oEWOD control as they enter the dispense region 16. The exit orifice 14 enables the constant and controlled delivery of microdroplets under EWOD or oEWOD control into the dispense region 16. The width of the exit orifice 14 is important because it allows for several parallel trains of microdroplets 18 to enter unimpeded. As shown in Figure 2, an area in the dispense region 16 contains a high-velocity carrier phase flow directed towards an outlet 24 in the chip 10. The dispense region 16 as disclosed in the present invention has an optimal shape and orientation such that there is a near-zero component of flow directed towards the exit orifice 14. The angle of the carrier phase inlets 26, shown in Figure 2, is optimised such that the carrier fluid flows are introduced into the dispense region 16 in the direction towards the outlet 24. The high velocity carrier fluid will carry the microdroplets 18 within the dispense region 16 towards the outlet 24 and out of the chip 10.

[0087] Optimally the angle a of the inlets 26 for introducing the carrier fluid flows into the dispense region 16 is between 60 to 70 degrees with respect to the exit orifice 14. Furthermore, the width of the carrier phase inlets 26 is optimally between 100pm and 200pm. The width of the inlets 26 may determine the peak velocity at the edge of the dispense region 16.

[0088] Referring to Figure 3, there is shown an alternative embodiment of an EWOD or oEWOD chip 10 according to the present invention. Figure 3 shows the EWOD or oEWOD chip 10 comprising a microfluidic space 12, an exit orifice 14 and a dispense region 16. The dispense region 16 can comprise a substrate (not shown in the accompanying drawing) in which a cavity 28 is provided on top of the substrate. The cavity 28 can have a depth of between 100 to 200pm. Typically, the cavity 28 can be an approximately 150pm deep space and ablated into the substrate, such as a glass substrate, of the EWOD or oEWOD chip 10.

[0089] In use, microdroplets 18 can be driven to the edge of the cavity 28 of the dispense region 16 and float under their buoyancy in the carrier fluid flow into the cavity 28. As shown in Figures 3 and 4, the carrier fluid flow enters the dispense region 16 through the carrier inlets 26 to move the microdroplets out of the chip 10 via an outlet 24.

[0090] As illustrated in Figure 4, the outlet 24 further comprises a conduit 30, such as a tube to control the direction of microdroplets 18 transportation out of the EWOD or oEWOD chip 10. The microdroplet 18 can exit the chip 10 via a hole 31 before entering the conduit 30. As shown in Figure 3, the carrier phase inlets 26 comprises an opening32 to introduce a carrier phase into the channel 26. The chip 10 can comprise more than one layer. The chip may be made of multi-layers or it can be made from a single layer. As illustrated in Figure 4, the chip 10 can be made from a first layer 33 and a second layer 34. The first and second layers 33, 34 can be made from the same material or they can be made from different materials.

[0091] The configuration shown in Figures 3 and 4 has the advantage that the buoyant force acts to propel microdroplets 18 out of the chip 10 more quickly than flow alone. Furthermore, less oil carrier fluid flow is required to move the microdroplets 18 out of the chip 10.

[0092] Referring to Figure 5, there is shown a chip 10, such as an EWOD or oEWOD chip, comprising a microfluidic space 12, an exit orifice 14 and a plurality of dispense regions 16. Each dispense region 16 has two symmetrical inlets for introducing a carrier fluid flow into the dispense region, such that micro-entities which may or may not be encapsulated in microdroplets are moved out of the chip under flow of the carrier fluid.

[0093] The arrangement of the plurality of dispense regions 16 provided on the chip 10, as shown in Figure 5, can be particularly useful for moving the micro-entities out of the chip 10 at high speed.

[0094] Referring to Figures 6A and 6B, there is shown a chip 10, such as an EWOD or oEWOD chip, comprising a microfluidic space 12, an exit orifice 14 and a plurality of dispense regions 16. In this embodiment, the dispense regions 16 comprise a cavity 28. The cavity 28 can have a depth of between 100 to 200pm. In use, a micro-entity, which may or may not be encapsulated in a microdroplet, can be driven to the edge of the cavity 28 of the dispense region 16 and float, as a result of their buoyancy in the carrier fluid flow, into the cavity 28.

[0095] A carrier fluid can then be subsequently introduced into the dispense region via the two inlets provided on either side of the dispense region 16 to drive the micro entities out of the dispense region through an outlet. The arrangement of the plurality of dispense regions 16 provided on the chip 10, as shown in Figures 6A and 6B, can be particularly useful for moving the micro-entities out of the chip 10 in a high throughput manner.

[0096] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0097] “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0098] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments, which are described.

[0099] It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments, it is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.

Claims

CLAIMS1 . A chip for manipulation of a plurality of micro-entities, the chip comprising: a microfluidic space configured to accommodate a plurality of micro-entities in a substantially planar array; wherein the height of the microfluidic space in the plane perpendicular to the array is sized to accommodate a single layer of micro-entities; wherein the microfluidic space contains a carrier fluid in which the array of microentities can be localised and manipulated using a force; wherein the microfluidic space has an exit orifice that extends a distance in the plane of the array that is at least double the height of the microfluidic space so that two or more micro-entities in parallel can be moved through the exit orifice simultaneously under the force; a dispense region configured to provide a flow of carrier fluid substantially non-parallel to the exit orifice so that the micro-entities are moved by the carrier fluid through the dispense region and out of the chip.

2. The chip according to claim 1 , wherein the dispense region is shaped and configured in order to minimise the flow of carrier fluid into the microfluidic space.

3. The chip according to any one of the preceding claims, wherein the dispense region is shaped and configured in order to maximise the rate at which micro-entities can be moved through the dispense region and out of the chip.

4. The chip according to any one of the preceding claims, wherein the microentities are microdroplets and the exit orifice extends a distance in the plane of the array that is at least five times the height of the microfluidic space so as to accommodate five or more microdroplets in parallel.

5. The chip according to any one of the preceding claims, wherein the exit orifice comprises a single gap; a series of gaps; a sieve; and / or a semi-permeable membrane.

6. The chip according to any one of the preceding claims, wherein the dispense region is configured to provide a flow of carrier fluid substantially at an angle of less than 70 degrees to the exit orifice.

7. The chip according to any one of the preceding claims, wherein the dispense region is configured to provide a flow of carrier fluid substantially at an angle of between 60 to 70 degrees to the exit orifice.

8. The chip according to any one of the preceding claims, further comprising a plurality of carrier fluid inlets for introducing the carrier fluid into the dispense region.

9. The chip according to claim 8, wherein the chip comprises two symmetrically positioned carrier fluid inlets either side of an outlet provided in the dispense region.

10. The chip according to any one of the preceding claims, wherein the movement of micro-entities through the dispense region and out of the chip is flow-driven.11 . The chip according to claims 1 to 9, wherein the dispense region comprises a cavity with a depth of between 100pm to 200pm.

12. The chip according to claim 11 , wherein the movement of micro-entities through the dispense region and out of the chip is buoyancy-driven.

13. The chip according to any one of claims 1 to 3, wherein each micro-entity is one of the following: a cell, a part of a cell, a bead, an organelle, an organism, a liposome, a biosynthetic structure or a vesicle.

14. The chip according to claim 13, wherein at least one micro-entity is localised in at least one sequestration pen.

15. The chip according to claim 4, wherein one or more micro-entities are encapsulated in at least a subset of the microdroplets.

16. The chip according to any one of the preceding claims, wherein the force is electrowetting, EWOD or oEWOD.

17. The chip according to any one of claims 1 to 16, comprising a plurality of dispense regions.

18. Use of a chip according to any one of the preceding claims.

19. A method of emptying a chip according to any one of claims 1 to 17, comprising the steps of: applying a force to move two or more micro-entities in parallel through the exit orifice; and providing a flow of carrier fluid substantially non-parallel to the exit orifice so that the micro-entities are moved by the carrier fluid through the dispense region and out of the chip.

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